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Exon-skipping therapy mediated by antisense oligonucleotides (ASOs) is expected to provide a therapeutic option for Duchenne muscular dystrophy (DMD). ASOs for exon skipping reported so far target a single continuous sequence in or around the target exon. In the present study, we investigated ASOs for exon 44 skipping (applicable to approximately 6% of all DMD patients) to improve activity by using a novel ASO design incorporating two connected sequences. Phosphorodiamidate morpholino oligomers targeting two separate sequences in exon 44 were created to simultaneously target two splicing regulators in exon 44, and their exon 44 skipping was measured. NS-089/NCNP-02 showed the highest skipping activity among the oligomers. NS-089/NCNP-02 also induced exon 44 skipping and dystrophin protein expression in cells from a DMD patient to whom exon 44 skipping is applicable. We also assessed thein vivoactivity of NS-089/NCNP-02 by intravenous administration to cynomolgus monkeys. NS-089/NCNP-02 induced exon 44 skipping in skeletal and cardiac muscle of cynomolgus monkeys. In conclusion, NS-089/NCNP-02, an ASO with a novel connected-sequence design, showed bothin vitroandin vivoexon-skipping activity.

Diabetic polyneuropathy (DPN) is the most common complication of diabetes, yet its pathophysiology has not been established. Accumulating evidence suggests that long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays pivotal roles in the regulation of cell growth and survival during diabetic complications. This study aimed to investigate the impact of MALAT1 silencing in dorsal root ganglion (DRG) sensory neurons, using an α-tocopherol–conjugated DNA/RNA heteroduplex oligonucleotide (Toc-HDO), on the peripheral nervous system of diabetic mice. We identified MALAT1 upregulation in the DRG of chronic diabetic mice that suggested either a pathological change or one that might be protective, and systemic intravenous injection of Toc-HDO effectively inhibited its gene expression. However, we unexpectedly noted that this intervention paradoxically exacerbated disease with increased thermal and mechanical nociceptive thresholds, indicating further sensory loss, greater sciatic-tibial nerve conduction slowing, and additional declines of intraepidermal nerve fiber density in the hind paw footpads. Serine/arginine-rich splicing factors, which are involved in pre-mRNA splicing by interacting with MALAT1, reside in nuclear speckles in wild-type and diabetic DRG neurons; MALAT1 silencing was associated with their disruption. The findings provide evidence for an important role that MALAT1 plays in DPN, suggesting neuroprotection and regulation of pre-mRNA splicing in nuclear speckles. This is also the first example in which a systemically delivered nucleotide therapy had a direct impact on DRG diabetic neurons and their axons.

Background: The co-administration of several therapeutic oligonucleotides targeting the same transcript is a beneficial approach. It broadens the target sites for diseases associated with various mutations or splice variants. However, little is known how a combination of antisense oligonucleotides (ASOs), which is one of the major modalities of therapeutic oligonucleotides, affects the potency. In this study, we aimed to elucidate the cocktail-effects of ASOs and the relationship between the target sites and potency of different combinations. Method and Results: We designed 113 ASOs targeting human superoxide dismutase 1 pre-mRNA and found 13 ASOs that had comparable silencing activity in vitro. An analysis of cocktail-effects on the silencing potency of 38 pairs of two ASOs on HeLa cells revealed that 30 pairs had comparable potency to that of two ASOs; on the other hand, eight pairs had reduced potency, indicating a negative impact on the activity. A reduced potency was seen in pairs targeting the same intron, exon-intron combination, or two different introns. The sequence distance of target sites was not the major determinant factor of cocktail-effects. In addition, a cocktail of three ASOs preserving the potency could be designed by avoiding two-ASO pairs, which had a reduced potency. Conclusions: This study revealed that more than half of the combinations retain their potency by paring two ASOs; in contrast, some pairs had a reduced potency. This could not be predicted only by the distance between the target sites.

Achieving regulation of endogenous gene expression in the central nervous system (CNS) with antisense oligonucleotides (ASOs) administered systemically would facilitate the development of ASO-based therapies for neurological diseases. We demonstrate that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol or α-tocopherol at the 5′ end of the RNA strand reach the CNS after subcutaneous or intravenous administration in mice and rats. The HDOs distribute throughout the brain, spinal cord and peripheral tissues and suppress the expression of four target genes by up to 90% in the CNS, whereas single-stranded ASOs conjugated to cholesterol have limited activity. Gene knockdown was observed in major CNS cell types and was greatest in neurons and microglial cells. Side effects, such as thrombocytopenia and focal brain necrosis, were limited by using subcutaneous delivery or by dividing intravenous injections. By crossing the blood–brain barrier more effectively, cholesterol-conjugated HDOs may overcome the limited efficacy of ASOs targeting the CNS without requiring intrathecal administration. Genes in the rodent brain are knocked down by DNA/RNA heteroduplexes injected intravenously.

DNA/RNA heteroduplex oligonucleotide (HDO), composed of DNA/locked nucleic acid (LNA) antisense oligonucleotide (ASO) and complementary RNA, is a next-generation antisense therapeutic agent. HDO is superior to the parental ASO in delivering to target tissues, and it exerts a more potent gene-silencing effect. In this study, we aimed to elucidate the intracellular trafficking mechanism of HDO-dependent gene silencing. HDO was more preferably transferred to the nucleus after transfection compared to the parental ASO. To determine when and where HDO is separated into the antisense strand (AS) and complementary strand (CS), we performed live-cell time-lapse imaging and fluorescence resonance energy transfer (FRET) assays. These assays demonstrated that HDO had a different intracellular trafficking mechanism than ASO. After endocytosis, HDO was separated in the early endosomes, and both AS and CS were released into the cytosol. AS was more efficiently transported to the nucleus than CS. Separation, endosomal release, and initiation of nuclear transport were a series of time-locked events occurring at a median of 30 s. CS cleavage was associated with efficient nuclear distribution and gene silencing in the nucleus. Understanding the unique intracellular silencing mechanisms of HDO will help us design more efficient drugs and might also provide insight into innate DNA/RNA cellular biology., Graphical Abstract, DNA/RNA heteroduplex oligonucleotide (HDO) is a newly developed antisense therapy. Here, Ono et al. demonstrated HDO was separated in and released from early endosomes and was efficiently transported into the nucleus. The double-stranded structure of HDO has dramatically changed the intracellular mechanism compared with its parent antisense oligonucleotide.

We recently reported the antisense properties of a DNA/RNA heteroduplex oligonucleotide consisting of a phosphorothioate DNA-gapmer antisense oligonucleotide (ASO) strand and its complementary phosphodiester RNA/phosphorothioate 2′-O-methyl RNA strand. When α-tocopherol was conjugated with the complementary strand, the heteroduplex oligonucleotide silenced the target RNA more efficiently in vivo than did the parent single-stranded ASO. In this study, we designed a new type of the heteroduplex oligonucleotide, in which the RNA portion of the complementary strand was replaced with phosphodiester DNA, yielding an ASO/DNA double-stranded structure. The ASO/DNA heteroduplex oligonucleotide showed similar activity and liver accumulation as did the original ASO/RNA design. Structure-activity relationship studies of the complementary DNA showed that optimal increases in the potency and the accumulation were seen when the flanks of the phosphodiester DNA complement were protected using 2′-O-methyl RNA and phosphorothioate modifications. Furthermore, evaluation of the degradation kinetics of the complementary strands revealed that the DNA-complementary strand as well as the RNA strand were completely cleaved in vivo. Our results expand the repertoire of chemical modifications that can be used with the heteroduplex oligonucleotide technology, providing greater design flexibility for future therapeutic applications., Graphical Abstract, Yokota, Seth, and colleagues have proposed a new concept of DNA/DNA double-stranded oligonucleotide, composed of a DNA-based antisense oligonucleotide and a DNA-based complementary strand. Systemic administration of this duplex conjugated with α-tocopherol significantly increased the target RNA-suppressing effect of the parent single-stranded antisense oligonucleotide following the degradation of complementary DNA in mouse liver.

Artificial nucleic acids are widely used in various technologies, such as nucleic acid therapeutics and DNA nanotechnologies requiring excellent duplex‐forming abilities and enhanced nuclease resistance. 2′‐O,4′‐C‐Methylene‐bridged nucleic acid/locked nucleic acid (2′,4′‐BNA/LNA) with 1,3‐diaza‐2‐oxophenoxazine (BNAP (BH)) was previously reported. Herein, a novel BH analogue, 2′,4′‐BNA/LNA with 9‐(2‐aminoethoxy)‐1,3‐diaza‐2‐oxophenoxazine (G‐clamp), named BNAP‐AEO (BAEO), was designed. The BAEO nucleoside was successfully synthesized and incorporated into oligodeoxynucleotides (ODNs). ODNs containing BAEO possessed up to 104‐, 152‐, and 11‐fold higher binding affinities for complementary (c) RNA than those of ODNs containing 2′‐deoxycytidine (C), 2′,4′‐BNA/LNA with 5‐methylcytosine (L), or 2′‐deoxyribonucleoside with G‐clamp (PAEO), respectively. Moreover, duplexes formed by ODN bearing BAEO with cDNA and cRNA were thermally stable, even under molecular crowding conditions induced by the addition of polyethylene glycol. Furthermore, ODN bearing BAEO was more resistant to 3′‐exonuclease than ODNs with phosphorothioate linkages., Stability by design: Artificial nucleic acids are widely used and in some applications require excellent duplex‐forming abilities and enhanced nuclease resistance. 2′‐O,4′‐C‐Methylene‐bridged nucleic acid/locked nucleic acid (2′,4′‐BNA/LNA) with G‐clamp (BNAP‐AEO) is synthesized and incorporated into oligodeoxynucleotides (ODNs). These modified ODNs show high thermal stabilities toward complementary DNA and RNA.

Cell-to-cell communications are critical determinants of pathophysiological phenotypes, but methodologies for their systematic elucidation are lacking. Herein, we propose an approach for the Systematic Elucidation and Assessment of Regulatory Cell-to-cell Interaction Networks (SEARCHIN) to identify ligand-mediated interactions between distinct cellular compartments. To test this approach, we selected a model of amyotrophic lateral sclerosis (ALS), in which astrocytes expressing mutant superoxide dismutase-1 (mutSOD1) kill wild-type motor neurons (MNs) by an unknown mechanism. Our integrative analysis that combines proteomics and regulatory network analysis infers the interaction between astrocyte-released amyloid precursor protein (APP) and death receptor-6 (DR6) on MNs as the top predicted ligand-receptor pair. The inferred deleterious role of APP and DR6 is confirmed in vitro in models of ALS. Moreover, the DR6 knockdown in MNs of transgenic mutSOD1 mice attenuates the ALS-like phenotype. Our results support the usefulness of integrative, systems biology approach to gain insights into complex neurobiological disease processes as in ALS and posit that the proposed methodology is not restricted to this biological context and could be used in a variety of other non-cell-autonomous communication mechanisms., Neuron-astrocyte communication plays a key role in pathophysiology, however systematic approaches to unveil it are limited. Here, the authors propose SEARCHIN, a multi-modal integrated workflow, as a tool to identify cross-compartment ligand-receptor interactions, applied to ALS models.

Purpose Pathological stimuli or injury to the peripheral nervous system can trigger neuropathic pain with common clinical features such as allodynia and hypersensitivity. Although various studies have identified molecules or genes related to neuropathic pain, the essential components are still unclear. Therefore, in this study, we investigated the molecular and genetic factors related to neuropathic pain. Methods We extracted candidate genes in the dorsal root ganglion (DRG) from three nerve injury mouse models and a sham-operated model (sciatic nerve ligation and resection, sural nerve resection, spared nerve injury [SNI], and sham) using DNA microarray to elucidate the genes responsible for the neuropathic pain mechanism in the SNI model, which exhibits hypersensitivity in the hindpaw of the preserved sural nerve area. We eliminated as many biases as possible. We then focused on an upregulated endogenous vasopressin receptor and clarified whether it is closely associated with traumatic neuropathic pain using a knockout mouse and drug-mediated suppression of the gene. Results Algorithm analysis of DNA microarray results identified 50 genes significantly upregulated in the DRG of the SNI model. Two independent genes-cyclin-dependent kinase-1 (CDK-1) and arginine vasopressin receptor 1A (V1a)-were subsequently identified as candidate SNI-specific genes in the DRG by quantitative PCR analysis. Administration of V1a agonist to wild-type SNI mice significantly alleviated neuropathic pain. However, V1a knockout mice did not exhibit higher hypersensitivity to mechanical stimulation than wild-type mice. In addition, V1a knockout mice showed similar pain behaviors after SNI to wild-type mice. Conclusion Through the DNA microarray analysis of several neuropathic models, we detected specific genes related to chronic pain. In particular, our results suggest that V1a in the DRG may partially contribute to the mechanism of neuropathic pain.

We present 850 $��$m polarimetric observations toward the Serpens Main molecular cloud obtained using the POL-2 polarimeter on the James Clerk Maxwell Telescope (JCMT) as part of the B-fields In STar-forming Region Observations (BISTRO) survey. These observations probe the magnetic field morphology of the Serpens Main molecular cloud on about 6000 au scales, which consists of cores and six filaments with different physical properties such as density and star formation activity. Using the histogram of relative orientation (HRO) technique, we find that magnetic fields are parallel to filaments in less dense filamentary structures where $N_{H_2} < 0.93\times 10^{22}$ cm$^{-2}$ (magnetic fields perpendicular to density gradients), while being perpendicular to filaments (magnetic fields parallel to density gradients) in dense filamentary structures with star formation activity. Moreover, applying the HRO technique to denser core regions, we find that magnetic field orientations change to become perpendicular to density gradients again at $N_{H_2} \approx 4.6 \times 10^{22}$ cm$^{-2}$. This can be interpreted as a signature of core formation. At $N_{H_2} \approx 16 \times 10^{22}$ cm$^{-2}$ magnetic fields change back to being parallel to density gradients once again, which can be understood to be due to magnetic fields being dragged in by infalling material. In addition, we estimate the magnetic field strengths of the filaments ($B_{POS} = 60-300~��$G)) using the Davis-Chandrasekhar-Fermi method and discuss whether the filaments are gravitationally unstable based on magnetic field and turbulence energy densities., 18 pages, accepted for publication in ApJ

Tao-Chung Ching et al., We present 850 μm dust polarization observations of the massive DR21 filament from the B-fields In STar-forming Region Observations (BISTRO) survey, using the POL-2 polarimeter and the SCUBA-2 camera on the James Clerk Maxwell Telescope. We detect ordered magnetic fields perpendicular to the parsec-scale ridge of the DR21 main filament. In the subfilaments, the magnetic fields are mainly parallel to the filamentary structures and smoothly connect to the magnetic fields of the main filament. We compare the POL-2 and Planck dust polarization observations to study the magnetic field structures of the DR21 filament on 0.1–10 pc scales. The magnetic fields revealed in the Planck data are well-aligned with those of the POL-2 data, indicating a smooth variation of magnetic fields from large to small scales. The plane-of-sky magnetic field strengths derived from angular dispersion functions of dust polarization are 0.6–1.0 mG in the DR21 filament and ∼0.1 mG in the surrounding ambient gas. The mass-to-flux ratios are found to be magnetically supercritical in the filament and slightly subcritical to nearly critical in the ambient gas. The alignment between column density structures and magnetic fields changes from random alignment in the low-density ambient gas probed by Planck to mostly perpendicular in the high-density main filament probed by James Clerk Maxwell Telescope. The magnetic field structures of the DR21 filament are in agreement with MHD simulations of a strongly magnetized medium, suggesting that magnetic fields play an important role in shaping the DR21 main filament and subfilaments., This work is supported by National Natural Science Foundation of China (NSFC) grant Nos. 11988101, U1931117, 11725313, and 12073061 and the CAS International Partnership Program of Chinese Academy of Sciences grant No. 114A11KYSB20160008. T.-C.C. is funded by Chinese Academy of Sciences Taiwan Young Talent Program grant No. 2018TW2JB0002. T.-C.C. and C.E. were supported by Special Funding for Advanced Users, budgeted and administrated by Center for Astronomical Mega-Science (CAMS), Chinese Academy of Sciences. K.P. is a Royal Society University Research Fellow, supported by grant No. URF\R1\211322. D.J. is supported by the National Research Council of Canada and by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant. P.M.K. is supported by the Ministry of Science and Technology (MoST) in Taiwan through grants 109-2112-M-001-022 and 110-2112-M-001-057. C.W.L. is supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2019R1A2C1010851), and by the Korea Astronomy and Space Science Institute grant funded by the Korea government (MSIT; Project No. 2022-1-840-05). T.H. is supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) through the Mid-career Research Program (2019R1A2C1087045). W.K. was supported by the NRF grant funded by the MSIT (2021R1F1A1061794). C.E. acknowledges the financial support from grant RJF/2020/000071 as a part of Ramanujan Fellowship awarded by the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Govt. of India. F.P. acknowledges support from the Spanish State Research Agency (AEI) under grant No. PID2019-105552RB-C43. M.T. is supported by JSPS KAKENHI grant Nos. 18H05442, 15H02063, and 22000005. J.K. is supported by JSPS KAKENHI grant No. 19K14775. L.F. and F.K. acknowledge the support by the MoST in Taiwan through grant 107-2119-M-001-031-MY3 and Academia Sinica through grant AS-IA-106-M03. L.F. acknowledges the support by the MoST in Taiwan through grants 111-2811-M-005-007 and 109-2112-M-005-003-MY3. C.L.H.H. acknowledges the support of the NAOJ Fellowship and JSPS KAKENHI grants 18K13586 and 20K14527. F.K. is supported by the Spanish program Unidad de Excelencia María de Maeztu CEX2020-001058-M, financed by MCIN/AEI/10.13039/501100011033. K.Q. is partially supported by National Key R&D Program of China No. 2022YFA1603100, and acknowledges the National Natural Science Foundation of China (NSFC) grant U1731237. S.P.L. acknowledges grants from the Ministry of Science and Technology of Taiwan 106-2119-M-007-021-MY3 and 109-2112-M-007-010-MY3. Y.D. acknowledges the support of JSPS KAKENHI grants 25247016 and 18H01250. Y.S.D. is supported by the National Key R&D Program of China for grant No. 2022YFA1605300, and NSFC grants Nos. 12273051, 11933003.

Hwang et al., We present and analyze observations of polarized dust emission at 850 μm toward the central 1 × 1 pc hub-filament structure of Monoceros R2 (Mon R2). The data are obtained with SCUBA-2/POL-2 on the James Clerk Maxwell Telescope (JCMT) as part of the B-fields in Star-forming Region Observations survey. The orientations of the magnetic field follow the spiral structure of Mon R2, which are well described by an axisymmetric magnetic field model. We estimate the turbulent component of the magnetic field using the angle difference between our observations and the best-fit model of the underlying large-scale mean magnetic field. This estimate is used to calculate the magnetic field strength using the Davis–Chandrasekhar–Fermi method, for which we also obtain the distribution of volume density and velocity dispersion using a column density map derived from Herschel data and the C18O (J = 3 − 2) data taken with HARP on the JCMT, respectively. We make maps of magnetic field strengths and mass-to-flux ratios, finding that magnetic field strengths vary from 0.02 to 3.64 mG with a mean value of 1.0 ± 0.06 mG, and the mean critical mass-to-flux ratio is 0.47 ± 0.02. Additionally, the mean Alfvén Mach number is 0.35 ± 0.01. This suggests that, in Mon R2, the magnetic fields provide resistance against large-scale gravitational collapse, and the magnetic pressure exceeds the turbulent pressure. We also investigate the properties of each filament in Mon R2. Most of the filaments are aligned along the magnetic field direction and are magnetically subcritical., J.H. is supported by the University of Science and Technology (UST) Overseas Training Program 2022, funded by the University of Science and Technology, Korea (No. 2022-017). K.P. is a Royal Society University Research Fellow, supported by grant number URF\R1\211322. C.W.L. was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF- 2019R1A2C1010851), and by the Korea Astronomy and Space Science Institute grant funded by the Korea government (MSIT; Project No. 2022-1-840-05). D.J. and J.d.F. are supported by the National Research Council of Canada and by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants. G.P. is supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF; 2020R1A6A3A01100208) W.K. was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT; NRF-2021R1F1A1061794). M.T. is supported by JSPS KAKENHI grant Nos. 18H05442, 15H02063, and 22000005. J.K. is supported by JSPS KAKENHI grant No. 19K14775. M.M. is supported by JSPS KAKENHI grant No. 20K03276. F.P. acknowledges support from the Spanish State Research Agency Agencia Estatal de Investigación (AEI) under grant number PID2019-105552RB-C43. S.P.L., H.Y.D., S.J.L., and M.Z.Y. acknowledge grants from the Ministry of Science and Technology of Taiwan 106-2119-M-007-021-MY3 and 109-2112-M-007-010-MY3. C.E. acknowledges the financial support from grant RJF/2020/000071 as a part of a Ramanujan Fellowship awarded by Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Govt. of India. L.F. and F.K. acknowledge support from the Ministry of Science and Technology of Taiwan, under grant No. MoST107-2119-M-001-031- MY3 and from Academia Sinica under grant No. AS-IA-106-M03. L.F. acknowledges the support by the MoST in Taiwan through grants 111-2811-M-005-007 and 109-2112-M-005-003-MY3. F.K. acknowledges support from the Spanish program Unidad de Excelencia María de Maeztu CEX2020-001058-M, financed by MCIN/AEI/10.13039/501100011033. Y.S.D. would like to acknowledge the support from NSFC grants 12273051 and 10878003. M.R. is supported by the international Gemini Observatory, a program of NSFs NOIRLab, which is managed by the Association of Universities for Research in Astronomy (AURA) under a cooperative agreement with the National Science Foundation, on behalf of the Gemini partnership of Argentina, Brazil, Canada, Chile, the Republic of Korea, and the United States of America. The JCMT is operated by the East Asian Observatory on behalf of The National Astronomical Observatory of Japan; Academia Sinica Institute of Astronomy and Astrophysics; the Korea Astronomy and Space Science Institute; the Operation, Maintenance and Upgrading Fund for Astronomical Telescopes and Facility Instruments, budgeted from the Ministry of Finance of China. Additional funding support is provided by the Science and Technology Facilities Council of the United Kingdom and participating universities and organizations in the United Kingdom, Canada, and Ireland. Additional funds for the construction of SCUBA-2 were provided by the Canada Foundation for Innovation.

For the simultaneous delivery of antisense oligonucleotides and their effector enzymes into cells, nanosized vesicular polyion complexes (PICs) were fabricated from oppositely charged polyion-pairs of oligonucleotides and poly(ethylene glycol) (PEG)-b-polypeptides. First, the polyion component structures were carefully designed to facilitate multimolecular (or secondary) association of unit PICs for non-covalent (or chemical crosslinking-free) stabilization of vesicular PICs. Chemically modified, single-stranded oligonucleotides (SSOs) dramatically stabilized the multimolecular associates under physiological conditions, compared to control SSOs without chemical modifications and duplex oligonucleotides. In addition, a high degree of guanidino groups in the polypeptide segment was also crucial for the high stability of multimolecular associates. Dynamic light scattering and transmission electron microscopy revealed the stabilized multimolecular associates to have a 100 nm-sized vesicular architecture with a narrow size distribution. The loading number of SSOs per nanovesicle was determined to be ~2,500 by using fluorescence correlation spectroscopic analyses with fluorescently labeled SSOs. Furthermore, the nanovesicle stably encapsulated ribonuclease H (RNase H) as an effector enzyme at ~10 per nanovesicle through simple vortex-mixing with preformed nanovesicles. Ultimately, the RNase H-encapsulated nanovesicle efficiently delivered SSOs with RNase H into cultured cancer cells, thereby eliciting the significantly higher gene knockdown compared with empty nanovesicles (without RNase H) or a mixture of nanovesicles with RNase H without encapsulation. These results demonstrate the great potential of non-covalently stabilized nanovesicles for the codelivery of two varying biomacromolecule payloads for ensuring their cooperative biological activity.

We present the results of simultaneous 450 $\mu$m and 850 $\mu$m polarization observations toward the massive star forming region NGC 2071IR, a target of the BISTRO (B-fields in Star-Forming Region Observations) Survey, using the POL-2 polarimeter and SCUBA-2 camera mounted on the James Clerk Maxwell Telescope. We find a pinched magnetic field morphology in the central dense core region, which could be due to a rotating toroidal disk-like structure and a bipolar outflow originating from the central young stellar object, IRS 3. Using the modified Davis-Chandrasekhar-Fermi method, we obtain a plane-of-sky magnetic field strength of 563$\pm$421 $\mu$G in the central $\sim$0.12 pc region from 850 $\mu$m polarization data. The corresponding magnetic energy density of 2.04$\times$10$^{-8}$ erg cm$^{-3}$ is comparable to the turbulent and gravitational energy densities in the region. We find that the magnetic field direction is very well aligned with the whole of the IRS 3 bipolar outflow structure. We find that the median value of polarization fractions, 3.0 \%, at 450 $\mu$m in the central 3 arcminute region, which is larger than the median value of 1.2 \% at 850 $\mu$m. The trend could be due to the better alignment of warmer dust in the strong radiation environment. We also find that polarization fractions decrease with intensity at both wavelengths, with slopes, determined by fitting a Rician noise model, of $0.59 \pm 0.03$ at 450 $\mu$m and $0.36 \pm 0.04$ at 850 $\mu$m, respectively. We think that the shallow slope at 850 $\mu$m is due to grain alignment at the center being assisted by strong radiation from the central young stellar objects., Comment: 17 pages, 11 figures, 4 tables

Microglial activation followed by recruitment of blood-borne macrophages into the central nervous system (CNS) aggravates neuroinflammation. Specifically, in multiple sclerosis (MS) as well as in experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, activated microglia and macrophages (Mg/Mφ) promote proinflammatory responses and expand demyelination in the CNS. However, a potent therapeutic approach through the systemic route for regulating their functions has not yet been developed. Here, we demonstrate that a systemically injected DNA/RNA heteroduplex oligonucleotide (HDO), composed of an antisense oligonucleotide (ASO) and its complementary RNA, conjugated to cholesterol (Chol-HDO) distributed more efficiently to demyelinating lesions of the spinal cord in EAE mice with significant gene silencing than the parent ASO. Importantly, systemic administration of Cd40-targeting Chol-HDO improved clinical signs of EAE with significant downregulation of Cd40 in Mg/Mφ. Furthermore, we successfully identify that macrophage scavenger receptor 1 (MSR1) is responsible for the uptake of Chol-HDO by Mg/Mφ of EAE mice. Overall, our findings demonstrate the therapeutic potency of systemically administered Chol-HDO to regulate activated Mg/Mφ in neuroinflammation.

We have obtained sensitive dust continuum polarization observations at 850 μm in the B213 region of Taurus using POL-2 on SCUBA-2 at the James Clerk Maxwell Telescope as part of the B-fields in STar-forming Region Observations (BISTRO) survey. These observations allow us to probe magnetic field (B-field) at high spatial resolution (∼2000 au or ∼0.01 pc at 140 pc) in two protostellar cores (K04166 and K04169) and one prestellar core (Miz-8b) that lie within the B213 filament. Using the Davis–Chandrasekhar–Fermi method, we estimate the B-field strengths in K04166, K04169, and Miz-8b to be 38 ± 14, 44 ± 16, and 12 ± 5 μG, respectively. These cores show distinct mean B-field orientations. The B-field in K04166 is well ordered and aligned parallel to the orientations of the core minor axis, outflows, core rotation axis, and large-scale uniform B-field, in accordance with magnetically regulated star formation via ambipolar diffusion taking place in K04166. The B-field in K04169 is found to be ordered but oriented nearly perpendicular to the core minor axis and large-scale B-field and not well correlated with other axes. In contrast, Miz-8b exhibits a disordered B-field that shows no preferred alignment with the core minor axis or large-scale field. We found that only one core, K04166, retains a memory of the large-scale uniform B-field. The other two cores, K04169 and Miz-8b, are decoupled from the large-scale field. Such a complex B-field configuration could be caused by gas inflow onto the filament, even in the presence of a substantial magnetic flux.

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.

AntimiR is an antisense oligonucleotide that has been developed to silence microRNA (miRNA) for the treatment of intractable diseases. Enhancement of its in vivo efficacy and improvement of its toxicity are highly desirable but remain challenging. We here design heteroduplex oligonucleotide (HDO)-antimiR as a new technology comprising an antimiR and its complementary RNA. HDO-antimiR binds targeted miRNA in vivo more efficiently by 12-fold than the parent single-stranded antimiR. HDO-antimiR also produced enhanced phenotypic effects in mice with upregulated expression of miRNA-targeting messenger RNAs. In addition, we demonstrated that the enhanced potency of HDO-antimiR was not explained by its bio-stability or delivery to the targeted cell, but reflected an improved intracellular potency. Our findings provide new insights into biology of miRNA silencing by double-stranded oligonucleotides and support the in vivo potential of this technology based on a new class of for the treatment of miRNA-related diseases.

Parkinson’s disease (PD) is a neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra. A characteristic pathological feature of PD is cytoplasmic accumulation of α-synuclein (SNCA) protein. Multiplication of the SNCA gene in familial PD and pathological accumulation of SNCA protein during progression of sporadic PD suggest that increased SNCA protein levels increase the risk of PD. Thus, reducing SNCA expression levels could delay PD onset or modify the disease course. For efficient knock down, we designed and synthesized an amido-bridged nucleic acids (AmNA)-modified antisense oligonucleotide (ASO) that targeted SNCA with improved stability and cellular uptake in vivo. AmNA-ASO efficiently downregulated SNCA at both the mRNA and protein level in vitro and in vivo. Notably, AmNA-ASO was efficiently delivered into the mouse brain by intracerebroventricular injection without the aid of additional chemicals. Furthermore, administration of AmNA-ASO ameliorated neurological defects in PD model mice expressing human wild type SNCA. Taken together, these findings suggest that AmNA-ASO is a promising therapeutic strategy for SNCA-associated pathology in PD.

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 [ p m o n o m e r ( P a ) ] and Ag3Br3 [ p t r i m e r ( P a ) ] were respectively fitted by the following linear equations over the temperature range 829.9–929.9 K: l n p m o n o m e r = - ( 23590 ± 519 ) / T + 25.22 ± 0.59 , l n ( p t r i m e r ) = - ( 19449 ± 509 ) / T + ( 20.42 ± 0.58 ) . The total vapor pressure of silver bromide [ p t o t a l ( P a ) ] fitted the following linear equation: l n ( p t o t a l ) = - ( 21653 ± 529 ) / T + ( 23.72 ± 0.60 ) .

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.