Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry.

Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities. To mitigate a broad spectrum of off-target interactions and enhance the safety profile of ASO drugs, we here devise a nanoarchitecture named BRace On a THERapeutic aSo (BROTHERS or BRO), which is composed of a standard gapmer ASO paired with a partially complementary peptide nucleic acid (PNA) strand. We show that these non-canonical ASO/PNA hybrids have reduced non-specific protein-binding capacity. The optimization of the structural and thermodynamic characteristics of this duplex system enables the operation of an in vivo toehold-mediated strand displacement (TMSD) reaction, effectively reducing hybridization with RNA off-targets. The optimized BROs dramatically mitigate hepatotoxicity while maintaining the on-target knockdown activity of their parent ASOs in vivo. This technique not only introduces a BRO class of drugs that could have a transformative impact on the extrahepatic delivery of ASOs, but can also help uncover the toxicity mechanism of ASOs.

Chemistry of Therapeutic Oligonucleotides That Drives Interactions with Biomolecules.

Oligonucleotide therapeutics that can modulate gene expression have been gradually developed for clinical applications over several decades. However, rapid advances have been made in recent years. Artificial nucleic acid technology has overcome many challenges, such as (1) poor target affinity and selectivity, (2) low in vivo stability, and (3) classical side effects, such as immune responses; thus, its application in a wide range of disorders has been extensively examined. However, even highly optimized oligonucleotides exhibit side effects, which limits the general use of this class of agents. In this review, we discuss the physicochemical characteristics that aid interactions between drugs and molecules that belong to living organisms. By systematically organizing the related data, we hope to explore avenues for symbiotic engineering of oligonucleotide therapeutics that will result in more effective and safer drugs.

Drug discovery and development scheme for liver-targeting bridged nucleic acid antisense oligonucleotides.

Antisense oligonucleotides (ASOs) containing bridged nucleic acids (BNAs) have been proven to be very powerful. However, ensuring a reliable discovery and translational development scheme for this class of ASOs with wider therapeutic windows remains a fundamental challenge. We here demonstrate the robustness of our scheme in the context of the selection of ASOs having two different BNA chemistries (2,'4'-BNA/locked nucleic acid [LNA] and amido-bridged nucleic acid [AmNA]) targeting human proprotein convertase subtilisin/kexin type 9 (PCSK9). The scheme features a two-step process, including (1) a unique and sensitive in vitro screening approach, called Ca2+ enrichment of medium (CEM) transfection, and (2) a ligand-targeted drug delivery approach to better reach target tissues, averting unintended accumulation of ASOs. Using CEM screening, we identified a candidate ASO that shows >70% cholesterol-lowering action in monkeys. An N-acetylgalactosamine (GalNAc) ligand then was appended to the candidate ASO to further broaden the therapeutic margin by altering the molecule's pharmacokinetics. The GalNAc conjugate, HsPCSK9-1811-LNA, was found to be at least ten times more potent in non-human primates (compared with the unconjugated counterpart), with reduced nephrotoxicity in rats. Overall, we successfully showed that our drug development scheme is better suited for selecting clinically relevant BNA-based ASOs, especially for the treatment of liver-associated diseases.

Programmed Instability of Ligand Conjugation Manifold for Efficient Hepatocyte Delivery of Therapeutic Oligonucleotides.

Ligand-targeted drug delivery (LTDD) has gained more attention in the field of nucleic acid therapeutics. To further elicit the potential of therapeutic oligonucleotides by means of LTDD, we newly developed (R)- and (S)-3-amino-1,2-propanediol (APD) manifold for ligand conjugation. N-acetylgalactosamine (GalNAc)/asialoglycoprotein receptor (ASGPr) system has been shown to be a powerful and robust paradigm of LTDD. Our novel APD-based GalNAc (GalNAcAPD) was shown to have intrinsic chemical instability that could play a role in better manipulation of active drug release. The APD manifold also enables facile production of conjugates through an on-support ligand cluster synthesis. We showed in a series of in vivo studies that while the knockdown activity of antisense oligonucleotides (ASOs) bearing 5'-GalNAcAPD was comparable to the conventional hydroxy-L-prolinol-linked GalNAc (GalNAcHP), 3'-GalNAcAPD elicited ASO activity by more than twice as much as the conventional 3'-GalNAcHP. This was ascribed partly to the GalNAcAPD's ideal susceptibility to nucleolytic digestion, which is expected to facilitate cytosolic internalization of ASO drugs. Moreover, an in vivo/ex vivo imaging study visualized the enhancement effect of monoantennary GalNAcAPD on liver localization of ASOs. This versatile manifold with chemical and biological instability would benefit therapeutic oligonucleotides that target both the liver and extrahepatic tissues.

Highly Potent GalNAc-Conjugated Tiny LNA Anti-miRNA-122 Antisense Oligonucleotides.

The development of clinically relevant anti-microRNA antisense oligonucleotides (anti-miRNA ASOs) remains a major challenge. One promising configuration of anti-miRNA ASOs called "tiny LNA (tiny Locked Nucleic Acid)" is an unusually small (~8-mer), highly chemically modified anti-miRNA ASO with high activity and specificity. Within this platform, we achieved a great enhancement of the in vivo activity of miRNA-122-targeting tiny LNA by developing a series of N-acetylgalactosamine (GalNAc)-conjugated tiny LNAs. Specifically, the median effective dose (ED50) of the most potent construct, tL-5G3, was estimated to be ~12 nmol/kg, which is ~300-500 times more potent than the original unconjugated tiny LNA. Through in vivo/ex vivo imaging studies, we have confirmed that the major advantage of GalNAc over tiny LNAs can be ascribed to the improvement of their originally poor pharmacokinetics. We also showed that the GalNAc ligand should be introduced into its 5' terminus rather than its 3' end via a biolabile phosphodiester bond. This result suggests that tiny LNA can unexpectedly be recognized by endogenous nucleases and is required to be digested to liberate the parent tiny LNA at an appropriate time in the body. We believe that our strategy will pave the way for the clinical application of miRNA-targeting small ASO therapy.

Understanding In Vivo Fate of Nucleic Acid and Gene Medicines for the Rational Design of Drugs.

Nucleic acid and genetic medicines are increasingly being developed, owing to their potential to treat a variety of intractable diseases. A comprehensive understanding of the in vivo fate of these agents is vital for the rational design, discovery, and fast and straightforward development of the drugs. In case of intravascular administration of nucleic acids and genetic medicines, interaction with blood components, especially plasma proteins, is unavoidable. However, on the flip side, such interaction can be utilized wisely to manipulate the pharmacokinetics of the agents. In other words, plasma protein binding can help in suppressing the elimination of nucleic acids from the blood stream and deliver naked oligonucleotides and gene carriers into target cells. To control the distribution of these agents in the body, the ligand conjugation method is widely applied. It is also important to understand intracellular localization. In this context, endocytosis pathway, endosomal escape, and nuclear transport should be considered and discussed. Encapsulated nucleic acids and genes must be dissociated from the carriers to exert their activity. In this review, we summarize the in vivo fate of nucleic acid and gene medicines and provide guidelines for the rational design of drugs.

Effect of modular conjugation strategy for N-acetylgalactosamine-targeted antisense oligonucleotides.

The asialoglycoprotein receptor (ASGPr) and N-acetylgalactosamine (GalNAc) is one of the most reliable receptor-ligand combinations for delivering antisense oligonucleotides (ASOs) to the liver. Here, we show that a modular GalNAc conjugation strategy allows us to reinforce the activity of the parent, naked 2',4'-BNA/LNA gapmer targeting apolipoprotein B. The conjugation partly reduced a possible hepatotoxicity of the parent ASO. The structure-activity study revealed the significance of the metabolic susceptibility of the GalNAc moiety to nucleolytic cleavage that results in exposure of the parent gapmer. The broad usefulness of our delivery strategy of ASOs to the liver has been demonstrated.

Synthesis of Monovalent N-Acetylgalactosamine Phosphoramidite for Liver-Targeting Oligonucleotides.

Ligand-targeted drug delivery (LTDD) has emerged as an attractive option in the field of oligonucleotide drugs following the great success of N-acetylgalactosamine (GalNAc)-conjugated siRNA and antisense oligonucleotides. GalNAc is a well-known ligand of the asialoglycoprotein receptor (ASGPR), and is classified as a C-type lectin associated with the metabolism of desialylated glycoproteins. This article describes the synthesis of a non-nucleosidic monovalent GalNAc phosphoramidite-a useful reagent for facilitating the conjugation of GalNAc epitopes into oligonucleotides using DNA synthesizers-together with some important caveats. The monomeric GalNAc consists of three parts: (1) a GalNAc moiety, (2) a linker moiety, and (3) a trans-4-hydroxyprolinol (tHP) branch point. The GalNAc moiety and the tHP moiety are coupled via a condensation reaction to prepare the monovalent GalNAc phosphoramidite. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of N-acetylgalactosamine ligand Basic Protocol 2: Preparation of trans-4-hydroxyprolinol building block Basic Protocol 3: Preparation of GalNAc phosphoramidite.

Osseointegration of Alkali-Modified NANOZR Implants: An In Vivo Study.

Ingredients and surface modification methods are being continually developed to improve osseointegration of dental implants and reduce healing times. In this study, we demonstrate in vitro that, by applying concentrated alkali treatment to NANOZR with strong bending strength and fracture toughness, a significant improvement in the bone differentiation of rat bone marrow cells can be achieved. We investigated the influence of materials modified with this treatment in vivo, on implanted surrounding tissues using polychrome sequential fluorescent labeling and micro-computer tomography scanning. NANOZR implant screws in the alkali-treated group and the untreated group were evaluated after implantation in the femur of Sprague⁻Dawley male rats, indicating that the amount of new bone in the alkali-modified NANOZR was higher than that of unmodified NANOZR. Alkali-modified NANOZR implants proved to be useful for the creation of new implant materials.

Effect of Amelogenin Coating of a Nano-Modified Titanium Surface on Bioactivity.

The interactions between implants and host tissues depend on several factors. In particular, a growing body of evidence has demonstrated that the surface texture of an implant influences the response of the surrounding cells. The purpose of this study is to develop new implant materials aiming at the regeneration of periodontal tissues as well as hard tissues by coating nano-modified titanium with amelogenin, which is one of the main proteins contained in Emdogain®. We confirmed by quartz crystal microbalance evaluation that amelogenin is easy to adsorb onto the nano-modified titanium surface as a coating. Scanning electron microscopy, scanning probe microscopy, X-ray photoelectron spectroscopy, and Fourier-transform infrared spectroscopy analyses confirmed that amelogenin coated the nano-modified titanium surface following alkali-treatment. In vitro evaluation using rat bone marrow and periodontal ligament cells revealed that the initial adhesion of both cell types and the induction of hard tissue differentiation such as cementum were improved by amelogenin coating. Additionally, the formation of new bone in implanted surrounding tissues was observed in in vivo evaluation using rat femurs. Together, these results suggest that this material may serve as a new implant material with the potential to play a major role in the advancement of clinical dentistry.

Synergistic effect of nanotopography and bioactive ions on peri-implant bone response.

Both bioactive ion chemistry and nanoscale surface modifications are beneficial for enhanced osseointegration of endosseous implants. In this study, a facile synthesis approach to the incorporation of bioactive Ca2+ ions into the interlayers of nanoporous structures (Ca-nano) formed on a Ti6Al4V alloy surface was developed by sequential chemical and heat treatments. Samples with a machined surface and an Na+ ion-incorporated nanoporous surface (Na-nano) fabricated by concentrated alkali and heat treatment were used in parallel for comparison. The bone response was investigated by microcomputed tomography assessment, sequential fluorescent labeling analysis, and histological and histomorphometric evaluation after 8 weeks of implantation in rat femurs. No significant differences were found in the nanotopography, surface roughness, or crystalline properties of the Ca-nano and Na-nano surfaces. Bone-implant contact was better in the Ca-nano and Na-nano implants than in the machined implant. The Ca-nano implant was superior to the Na-nano implant in terms of enhancing the volume of new bone formation. The bone formation activity consistently increased for the Ca-nano implant but ceased for the Na-nano implant in the late healing stage. These results suggest that Ca-nano implants have promising potential for application in dentistry and orthopedics.

Ecological correlates and determinants in the geographical variation of deer morphology.

Previous studies on patterns in ungulate size variations have emphasized the effect of a particular environmental factor such as Bergmann's rule and the island rule. However, although multiple environmental factors may influence the body size, these studies focused on a single factor, and various measurements that may be influenced by different environmental factors (at least partly) were used as indices of body size. In this study, we used several skull and limb measurements to examine size variations among island populations of sika deer (Cervus nippon) in southern Japan considering the effects of multiple environmental factors. We found that all measurements differed markedly between populations. We focused on the skull and limb condylobasal length (CBL) and metacarpal length because they had the most important variations among the populations and the largest sample sizes. The common environmental factors influencing CBL and metacarpal length were island area and precipitation. Since these environmental factors reflect the availability of food resources, the causal factor of body size variation may be food resources. Interpopulation variation in metacarpal length was greater than that of CBL, indicating that metacarpal length may be affected by additional factors besides the common factors shared with CBL. Specific environmental factors influencing relative (CBL adjusted) metacarpal length were precipitation and slope. A common direct cause of those environmental factors was discussed in relation to topography. Analyses of phenotypic variation using multiple measurements with multiple environmental factors are useful to gain insight into underlying causes and can lead to identification of a measurement-specific variation with a specific driving force.