Transient Cyclic Structured Oligonucleotide Designs for Therapeutic Applications.

Oligonucleotide therapeutics are attracting attention as a new treatment modality for a range of diseases that have been difficult to target using conventional approaches. Technical advances in chemical modification and drug delivery systems have led to the generation of compounds with excellent profiles as pharmaceuticals, and 16 oligonucleotide therapeutics have been marketed to date. There is a growing need to develop optimal and efficient approaches to evaluate drug metabolism and pharmacokinetics (DMPK) and drug–drug interactions (DDIs) of oligonucleotide therapeutics. The DMPK/DDI profiles of small molecule drugs are highly diverse depending on their structural and physicochemical characteristics, whereas oligonucleotide therapeutics share similar DMPK profiles within each chemistry type. Most importantly, the mechanisms and molecules involved in the distribution and metabolism of oligonucleotides differ from those of small molecules. In addition, there are considerations regarding experimental approaches in the evaluation of oligonucleotides, such as bioanalytical challenges, the use of radiolabeled tracers, materials for in vitro metabolism/DDI studies, and methods to study biodistribution. In this review, we attempt to summarize the DMPK characteristics of antisense oligonucleotide (ASO) therapeutics and discuss some of the issues regarding how to optimize the evaluation and prediction of the DMPK and DDI of ASOs.

Nanoparticle-based delivery systems have emerged as promising tools in oligonucleotide therapeutics, facilitating precise and targeted delivery to address several disease conditions. The multifaceted landscape of nanoparticle-based oligonucleotide delivery encompasses the fundamental aspects of nanotechnology in delivery systems, various classes of oligonucleotides, and the growing field of ON-based therapeutics. These ON-based therapeutics are utilized to target specific genetic sequences within cells, offering promising avenues for treating various diseases by regulating gene expression or interfering with specific cellular processes. The integration of nanotechnology in delivery systems offers several advantages, given their intricate systems. Being a diverse class of agents, oligonucleotides provide a wide range of potential owed to each class of agents that support therapeutic interventions. Oligonucleotide-based platforms have demonstrated their versatility in molecular targeting and intervention strategies. Moreover, the complexities and delivery challenges in oligonucleotide therapeutics are expected to be overcome by the application of nanotechnology-based platforms.Because nanoparticles can overcome biological barriers and improve bioavailability, stability, and specificity, their role in developing oligonucleotide delivery systems is greatly valued. The innovative solutions facilitated by nanoparticles are efficient strategies to address the arduous barriers. These strategies beat obstacles like enzymatic degradation, cellular uptake, and immune response, which in turn paves the way for enhanced therapeutic efficacy. This review paper intends to explore the various applications of nanoparticle-mediated oligonucleotide delivery in a variety of diseases. It outlines the promising growth of therapies enabled by these systems, extending from cancer to genetic disorders, neurodegenerative diseases, etc. We have underscored the pivotal role of nanoparticle-based delivery systems in uncovering the full potential of oligonucleotide therapeutics, thereby fostering advancements in precision medicine and targeted therapies.

In the beginning of the 21st century, therapeutic oligonucleotides have shown great potential for the treatment of many life-threatening diseases. However, effective delivery of therapeutic oligonucleotides to the targeted location in vivo remains a major issue. As an emerging field, DNA nanotechnology is applied in many aspects including bioimaging, biosensing, and drug delivery. With sequence programming and optimization, a series of DNA nanostructures can be precisely engineered with defined size, shape, surface chemistry, and function. Simply with hybridization, therapeutic oligonucleotides including unmethylated cytosine-phosphate-guanine dinucleotide oligos, small interfering RNA (siRNA) or antisense RNA, single guide RNA of the regularly interspaced short palindromic repeat-Cas9 system, and aptamers, are successfully loaded on DNA nanostructures for delivery. In this progress report, the development history of DNA nanotechnology is first introduced, and then the mechanisms and means for cellular uptake of DNA nanostructures are discussed. Next, current approaches to deliver therapeutic oligonucleotides with DNA nanovehicles are summarized. In the end, the challenges and opportunities for DNA nanostructure-based systems for the delivery of therapeutic oligonucleotides are discussed.

Watson–Crick base pairs are crucial in the formation of double‐helical deoxyribonucleic acid (DNA) and in the storage of genetic information. They comprise two of the most common geometries involving the pairing of deoxyadenosine (dA) with deoxythymidine (dT) and deoxyguanosine (dG) with deoxycytidine (dC) through the formation of two and three hydrogen bonds, respectively. The complementarity of the antiparallel strands forming a double helix is crucial in DNA replication since the complementary strand is synthesised by adding the Watson–Crick complementary bases of the parent strand. The hybridisation of DNA or DNA–RNA ( ribonucleic acid ) strands is widely used in biotechnology and molecular biology for multiple purposes. The stability of a nucleic acid helix depends on its sequence, conformation, hydration and solution conditions, and it is measured by temperature‐dependent techniques, such as UV (ultraviolet) melting. Examples of oligonucleotide helices with chemical modifications are provided to indicate how these modifications affect the overall stability of a DNA molecule. Key Concepts: Base pairs are two nucleotide bases interacting via hydrogen bonding. Bulge is a nonpaired nucleotide base in a double‐stranded nucleic acid. Hybridisation is a process where two complementary strands interact via base pairing. Nucleic acid conformation is the three‐dimensional structural arrangement of a double‐helical nucleic acid. Nucleic acid modification is the incorporation, removal or substitution of a chemical group on the natural occurring nucleic acids. Melting curves are the optical changes of a nucleic acid or protein‐like molecules as a function of temperature. Stability of nucleic acids is the resistance of nucleic acid duplex to undergo unfolding, proportional to the magnitude of Δ G . Thermal stability is the ability of a nucleic acid duplex to resist temperature denaturation. Unfolding thermodynamic profile is the free energy (Δ G ∘ ), enthalpy (Δ H ) and entropy (Δ S ) parameters associated with the unfolding of a nucleic acid or protein molecule. Cooperativity refers to the number of base pairs that are broken simultaneously in the unfolding of a nucleic acid or protein.

586. Dimerization Inhibition of HIV-1 RNA by 2’-Deoxy-2’-fluoro-beta-D-arabinose Nucleic Acid (2’-FANA) Modified Antisense Oligonucleotides Results in Potential Inhibition of Viral Expression

Secondary structure of poly-5-methylcytidylic acid

<p>Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), is the leading cause of death and disease by a bacterial pathogen worldwide. The growing incidence of drug resistant TB, especially multi-drug resistant TB highlights the need for new drugs with novel modes of action. Current treatment of TB involves a multi-drug regimen of four drugs including isoniazid and rifampicin, both of which were discovered over 40 years ago. Bedaquiline is one of the first novel TB drugs to enter clinical trials since the discovery of rifampicin, and has shown excellent activity against drug resistant TB. Although isoniazid and rifampicin are well established anti-TB drugs, significant gaps in knowledge regarding their modes of action exist. Furthermore, little information on the mode of action of the novel drug bedaquiline is known beyond its primary target. Characterisation of drug mode of action facilitates rational modifications of drugs to improve the treatment of TB.</p> <p><br></p> <p>The aim of this study was to identify novel aspects of the modes of action of isoniazid, rifampicin, and bedaquiline by characterising drug hypersensitive mutants of M. smegmatis mc²155. A sub-saturated M. smegmatis mc²155 transposon mutant collection with 1.1-fold genome coverage (7680 mutants) was constructed, with this collection estimated to contain mutations in 73.2% of all genes capable of maintaining a transposon insertion (non-essential genes). A high-throughput assay was developed for screening the collection, and mutants related to known drug mode of action were identified for isoniazid (ahpC and eccCa₁) and bedaquiline (atpB). Additionally, known mechanisms of drug inactivation were identified for isoniazid (nudC), rifampicin (arr and lspA), and bedaquiline (mmpL5). The finding that transposon mutants of nudC are hypersensitive to isoniazid independently validated the recent discovery of the role of NudC in basal isoniazid resistance by Wang et al. (2011). The remaining genes identified in this thesis represent potentially novel aspects of the modes of action or resistance mechanisms of these drugs.</p> <p><br></p> <p>Cross-sensitivity to other drugs indicated that the mechanism of sensitivity was drug specific for the mutants examined. Differential-sensitivity testing against drug analogues revealed that Arr is involved in resistance to the rifampicin analogue rifapentine as well, indicating that Arr can detoxify rifapentine similar to rifampicin. The nudC mutant showed increased sensitivity to a range of isoniazid analogues, indicating that it can detoxify these analogues similar to the parent compound. Interestingly six analogues were found to be less active against the nudC mutant than expected. A number of overexpression strains were tested against these six analogues; a nudC overexpression strain, and a strain overexpressing inhA, the primary target for isoniazid. Overexpression of nudC as well inhA increased the resistance of WT to isoniazid, but failed to increase resistance to three of the analogues, NSC27607, NSC33759, and NSC40350. Isoniazid is a prodrug and is activated by the peroxidase/catalase enzyme KatG. Overexpression of katG resulted in increased isoniazid sensitivity, as well as increased sensitivity to NSC27607, NSC33759, and NSC40350. Together these results suggest that NSC27607, NSC33759, and NSC40350 are activated by KatG, but that InhA is not the primary target. Additionally the inability of NudC overexpression to confer resistance suggests these analogues are not acting via a NAD adduct, the mechanism by which isoniazid inhibits InhA. These results suggest that there are other toxic metabolites being produced by KatG activation of these three analogues. </p> <p><br></p> <p>In conclusion, characterisation of mutants identified in a high-throughput assay for drug hypersensitivity identified genes involved in the modes of action or resistance mechanisms for isoniazid, rifampicin, and bedaquiline. Additionally, a number of novel genes were identified that have no known connections to the known modes of action or resistance mechanisms for these drugs. Further testing of a nudC mutant revealed three isoniazid analogues that appear to inhibit growth of M. smegmatis mc²155 independent of InhA, the primary target of isoniazid. This study has successfully demonstrated that screening for drug hypersensitivity can generate novel information on drug mode of action and resistance mechanisms. This information can ultimately be used to help drive the development of new drugs, and improve treatment of TB.</p>

Chronic liver disease is an important cause of morbidity and mortality in the United States. Despite significant advances, the treatment options for many chronic liver diseases are limited. Recently, oligonucleotide-based therapeutics emerged as a novel treatment approach for a number of liver diseases. Therapeutic oligonucleotides comprised of 15 to 30 nucleotides in length that work in complementary to a specific region of a messenger RNA (mRNA) encoding a disease-related protein or a regulatory non-coding RNA and interfere with the target gene expression. These oligonucleotides can bind with high affinity and specificity through RNA-RNA or RNA-DNA base-pairing and targets the expression of mRNA leading to the disease control. A large number of druggable targets including miR-122, miR-132, miR-34, HSP 47, PNPLA3, STK25 etc., have emerged as susceptible molecular targets in the liver to treat various diseases. Thus, chronic liver diseases may be treated effectively by modulating these susceptible molecular targets using novel therapeutic oligonucleotides. Currently, several preclinical and clinical studies are underway to evaluate the clinical effectiveness and safety of different oligonucleotide therapies in the treatment of chronic liver diseases. In order to optimize its clinical effectiveness, various strategies have been explored including chemical modifications of the oligonucleotides and developing effective drug delivery systems such as oligonucleotide -ligand conjugates, oligonucleotide-polymer conjugates, and lipid or polymer-based nano-carriers. In this review, we provide an overview of the historical perspectives, the mechanism of action, delivery technologies, and clinical experience of using oligonucleotide-based therapies in treating liver diseases.

The protein corona is a protein layer formed on the surface of nanoparticles administered in vivo and considerably affects the in vivo fate of nanoparticles. Although it is challenging to control protein adsorption on nanoparticles precisely, the protein corona may be harnessed to develop a targeted drug delivery system if the nanoparticles are decorated with a ligand with enhanced affinity to target tissue- and cell-homing proteins. Here, we prepared a DNA tetrahedron with trivalent cholesterol conjugation (Chol3-Td) that can induce enhanced interaction with lipoproteins in serum, which in situ generates the lipoprotein-associated protein corona on a DNA nanostructure favorable for cells abundantly expressing lipoprotein receptors in the liver, such as hepatocytes in healthy mice and myofibroblasts in fibrotic mice. Chol3-Td was further adopted for liver delivery of antisense oligonucleotide (ASO) targeting TGF-β1 mRNA to treat liver fibrosis in a mouse model. The potency of ASO@Chol3-Td was comparable to that of ASO conjugated with the clinically approved liver-targeting ligand, trivalent N-acetylgalactosamine (GalNAc3), demonstrating the potential of Chol3-Td as a targeted delivery system for oligonucleotide therapeutics. This study suggests that controlled seeding of the protein corona on nanomaterials can provide a way to steer nanoparticles into the target area.

Modified siRNA Structure With a Single Nucleotide Bulge Overcomes Conventional siRNA-mediated Off-target Silencing

Mucosal sites represent the primary routes of HIV transmission, yet the oral mucosa appears uniquely resistant to HIV-1 infection. Since the oral cavity is not conducive to either infection or transmission of HIV, characterizing the responsible resistance factors, particularly components of innate immunity, and their mechanisms of action may identify new opportunities for interference with viral acquisition at other mucosal sites. Although many of the innate immune factors described to date exhibit both antibacterial and antiviral activities, molecules that inhibit HIV-1 are largely HIV-1 specific. Nonetheless, if these oral innate factors can be identified, characterized, and harnessed, they may be useful in protection and/or intervention in acute HIV-1 infection. Endogenous molecules acting independently or synergistically, given their lack of substantive side effects, may represent an alternative novel approach for reducing HIV risk and suppressing infection. While HIV infection via breast milk does involve oral trafficking and is an important area of study, it will only be briefly discussed here. The major emphasis of this article is on the oral cavity and salivary substances that can influence HIV infection Epidemiology of oral transmission of HIV More than twenty-five years into the global HIV/AIDS epidemic, it is well established that in addition to blood, the reproductive and/or rectal mucosae represent the primary portals of HIV entry in adults. There is less definitive evidence regarding whether other mucosal sites serve as viral conduits and in particular, uncertainty remains as to the possibility of transmission of HIV-1 via the oral entrance to the gastrointestinal (GI) tract. This is not a trivial concern, since despite the successes of combination therapy for HIV/AIDS, infection rates remain high, compounded by persistence of latent viral reservoirs, antiretroviral drug resistance, and the lack of an effective vaccine or potential cure. Although it is often accepted that oral transmission is of little or no consequence, we have failed to capitalize on that information to identify the mechanism(s) of protection at the oral mucosal surface. These mechanisms could provide clues for novel strategies of prevention/intervention at other more vulnerable viral entry sites. Early in the epidemic, evidence of the presence of HIV-1 in the oral cavity suggested the possibility of transmissibility via this route [1, 2]. Whereas in some early studies [3–7], oral transmission could not be established, a few studies implicated this route as a potential, even likely mode of transmission (e.g., [8]). In larger cohorts of individuals and in well-designed studies to monitor oral transmission, the rarity of HIV-1 infection by this route was substantiated [9]. Nonetheless, potential shortcomings in these studies, including accurately documenting the definitive route of infection, underscore our lack of certainty regarding the relative risk of oral transmission. (Reviews: [10, 11]). The low frequency of documented oral transmission of HIV-1 could reflect low titers of infectious virus in saliva. While high levels of HIV-1 RNA and antigens can routinely be measured in oral fluids, infectious virus was not detected in early studies attempting to isolate infectious virus from saliva [12, 13] or more recently in large cohort studies, despite plasma viremia [14, 15].

A meeting on the health effects of arsenic (As), its modes of action, and areas in need of future research was held in Hunt Valley, Maryland, on 22-24 September 1997. Exposure to As in drinking water has been associated with the development of skin and internal cancers and noncarcinogenic effects such as diabetes, peripheral neuropathy, and cardiovascular diseases. There is little data on specific mechanism(s) of action for As, but a great deal of information on possible modes of action. Although arsenite [As(III)] can inhibit more than 200 enzymes, events underlying the induction of the noncarcinogenic effects of As are not understood. With respect to carcinogenicity, As can affect DNA repair, methylation of DNA, and increase radical formation and activation of the protooncogene c-myc, but none of these potential pathways have widespread acceptance as the principal etiologic event. In addition, there are no accepted models for the study of As-induced carcinogenesis. At the final meeting session we considered research needs. Among the most important areas cited were a) As metabolism and its interaction with cellular constituents; b) possible bioaccumulation of As; c) interactions with other metals; d) effects of As on genetic material; e) development of animal models and cell systems to study effects of As; and f) a better characterization of human exposures as related to health risks. Some of the barriers to the advancement of As research included an apparent lack of interest in the United States on As research; lack of relevant animal models; difficulty with adoption of uniform methodologies; lack of accepted biomarkers; and the need for a central storage repository for stored specimens.

Therapeutic oligonucleotides, such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs), hold promise for suppressing pathogenic genes, including mutated KRAS, a key driver in many cancers. However, their systemic administration is challenged by rapid degradation and poor cellular uptake. Existing delivery systems, like lipid nanoparticles, face limitations in safety, extrahepatic delivery, and evading endolysosomal degradation. To address these limitations, we developed and evaluated an extracellular vesicle (EV)-based delivery platform for CD47-mediated oligonucleotide delivery in vivo. EVs, known for their biocompatibility, were engineered to display the SIRPα membrane protein on their surface, creating CD47-targeting SIRPα-EVs (S-EVs). These S-EVs were designed to preferentially accumulate in CD47-overexpressing pathological tissues, such as inflamed and tumor sites, as confirmed by in vivo imaging systems, flow cytometry, and intravital live imaging. To ensure consistent oligonucleotide encapsulation and batch-to-batch stability, we employed a cholesterol-based exogenous loading technique. Mechanistically, S-EVs entered target cells through CD47-mediated endocytosis, bypassing endolysosomal degradation and delivering oligonucleotides efficiently. Our results demonstrated that S-EVs accumulated selectively in CD47 overexpressing tissues and delivered oligonucleotides more efficiently than lipid-based transfection reagents or naïve EVs, significantly enhancing therapeutic efficacy. When administered systemically in an LPS-induced inflammatory model, S-EVs loaded with NLRP3-ASOs effectively reduced inflammation, outperforming naïve EVs. In a KRAS-mutated tumor model, KRAS siRNA-loaded S-EVs (siKRAS-S-EVs) suppressed KRAS expression in tumor tissues and increased CD8+ T cell infiltration into the tumor microenvironment, demonstrating significant antitumor effects. When combined with PD-1 blockade therapy, siKRAS-S-EVs led to substantial survival improvements, with 60% of treated animals achieving complete tumor remission. These findings emphasize the advantages of CD47-mediated EV delivery, enabling selective targeting of pathological tissues and efficient intracellular delivery of therapeutic oligonucleotides by bypassing endolysosomal degradation. This study highlights the potential of EV-based CD47-mediated delivery platforms as next-generation systems for treating cancers and other CD47-overexpressing diseases, addressing the limitations of current therapeutic approaches. Citation Format: Min Kyoung Jo, Yewon Jeong, Yoon Kyoung Kim, Seok Ho Song, Hyemin Joo, Yoonjeong Choi, Inkyu Lee, Minjeong Kwon, Gi Beom Kim, In-San Kim, Gi-Hoon Nam. In vivo CD47-mediated targeted oligonucleotide delivery for endolysosomal escape in cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 476.

Anti-PF4 antibodies are a potential mediator of antisense oligonucleotide (ASO)-induced thrombocytopenia.

ASP210 - a Potent Oligonucleotide-Based Inhibitor for Combating Both BCR-ABL1 Dependent and Independent TKI-Resistant CML Cells