A promising therapeutic approach for Duchenne muscular dystrophy (DMD) is exon skipping using antisense oligonucleotides (AOs). In-frame deletions of the hinge 3 region of the dystrophin protein, ...which is encoded by exons 50 and 51, are predicted to cause a variety of phenotypes. Here, we performed functional analyses of muscle in the exon 52–deleted mdx (mdx52) mouse, to predict the function of in-frame dystrophin following exon 51-skipping, which leads to a protein lacking most of hinge 3. A series of AOs based on phosphorodiamidate morpholino oligomers was screened by intramuscular injection into mdx52 mice. The highest splicing efficiency was generated by a two-oligonucleotide cocktail targeting both the 5′ and 3′ splice sites of exon 51. After a dose-escalation study, we systemically delivered this cocktail into mdx52 mice seven times at weekly intervals. This induced 20–30% of wild-type (WT) dystrophin expression levels in all muscles, and was accompanied by amelioration of the dystrophic pathology and improvement of skeletal muscle function. Because the structure of the restored in-frame dystrophin resembles human dystrophin following exon 51-skipping, our results are encouraging for the ongoing clinical trials for DMD. Moreover, the therapeutic dose required can provide a suggestion of the theoretical equivalent dose for humans.
Downsizing nanocarriers is a promising strategy for systemically targeting fibrotic cancers, such as pancreatic cancer, owing to enhanced tissue permeability. We recently developed a small ...oligonucleotide nanocarrier called a unit polyion complex (uPIC) using a single oligonucleotide molecule and one or two molecule(s) of two-branched poly(ethylene glycol)-b-poly(l-lysine) (bPEG-PLys). The uPIC is a dynamic polyion-pair equilibrated with free bPEG-PLys, and thus, is highly stabilized in the presence of excess amounts of free bPEG-PLys in the bloodstream. However, the dynamic polyion-pairing behavior of uPICs needs to be further investigated for longevity in the bloodstream, especially under lower amounts of free bPEG-PLys. Herein, the polyion-pairing behavior of uPICs was investigated by highlighting oligonucleotide stability and negative charge number. To this end, small interfering RNA (siRNA) and antisense oligonucleotides (ASO) were chemically modified to acquire nuclease resistance, and the ASO was hybridized with complementary RNA (cRNA) to form a hetero-duplex oligonucleotide (HDO) with twice the negative charges. While all oligonucleotides similarly formed sub-20 nm-sized uPICs from a single oligonucleotide molecule, the association number of bPEG-PLys (ANbPEG-PLys) in uPICs varied based on the negative charge number of oligonucleotides (N−), that is, ANbPEG-PLys = ~2 at N− = ~40 (i.e., siRNA and HDO) and ANbPEG-PLys = ~1 at N− = 20 (i.e., ASO), presumably because of the balanced charge neutralization between the oligonucleotide and bPEG-PLys with a positive charge number (N+) of ~20. Ultimately, the uPICs prepared from the chemically modified oligonucleotide with higher negative charges showed considerably longer blood retention than those from the control oligonucleotides without chemical modifications or with lower negative charges. The difference in the blood circulation properties of uPICs was more pronounced under lower amounts of free bPEG-PLys. These results demonstrate that the chemical modification and higher negative charge in oligonucleotides facilitated the polyion-pairing between the oligonucleotide and bPEG-PLys under harsh biological conditions, facilitating enhanced blood circulation of uPICs.
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•A single oligonucleotide molecule was precisely loaded into an ultra-small nanocarrier with Y-shaped block copolymer(s).•Chemical modifications of oligonucleotides dramatically enhanced the blood circulation property of the ultra-small nanocarrier, especially in the presence of relatively lower amounts of Y-shaped block copolymers.•A double-stranded oligonucleotide also prolonged the blood circulation of the ultra-small nanocarrier, compared with a single-stranded one.•The oligonucleotide structures played a crucial role for the pharmacokinetics of the ultra-small nanocarriers.
•Vapor pressures of AgBr were measured by the Knudsen effusion method.•The vapor species of AgBr were considered to be monomers and trimers.•The partial pressures of AgBr and Ag3Br3 were fitted by ...linear equations.
The vapor pressure of silver bromide (AgBr) liquid was measured by the Knudsen effusion method. The ratio of the vapor species of silver bromide was calculated from the literature Hildenbrand and Lau, Pankratz 3,6, and the vapor pressures were calculated on the basis of the ratio. As reported in the literature Hildenbrand and Lau 3, we considered the vapor species to be monomers (AgBr) and trimers (Ag3Br3). The partial pressures of AgBr pmonomer(Pa) and Ag3Br3 ptrimer(Pa) were respectively fitted by the following linear equations over the temperature range 829.9–929.9 K:
lnpmonomer= -(23590 ± 519) / T + 25.22 ± 0.59,
ln(ptrimer) = -(19449 ± 509) / T + (20.42 ± 0.58).
The total vapor pressure of silver bromide ptotal(Pa) fitted the following linear equation:
ln(ptotal) = -(21653 ± 529) / T + (23.72 ± 0.60).
Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of ...frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45–55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45–55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45–55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45–55 skipping in a dystrophin-deficient animal model.
Abstract
A new method for measuring the global magnetic field structure of the Galactic plane is presented. We have determined the near-infrared polarization of field stars around 52 Cepheids found ...in recent surveys toward the Galactic plane. The Cepheids are located at the galactic longitudes $-10^{\circ} \le l \le +10.^{\!\!\!\circ }5$ and latitudes $-0.^{\!\!\!\circ }22\le b \le +0.^{\!\!\!\circ }45$, and their distances are mainly in the range of 10 to 15 kpc from the Sun. Simple classification of the sightlines is made with the polarization behavior vs. $H-K_{\mathrm{S}}$ color of field stars, and typical examples of three types are presented. Then, division of the field stars in each line of sight into (a) foreground, (b) bulge, and (c) background is made with the Gaia DR2 catalog, the peak of the $H-K_{\mathrm{S}}$ color histogram, and $H-K_{\mathrm{S}}$ colors consistent with the distance of the Cepheid in the center, respectively. Differential analysis between them enables us to examine the magnetic field structure more definitely than just relying on the $H-K_{\mathrm{S}}$ color difference. In one line of sight, the magnetic field is nearly parallel to the Galactic plane and well aligned all the way from the Sun to the Cepheid position on the other side of the Galactic center. Contrary to our preconceived ideas, however, sightlines having such well-aligned magnetic fields in the Galactic plane are rather small in number. At least 36 Cepheid fields indicate random magnetic field components are significant. Two Cepheid fields indicate that the magnetic field orientation changes more than $45^{\circ }$ in the line of sight. The polarization increase per color change $\Delta P/\Delta (H-K_{\mathrm{S}})$ varies from region to region, reflecting the change in the ratio of the magnetic field strength and the turbulence strength.
Duchenne muscular dystrophy (DMD) and the less severe Becker muscular dystrophy (BMD) are due to mutations in the DMD gene. Previous reports show that in-frame deletion of exons 45–55 produces an ...internally shorted, but functional, dystrophin protein resulting in a very mild BMD phenotype. In order to elucidate the molecular mechanism leading to this phenotype, we generated exon 45–55 deleted dystrophin transgenic/mdx (Tg/mdx) mice. Muscular function of Tg/mdx mice was restored close to that of wild type (WT) mice but the localization of the neuronal type of nitric oxide synthase was changed from the sarcolemma to the cytosol. This led to hyper-nitrosylation of the ryanodine receptor 1 causing increased Ca2+ release from the sarcoplasmic reticulum. On the other hand, Ca2+ reuptake by the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) was restored to the level of WT mice, suggesting that the Ca2+ dysregulation had been compensated by SERCA activation. In line with this, expression of sarcolipin (SLN), a SERCA-inhibitory peptide, was upregulated in mdx mice, but strongly reduced in Tg/mdx mice. Furthermore, knockdown of SLN ameliorated the cytosolic Ca2+ homeostasis and the dystrophic phenotype in mdx mice. These findings suggest that SLN may be a novel target for DMD therapy.
Abstract
Antisense oligonucleotide (ASO)-based therapy is one of the next-generation therapy, especially targeting neurological disorders. Many cases of ASO-dependent gene expression suppression have ...been reported. Recently, we developed a tocopherol conjugated DNA/RNA heteroduplex oligonucleotide (Toc-HDO) as a new type of drug. Toc-HDO is more potent, stable, and efficiently taken up by the target tissues compared to the parental ASO. However, the detailed mechanisms of Toc-HDO, including its binding proteins, are unknown. Here, we developed native gel shift assays with fluorescence-labeled nucleic acids samples extracted from mice livers. These assays revealed two Toc-HDO binding proteins, annexin A5 (ANXA5) and carbonic anhydrase 8 (CA8). Later, we identified two more proteins, apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) and flap structure-specific endonuclease 1 (FEN1) by data mining. shRNA knockdown studies demonstrated that all four proteins regulated Toc-HDO activity in Hepa1–6, mouse hepatocellular cells. In vitro binding assays and fluorescence polarization assays with purified recombinant proteins characterized the identified proteins and pull-down assays with cell lysates demonstrated the protein binding to the Toc-HDO and ASO in a biological environment. Taken together, our findings provide a brand new molecular biological insight as well as future directions for HDO-based disease therapy.
Abstract
The star S0-2, orbiting the Galactic central massive black hole candidate Sgr A$^\ast$, passed its pericenter in 2018 May. This event is the first chance to detect the general relativistic ...(GR) effect of a massive black hole, free from non-gravitational physics. The observable GR evidence in the event is the difference between the GR redshift and the Newtonian redshift of photons coming from S0-2. Within the present observational precision, the first post-Newtonian (1PN) GR evidence is detectable. In this paper, we give a theoretical analysis of the time evolution of the 1PN GR evidence, under a presupposition that is different from used in previous papers. Our presupposition is that the GR/Newtonian redshift is always calculated with the parameter values (the mass of Sgr A$^\ast$, the initial conditions of S0-2, and so on) determined by fitting the GR/Newtonian motion of S0-2 with the observational data. It is then revealed that the difference of the GR redshift and the Newtonian one shows two peaks before and after the pericenter passage. This double-peak appearance is due to our presupposition, and reduces to a single peak if the same parameter values are used in both GR and Newtonian redshifts as considered in previous papers. In addition to this theoretical discussion, we report our observational data obtained with the Subaru telescope by 2018. The quality and the number of Subaru data in 2018 are not sufficient to confirm the detection of the double-peak appearance.
Antisense oligonucleotide (ASO) is a major tool used for silencing pathogenic genes. For stroke in the hyperacute stage, however, the ability of ASO to regulate genes is limited by its poor delivery ...to the ischemic brain owing to sudden occlusion of the supplying artery. Here we show that, in a mouse model of permanent ischemic stroke, lipid-ligand conjugated DNA/RNA heteroduplex oligonucleotide (lipid-HDO) was unexpectedly delivered 9.6 times more efficiently to the ischemic area of the brain than to the contralateral non-ischemic brain and achieved robust gene knockdown and change of stroke phenotype, despite a 90% decrease in cerebral blood flow in the 3 h after occlusion. This delivery to neurons was mediated via receptor-mediated transcytosis by lipoprotein receptors in brain endothelial cells, the expression of which was significantly upregulated after ischemia. This study provides proof-of-concept that lipid-HDO is a promising gene-silencing technology for stroke treatment in the hyperacute stage.
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Takanori Yokota and colleagues demonstrated that systemic administration of α-tocopherol-ligand conjugated DNA/RNA heteroduplex oligonucleotide is selectively delivered into the ischemic brain region, even in the hyperacute phase after ischemic stroke, achieved significant gene-silencing efficacy and changed stroke phenotype in mice.
Observational data for the hourglass-like magnetic field toward the starless dense core FeSt 1-457 were compared with a flux freezing magnetic field model. Fitting of the observed plane-of-sky ...magnetic field using the flux freezing model gave a residual angle dispersion comparable to the results based on a simple 3D parabolic model. The best-fit parameters for the flux freezing model were a line-of-sight magnetic inclination angle of γmag = 35° 15° and a core center to ambient (background) density contrast of c/ bkg = 75. The initial density for core formation ( 0) was estimated to be c/75 = 4670 cm−3, which is about one order of magnitude higher than the expected density (∼300 cm−3) for the interclump medium of the Pipe Nebula. FeSt 1-457 is likely to have been formed from the accumulation of relatively dense gas, and the relatively dense background column density of AV 5 mag supports this scenario. The initial radius (core formation radius) R0 and the initial magnetic field strength B0 were obtained to be 0.15 pc (1.64R) and 10.8-14.6 G, respectively. We found that the initial density 0 is consistent with the mean density of the nearly critical magnetized filament with magnetic field strength B0 and radius R0. The relatively dense initial condition for core formation can be naturally understood if the origin of the core is the fragmentation of magnetized filaments.
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