Antisense Nucleic Acids Research Articles

The West Nile virus genome harbors essential riboregulatory elements with conserved and host-specific functional roles

West Nile virus (WNV) is an arthropod-borne, positive-sense RNA virus that poses an increasing global threat due to warming climates and lack of effective therapeutics. Like other enzootic viruses, little is known about how host context affects the structure of the full-length RNA genome. Here, we report a complete secondary structure of the entire WNV genome within infected mammalian and arthropod cell lines. Our analysis affords structural insights into multiple, conserved aspects of flaviviral biology. We show that the WNV genome folds with minimal host dependence, and we prioritize well-folded regions for functional validation using structural homology between hosts as a guide. Using structure-disrupting, antisense locked nucleic acids, we then demonstrate that the WNV genome contains riboregulatory structures with conserved and host-specific functional roles. These results reveal promising RNA drug targets within flaviviral genomes, and they highlight the therapeutic potential of ASO-LNAs as both WNV-specific and pan-flaviviral therapeutic agents.

Novel antiviral approaches for Marburg: a promising therapeutics in the pipeline.

Marburg virus disease (MVD) presents a significant global health threat, lacking effective antivirals and with current supportive care offering limited therapeutic options. This mini review explores the emerging landscape of novel antiviral strategies against MVD, focusing on promising therapeutics currently in the development pipeline. We delve into direct-acting antiviral approaches, including small molecule inhibitors targeting viral entry, replication, and assembly, alongside nucleic acid antisense and RNA interference strategies. Host-targeting antivirals are also considered, encompassing immune modulators like interferons and cytokine/chemokine modulators, broad-spectrum antivirals, and convalescent plasma and antibody-based therapies. The paper then examines preclinical and clinical development for the novel therapeutics, highlighting in vitro and in vivo models for antiviral evaluation, safety and efficacy assessments, and the critical stages of clinical trials. Recognizing the challenges of drug resistance and viral escape, the mini review underscores the potential of combination therapy strategies and emphasizes the need for rapid diagnostic tools to optimize treatment initiation. Finally, we discuss the importance of public health preparedness and equitable access to these promising therapeutics in achieving effective MVD control and global health security. This mini review presents a comprehensive overview of the burgeoning field of MVD antivirals, highlighting the potential of these novel approaches to reshape the future of MVD treatment and prevention.

Antisense and Functional Nucleic Acids in Rational Drug Development.

This review is focused on antisense and functional nucleic acid used for completely rational drug design and drug target assessment, aiming to reduce the time and money spent and increase the successful rate of drug development. Nucleic acids have unique properties that play two essential roles in drug development as drug targets and as drugs. Drug targets can be messenger, ribosomal, non-coding RNAs, ribozymes, riboswitches, and other RNAs. Furthermore, various antisense and functional nucleic acids can be valuable tools in drug discovery. Many mechanisms for RNA-based control of gene expression in both pro-and-eukaryotes and engineering approaches open new avenues for drug discovery with a critical role. This review discusses the design principles, applications, and prospects of antisense and functional nucleic acids in drug delivery and design. Such nucleic acids include antisense oligonucleotides, synthetic ribozymes, and siRNAs, which can be employed for rational antibacterial drug development that can be very efficient. An important feature of antisense and functional nucleic acids is the possibility of using rational design methods for drug development. This review aims to popularize these novel approaches to benefit the drug industry and patients.

A novel universal fluorescence detection platform based on target binding-mediated primer exchange reaction strategy for sensitive and accurate detection of multiple biomolecules

This study introduces a universal fluorescent detection platform based on the binding-mediated primer exchange reaction strategy, designed for highly sensitive and accurate detection of multiple biomolecules. This method uses an intact DNA hybridization probe as the initial target recognition site, forming a stable probe-target complex upon binding to the target. This complex drives the primer exchange reaction to amplify the signal, and then to achieve signal output through fluorescence resonance energy transfer (FRET). The reaction process is initiated directly by the catalytic target binding medium, ensuring method specificity. The versatile platform, equipped with various recognition elements such as antisense nucleic acids, biotin, and split aptamers, demonstrates exceptional universality in detecting a range of biomolecules, including virus nucleic acid fragments, streptavidin, and ATP. The method exhibits strong linear relationships with detection limits of 1.02 pM for virus nucleic acid fragments, 1.08 nM for streptavidin, and 15.7 μM for ATP, respectively. Moreover, the method displays excellent selectivity and outstanding performance in complex biological samples. This promising biosensing platform is believed to find numerous applications spanning bioanalysis, disease diagnosis, and biomedicine.

A Modular Albumin-Oligonucleotide Biomolecular Assembly for Delivery of Antisense Therapeutics.

Antisense nucleic acid drugs are susceptible to nuclease degradation, rapid renal clearance, and short circulatory half-life. In this work, we introduce a modular-based recombinant human albumin-oligonucleotide (rHA-cODN) biomolecular assembly that allows incorporation of a chemically stabilized therapeutic gapmer antisense oligonucleotide (ASO) and FcRn-driven endothelial cellular recycling. A phosphodiester ODN linker (cODN) was conjugated to recombinant human albumin (rHA) using maleimide chemistry, after which a complementary gapmer ASO, targeting ADAMTS5 involved in osteoarthritis pathogenesis, was annealed. The rHA-cODN/ASO biomolecular assembly production, fluorescence labeling, and purity were confirmed using polyacrylamide gel electrophoresis. ASO release was triggered by DNase-mediated degradation of the linker strand, reaching 40% in serum after 72 h, with complete release observed following 30 min of incubation with DNase. Cellular internalization and trafficking of the biomolecular assembly using confocal microscopy in C28/I2 cells showed higher uptake and endosomal localization by increasing incubation time from 4 to 24 h. FcRn-mediated cellular recycling of the assembly was demonstrated in FcRn-expressing human microvascular endothelial cells. ADAMTS5 in vitro silencing efficiency reached 40%, which was comparable to free gapmer after 72 h incubation with human osteoarthritis patients' chondrocytes. This work introduces a versatile biomolecular modular-based "Plug-and-Play" platform potentially applicable for albumin-mediated half-life extension for a range of different types of ODN therapeutics.

Development of antisense peptide-peptide nucleic acids against fluoroquinolone-resistant Escherichia coli.

Fluoroquinolones (FQs) are potent and broad-spectrum antibiotics commonly used to treat MDR bacterial infections, but bacterial resistance to FQs has emerged and spread rapidly around the world. The mechanisms for FQ resistance have been revealed, including one or more mutations in FQ target genes such as DNA gyrase (gyrA) and topoisomerase IV (parC). Because therapeutic treatments for FQ-resistant bacterial infections are limited, it is necessary to develop novel antibiotic alternatives to minimize or inhibit FQ-resistant bacteria. To examine the bactericidal effect of antisense peptide-peptide nucleic acids (P-PNAs) that can block the expression of DNA gyrase or topoisomerase IV in FQ-resistant Escherichia coli (FRE). A set of antisense P-PNA conjugates with a bacterial penetration peptide were designed to inhibit the expression of gyrA and parC and were evaluated for their antibacterial activities. Antisense P-PNAs, ASP-gyrA1 and ASP-parC1, targeting the translational initiation sites of their respective target genes significantly inhibited the growth of the FRE isolates. In addition, ASP-gyrA3 and ASP-parC2, which bind to the FRE-specific coding sequence within the gyrA and parC structural genes, respectively, showed selective bactericidal effects against FRE isolates. Our results demonstrate the potential of targeted antisense P-PNAs as antibiotic alternatives against FQ-resistance bacteria.

The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

Epigenetic mechanisms oversee epidermal homeostasis and oncogenesis. The identification of kinases controlling these processes has direct therapeutic implications. We show that ULK3 is a nuclear kinase with elevated expression levels in squamous cell carcinomas (SCCs) arising in multiple body sites, including skin and Head/Neck. ULK3 loss by gene silencing or deletion reduces proliferation and clonogenicity of human keratinocytes and SCC-derived cells and affects transcription impinging on stem cell-related and metabolism programs. Mechanistically, ULK3 directly binds and regulates the activity of two histone arginine methyltransferases, PRMT1 and PRMT5 (PRMT1/5), with ULK3 loss compromising PRMT1/5 chromatin association to specific genes and overall methylation of histone H4, a shared target of these enzymes. These findings are of translational significance, as downmodulating ULK3 by RNA interference or locked antisense nucleic acids (LNAs) blunts the proliferation and tumorigenic potential of SCC cells and promotes differentiation in two orthotopic models of skin cancer.

An antisense amido-bridged nucleic acid gapmer oligonucleotide targeting SRRM4 alters REST splicing and exhibits anti-tumor effects in small cell lung cancer and prostate cancer cells

BackgroundAntisense oligonucleotide (ASO) medicine for clinical applications has been becoming a reality. We previously developed a gapmer ASO targeting Ser/Arg repetitive matrix 4 (SRRM4) that is abnormally expressed in small cell lung cancer (SCLC). However the detailed mechanism of ASO through repressing SRRM4 has not been completely elucidated. Further, effectiveness of SRRM4 ASO to prostate cancer (PCa) cells expressing SRRM4 similar to SCLC remains to be elucidated. RE1-silencing transcription factor (REST) is a tumor suppressor, and its splicing isoform (sREST) is abnormally expressed by SRRM4 and causes carcinogenesis with neuroendocrine phenotype in SCLC. The present study aimed to understand the contribution of REST splicing by SRRM4 ASO administration.MethodsSRRM4 expression and REST splicing were analyzed by RT-qPCR and conventional RT-PCR after treating SRRM4 ASO, and cell viability was analyzed in vitro. Exogenous reconstitution of Flag-tagged REST plasmid in SCLC cells and the splice-switching oligonucleotide (SSO) specific for REST was analyzed for cell viability. Furthermore, we expanded the application of SRRM4 ASO in PCa cells abnormally expressing SRRM4 mRNA in vitro.ResultsSRRM4 ASO successfully downregulated SRRM4 expression, followed by repressed cell viability of SCLC and PCa cells in a dose-dependent manner. Administration of SRRM4 ASO then modified the alternative splicing of REST, resulting reduced cell viability. REST SSO specifically modified REST splicing increased REST expression, resulting in reduced cell viability.ConclusionsOur data demonstrate that a gapmer ASO targeting SRRM4 (SRRM4 ASO) reduces cell viability through splicing changes of REST, followed by affecting REST-controlled genes in recalcitrant tumors SCLC and PCa cells.

Restoring Dystrophin Expression with Duchenne Muscular Dystrophy Exon 45 Skipping in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Induced pluripotent stem cell (iPSC)-based disease model is a useful tool that can represent the pathophysiology of patient organs that are inaccessible due to invasiveness. Here, we present a method to induce differentiation of Duchenne muscular dystrophy (DMD) patient-derived iPSCs into cardiomyocytes and restore dystrophin expression by exon skipping using antisense nucleic acids. This involves a 20-day multi-step culture process for differentiation to cardiomyocytes, followed by exon-skipping experiments. Additionally, RT-PCR, western blotting, and immunocytochemistry are used to confirm the restoration of dystrophin expression.

Boron Clusters as Enhancers of RNase H Activity in the Smart Strategy of Gene Silencing by Antisense Oligonucleotides.

Boron cluster-conjugated antisense oligonucleotides (B-ASOs) have already been developed as therapeutic agents with “two faces”, namely as potential antisense inhibitors of gene expression and as boron carriers for boron neutron capture therapy (BNCT). The previously observed high antisense activity of some B-ASOs targeting the epidermal growth factor receptor (EGFR) could not be rationally assigned to the positioning of the boron cluster unit: 1,2-dicarba-closo-dodecaborane (0), [(3,3′-Iron-1,2,1′,2′-dicarbollide) (1-), FESAN], and dodecaborate (2-) in the ASO chain and its structure or charge. For further understanding of this observation, we performed systematic studies on the efficiency of RNase H against a series of B-ASOs models. The results of kinetic analysis showed that pyrimidine-enriched B-ASO oligomers activated RNase H more efficiently than non-modified ASO. The presence of a single FESAN unit at a specific position of the B-ASO increased the kinetics of enzymatic hydrolysis of complementary RNA more than 30-fold compared with unmodified duplex ASO/RNA. Moreover, the rate of RNA hydrolysis enhanced with the increase in the negative charge of the boron cluster in the B-ASO chain. In conclusion, a “smart” strategy using ASOs conjugated with boron clusters is a milestone for the development of more efficient antisense therapeutic nucleic acids as inhibitors of gene expression.

Satellite repeat RNA expression in epithelial ovarian cancer associates with a tumor-immunosuppressive phenotype.

Aberrant expression of viral-like repeat elements is a common feature of epithelial cancers, and the substantial diversity of repeat species provides a distinct view of the cancer transcriptome. Repeatome profiling across ovarian, pancreatic, and colorectal cell lines identifies distinct clustering independent of tissue origin that is seen with coding gene analysis. Deeper analysis of ovarian cancer cell lines demonstrated that human satellite II (HSATII) satellite repeat expression was highly associated with epithelial-mesenchymal transition (EMT) and anticorrelated with IFN-response genes indicative of a more aggressive phenotype. SATII expression — and its correlation with EMT and anticorrelation with IFN-response genes — was also found in ovarian cancer RNA-Seq data and was associated with significantly shorter survival in a second independent cohort of patients with ovarian cancer. Repeat RNAs were enriched in tumor-derived extracellular vesicles capable of stimulating monocyte-derived macrophages, demonstrating a mechanism that alters the tumor microenvironment with these viral-like sequences. Targeting of HSATII with antisense locked nucleic acids stimulated IFN response and induced MHC I expression in ovarian cancer cell lines, highlighting a potential strategy of modulating the repeatome to reestablish antitumor cell immune surveillance.

Identification and characterization of a MAPT-targeting locked nucleic acid antisense oligonucleotide therapeutic for tauopathies

Tau is a microtubule-associated protein (MAPT, tau) implicated in the pathogenesis of tauopathies, a spectrum of neurodegenerative disorders characterized by accumulation of hyperphosphorylated, aggregated tau. Because tau pathology can be distinct across diseases, a pragmatic therapeutic approach may be to intervene at the level of the tau transcript, as it makes no assumptions to mechanisms of tau toxicity. Here we performed a large library screen of locked-nucleic-acid (LNA)-modified antisense oligonucleotides (ASOs), where careful tiling of the MAPT locus resulted in the identification of hot spots for activity in the 3′ UTR. Further modifications to the LNA design resulted in the generation of ASO-001933, which selectively and potently reduces tau in primary cultures from hTau mice, monkey, and human neurons. ASO-001933 was well tolerated and produced a robust, long-lasting reduction in tau protein in both mouse and cynomolgus monkey brain. In monkey, tau protein reduction was maintained in brain for 20 weeks post injection and corresponded with tau protein reduction in the cerebrospinal fluid (CSF). Our results demonstrate that LNA-ASOs exhibit excellent drug-like properties and sustained efficacy likely translating to infrequent, intrathecal dosing in patients. These data further support the development of LNA-ASOs against tau for the treatment of tauopathies.

Host-Directed Targeting of LincRNA-MIR99AHG Suppresses Intracellular Growth of Mycobacterium tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) kills 1.6 million people worldwide every year, and there is an urgent need for targeting host-pathogen interactions as a strategy to reduce mycobacterial resistance to current antimicrobials. Non-coding RNAs are emerging as important regulators of numerous biological processes and avenues for exploitation in host-directed therapeutics. Although long non-coding RNAs (lncRNAs) are abundantly expressed in immune cells, their functional role in gene regulation and bacterial infections remains under-studied. Here, we identify an immunoregulatory long intergenic non-coding RNA, lincRNA-MIR99AHG, which is upregulated in mouse and human macrophages upon IL-4/IL-13 stimulation and downregulated after clinical Mtb HN878 strain infection and in peripheral blood mononuclear cells (PBMC) from active TB patients. To evaluate the functional role of lincRNA-MIR99AHG, we employed antisense LNA GapmeR-mediated antisense oligonucleotide (ASO) lncRNA knockdown experiments. Knockdown of lincRNA-MIR99AHG with ASOs significantly reduced intracellular Mtb growth in mouse and human macrophages and reduced proinflammatory cytokine production. In addition, in vivo treatment of mice with MIR99AHG ASOs reduced the mycobacterial burden in the lung and spleen. Further, in macrophages, lincRNA-MIR99AHG is translocated to the nucleus and interacts with high affinity to hnRNPA2/B1 following IL-4/IL-13 stimulation and Mtb HN878 infection. Together, these findings identify lincRNA-MIR99AHG as a positive regulator of inflammation and macrophage polarization to promote Mtb growth and a possible target for adjunctive host-directed therapy against TB.

Effects of LPS Composition in Escherichia coli on Antibacterial Activity and Bacterial Uptake of Antisense Peptide-PNA Conjugates.

The physical and chemical properties of the outer membrane of Gram-negative bacteria including Escherichia coli have a significant impact on the antibacterial activity and uptake of antibiotics, including antimicrobial peptides and antisense peptide-peptide nucleic acid (PNA) conjugates. Using a defined subset of E. coli lipopolysaccharide (LPS) and envelope mutants, components of the LPS-core, which provide differential susceptibility toward a panel of bacterial penetrating peptide (BPP)-PNA conjugates, were identified. Deleting the outer core of the LPS and perturbing the inner core only sensitized the bacteria toward (KFF)3K-PNA conjugates, but not toward conjugates carrying arginine-based BPPs. Interestingly, the chemical composition of the outer LPS core as such, rather than overall hydrophobicity or surface charge, appears to determine the susceptibility to different BPP-PNA conjugates thereby clearly demonstrating the complexity and specificity of the interaction with the LPS/outer membrane. Notably, mutants with outer membrane changes conferring polymyxin resistance did not show resistance toward the BPP-PNA conjugates, thereby eliminating one possible route of resistance for these molecules. Finally, envelope weakening, through deletion of membrane proteins such as OmpA as well as some proteins previously identified as involved in cationic antimicrobial peptide uptake, did not significantly influence BPP-PNA conjugate activity.

The microRNA-29 family: role in metabolism and metabolic disease.

The microRNA-29 family members miR-29a-3p, miR-29b-3p, and miR-29c-3p are ubiquitously expressed and consistently increased in various tissues and cell types in conditions of metabolic disease, obesity, insulin resistance, and type 2 diabetes. In pancreatic β cells, miR-29a is required for normal exocytosis, but increased levels are associated with impaired β-cell function. Similarly, in liver, miR-29 species are higher in models of insulin resistance and type 2 diabetes, and either knock-out or depletion using a microRNA inhibitor improves hepatic insulin resistance. In skeletal muscle, miR-29 family upregulation is associated with insulin resistance and altered substrate oxidation, and similarly, in adipocytes, overexpression of miR-29a leads to insulin resistance. Blocking miR-29a using nucleic acid antisense therapeutics show promising results in preclinical animal models of obesity and type 2 diabetes, although the widespread expression pattern of miR-29 family members complicates the exploration of single target tissues. However, in fibrotic diseases, such as in late complications of diabetes and metabolic disease (diabetic kidney disease, nonalcoholic steatohepatitis), miR-29 species expression is suppressed by TGF-β allowing increased extracellular matrix collagen to form. In the clinical setting, circulating levels of miR-29a and miR-29b are consistently increased in type 2 diabetes and in gestational diabetes and are also possible prognostic markers for deterioration of glucose tolerance. In conclusion, miR-29 family miRNAs play an essential role in various organs relevant to intermediary metabolism and its upregulation contributes to impaired glucose metabolism, whereas it suppresses fibrosis development. Thus, a correct balance of levels of miR-29 family miRNA seems important for cellular and organ homeostasis in metabolism.

S100A8 inhibition in leukemic lymphoblasts induces sensitivity to chemotherapy and inhibition of disease relapse.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and relapsed B-ALL is the leading cause of mortality in children with leukemia due to a lack of response to treatment. S100A8 is a low molecular weight calcium-binding intracellular protein that is expressed in certain cells, and its increased expression is seen in most tumors as well as in relapsed childhood B-ALL cases. The present study indicates the important role of S100A8 in improving viability and resistance to chemotherapy in relapsed B-ALL lymphoblasts. S100A8 levels were compared in B-ALL and relapsed B-ALL lymphoblasts that were sensitive and resistant to Vincristine, respectively. S100A8 was inhibited in the lymphoblasts of two patients by antisense locked nucleic acid (LNA) GapmeRs and the decreased expression of S100A8 was evaluated using quantitative real-time PCR and ELISA. Then, the S100A8 antisense LNA GapmeRs-transfected cells were treated with Vincristine and the expression levels of S100A8 mRNA and S100A8 protein were re-determined. At all of these stages, cell viability and LC50 were assessed by MTT assay. The results showed that S100A8 levels in relapsed B-ALL lymphoblasts were significantly higher than B-ALL lymphoblasts. Moreover, the increase in S100A8 expression was proportionate to the increase in Vincristine resistance in these cells. The S100A8 knockdown procedure using antisense LNA GapmeRs decreased the cell viability and increased vincristine sensitivity in lymphoblasts of two patients, and it also increased the sensitivity to chemotherapy in relapsed B-ALL lymphoblasts. According to the findings of the present study, S100A8 is effective in developing lymphoblast resistance to chemotherapy, and its enhanced expression may contribute to shifting B-ALL into the relapse phase of the illness. As a result, S100A8 may be a valuable target for managing and improving relapses B-ALL.

Absorption, Distribution, Metabolism, and Excretion of FDA‐approved Antisense Oligonucleotide Drugs

Absorption, distribution, metabolism, and excretion (ADME) are key biological processes for determination of a drug’s pharmacokinetic parameters, which have direct impacts on therapeutic efficacy and adverse drug reactions (ADRs). Drug structures, formulations, and administration sites and routes cause different classes of drugs to have various ADME characteristics and consequent impacts upon efficacy and ADRs. For example, orally administered small molecule drugs are usually absorbed and distributed by uptake transporters, biotransformed by phase I/II drug‐metabolizing enzymes, and excreted by efflux transporters. However, newly developed large molecule biological drugs, including antisense oligonucleotide (ASO) drugs, have substantially different ADME parameters. ASO drugs are single‐stranded antisense nucleic acids with diverse modes of drug action that induce mRNA degradation, exon skipping, and ASO‐protein interactions. ASOs have a great potential to treat certain human diseases that have not been treatable with small molecule drugs. The ADME behaviors of ASO drugs contribute to their unique set of ADRs and toxicity. To better understand their ADME features, ten FDA‐approved ASO drugs were selected, including Fomivirsen, Pegaptanib, Nusinersen, Mipomersen, Inotersen, Defibrotide, Eteplirsen, Golodirsen, Viltolarsen, and Casimersen. A comprehensive meta‐analysis was conducted on their formulation, dosage, sites of administration, local and systematic distribution, metabolism, degradation, and excretion. Membrane permeabilization and nucleotide degradation by endonucleases and exonucleases are major contributors to ADME features of the ASO drugs and these differ from the roles of transporters and metabolizing enzymes of small molecule drugs. The summarized information provides the most updated knowledge of ADME characteristics of these ASO drugs, leading to a better understanding of their therapeutic efficacy and potential ADRs and toxicity. Numerous knowledge gaps, particularly on cellular uptake and subcellular trafficking and distributions are identified and future perspective and directions are discussed.

Mycobacterium tuberculosis Induced Osteoblast Dysregulation Involved in Bone Destruction in Spinal Tuberculosis.

Disturbance of bone homeostasis caused by Mycobacterium tuberculosis (Mtb) is a key clinical manifestation in spinal tuberculosis (TB). However, the complete mechanism of this process has not been established, and an effective treatment target does not exist. Increasing evidence shows that abnormal osteoclastogenesis triggered by an imbalance of the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) axis may play a key role in the disturbance of bone homeostasis. Previous studies reported that RANKL is strongly activated in patients with spinal TB; however, the OPG levels in these patients were not investigated in previous studies. In this study, we investigated the OPG levels in patients with spinal TB and the dysregulation of osteoblasts caused by Mtb infection. Inhibition of the Mce4a gene of Mtb by an antisense locked nucleic acid (LNA) gapmer (Mce4a-ASO) was also investigated. Analysis of the serum OPG levels in clinical samples showed that the OPG levels were significantly decreased in patients with spinal TB compared to those in the group of non-TB patients. The internalization of Mtb in osteoblasts, the known major source of OPG, was investigated using the green fluorescent protein (GFP)-labeled Mycobacterium strain H37Ra (H37RaGFP). The cell-associated fluorescence measurements showed that Mtb can efficiently enter osteoblast cells. In addition, Mtb infection caused a dose-dependent increase of the CD40 mRNA expression and cytokine (interleukin 6, IL-6) secretion in osteoblast cells. Ligation of CD40 by soluble CD154 reversed the increased secretion of IL-6. This means that the induced CD40 is functional. Considering that the interaction between CD154-expressing T lymphocytes and bone-forming osteoblast cells plays a pivotal role in bone homeostasis, the CD40 molecule might be a strong candidate for mediating the target for treatment of bone destruction in spinal TB. Additionally, we also found that Mce4a-ASO could dose-dependently inhibit the Mce4a gene of Mtb and reverse the decreased secretion of IL-6 and the impaired secretion of OPG caused by Mtb infection of osteoblast cells. Taken together, the current finding provides breakthrough ideas for the development of therapeutic agents for spinal TB.

Synthesis of 4′-C-(aminoethyl)thymidine and 4′-C-[(N-methyl)aminoethyl]thymidine by a new synthetic route and evaluation of the properties of the DNAs containing the nucleoside analogs

A gapmer-type antisense oligonucleotide is an oligonucleotide therapeutic that targets pathogenic mRNA directly, and it is expected to be a next-generation therapeutic drug. In this study, we designed and synthesized 4′-C-[(N-methyl)aminoethyl]-thymidine (4′-MAE-T) as a novel nucleoside analog and compared its properties with those of 4′-C-aminoethyl-thymidine (4′-AE-T). Furthermore, we designed a new synthetic route for 4′-C-aminoethyl-modified nucleosides and accomplished the synthesis of 4′-AE-T via a novel pathway with high total yield. DNA containing 4′-MAE-T analogs decreased RNA affinity slightly more than unmodified DNA and DNA containing 4′-AE-T, but significantly improved nuclease resistance compared to unmodified DNA in a solution containing bovine serum. In addition, the impact of 4′-MAE-T on DNA stability was higher than that of 4′-AE-T. Also, DNA containing these analogs can activate Escherichia coli-derived RNase H. Thus, 4′-MAE-T has the potential to be used in gapmer-type antisense nucleic acids as a suitable candidate for the development of therapeutic antisense oligonucleotides.

Absorption, Distribution, Metabolism, and Excretion of US Food and Drug Administration-Approved Antisense Oligonucleotide Drugs.

Absorption, distribution, metabolism, and excretion (ADME) are the key biologic processes for determination of a drug's pharmacokinetic parameters, which have direct impacts on efficacy and adverse drug reactions (ADRs). The chemical structures, dosage forms, and sites and routes of administration are the principal determinants of ADME profiles and consequent impacts on their efficacy and ADRs. Newly developed large molecule biologic antisense oligonucleotide (ASO) drugs have completely unique ADME that is not fully defined. ASO-based drugs are single-stranded synthetic antisense nucleic acids with diverse modes of drug actions from induction of mRNA degradation, exon skipping and restoration, and interactions with proteins. ASO drugs have a great potential to treat certain human diseases that have remained untreatable with small molecule-based drugs. The ADME of ASO drugs contributes to their unique set of ADRs and toxicity. In this review, to better understand their ADME, the 10 US Food and Drug Administration (FDA)-approved ASO drugs were selected: fomivirsen, pegaptanib, mipomersen, nusinersen, inotersen, defibrotide, eteplirsen, golodirsen, viltolarsen, and casimersen. A meta-analysis was conducted on their formulation, dosage, sites of administration, local and systematic distribution, metabolism, degradation, and excretion. Membrane permeabilization through endocytosis and nucleolytic degradation by endonucleases and exonucleases are major ADME features of the ASO drugs that differ from small-molecule drugs. The information summarized here provides comprehensive ADME characteristics of FDA-approved ASO drugs, leading to a better understanding of their therapeutic efficacy and their potential ADRs and toxicity. Numerous knowledge gaps, particularly on cellular uptake and subcellular trafficking and distribution, are identified, and future perspectives and directions are discussed. SIGNIFICANCE STATEMENT: Through a systematic analysis of the existing information of absorption, distribution, metabolism, and excretion (ADME) parameters for 10 US Food and Drug Administration (FDA)-approved antisense oligonucleotide (ASO) drugs, this review provides an overall view of the unique ADME characteristics of ASO drugs, which are distinct from small chemical drug ADME. This knowledge is useful for discovery and development of new ASO drugs as well as clinical use of current FDA-approved ASO drugs.