Extravascular administration of mRNA lipid nanoparticles (mRNA-LNPs) offers practical and clinical advantages but is limited by tissue barriers that restrict dispersion, lymphatic access, systemic exposure, and ultimately, target tissue expression. Recombinant human hyaluronidase PH20 (rHuPH20) transiently modifies the hyaluronan-rich extracellular matrix in the subcutaneous (SC) space, enhancing SC delivery of protein therapeutics; however, its effect on mRNA-LNP delivery has not been systematically evaluated. Here, the impact of rHuPH20 on extravascular mRNA-LNP delivery was assessed across routes of administration and formulations. In a coordinated series of mouse and minipig studies, mRNA-LNP expression, biodistribution, lymphatic trafficking, systemic exposure, tissue deposition, and inflammatory responses were evaluated following SC, intramuscular (IM), and intravenous (IV) administration. In mice, co-administration with rHuPH20 increased local expression after IM dosing of luciferase mRNA-LNPs up to 7-fold and whole-body expression after SC dosing up to 38-fold, relative to the corresponding route without rHuPH20. In minipigs, rHuPH20-enabled SC administration of huEPO mRNA-LNPs produced serum huEPO exposure that approached IV benchmarks. rHuPH20 also increased hepatic expression following extravascular delivery, demonstrated consistent effects across ionizable lipid chemistries, and was associated with reduced pro-inflammatory cytokine responses following repeat-dosing. Collectively, these findings support rHuPH20 as an effective enabler of extravascular mRNA-LNP delivery.
Rondaptivon pegol (BT200) is a PEGylated RNA aptamer that prolongs von Willebrand factor (VWF) and factor VIII half-lives in patients with hemophilia A and von Willebrand disease (VWD) type 2B. Embryo-fetal safety is particularly relevant given the disproportionate disease burden in women with VWD. Data on placental transfer and developmental safety of PEGylated aptamers remain limited. An embryo-fetal development study was conducted in pregnant rabbits. Animals received rondaptivon pegol subcutaneously (0, 1.5, 5, or 15 mg/kg/day) from gestational days (GDs) 7-19. Maternal toxicokinetic samples and pooled fetal plasma concentrations were collected during gestation to quantify transplacental exposure. Rondaptivon pegol was well tolerated, with no maternal toxicity, no effects on implantation, and no treatment-related fetal malformations. Dose-related reductions in mean fetal body weight (<11%) were considered adverse only at the highest dose level. Maternal systemic exposure was supratherapeutic, with mean Cmax ranging from 133 to 807 µg/mL on GD19, far exceeding the human target concentration (∼1.2 µg/mL). Fetal plasma concentrations were <0.25% of maternal levels across all dose groups, confirming minimal placental transfer. The no-observed-adverse-effect level was 5 mg/kg/day. At maternal systemic exposures ranging from 110- to 670-fold above anticipated clinical levels, rondaptivon pegol did not adversely affect implantation, embryonic viability, or fetal development. These findings provide the first quantitative maternal-fetal pharmacokinetic and developmental safety data for a PEGylated aptamer, supporting further clinical evaluation of rondaptivon pegol in women of reproductive age.
Antisense oligonucleotides (ASOs) are a rapidly growing therapeutic modality that directly modulate splicing or expression of disease-causing genes. ASOs are internalized through various endocytic mechanisms that converge on the endolysosomal pathway. Our work here aims to evaluate changes to the endolysosomal system following repeated ASO exposure. Histological examinations of nonhuman primates following repeated intrathecal administration of ASOs reveal dose-related neuronal microvesicular vacuolation in the hippocampus, cortex, and spinal cord. These changes are not associated with any neuronal degenerative changes or glial activation. Examination by electron microscopy reveals lysosomes containing stacked membranous material. We established an induced pluripotent stem cell-derived motor neuron (iPSC-MN) model that recapitulates these lysosome changes. ASO exposure did not cause any changes in iPSC-MN viability. To characterize lysosomal changes, we isolated lysosomes from iPSC-MNs after ASO treatment and quantified their protein and lipid contents by liquid chromatography-mass spectrometry. Our lipidomics studies documented increases in bis(monoacylglycerol)phosphate and lactosylceramide following ASO administration; proteomic analysis showed changes in several proteins, including decreases in four lysosomal hydrolases (Carboxypeptidase Q, ß-galactosidase, Cathepsin A, and α-l-Fucosidase). Altogether, this work advances our understanding of the cellular consequences following prolonged ASO administration and may guide further investigations to characterize these effects.
Phosphorothioate-based antisense oligonucleotides (PS-ASO) are a common class of ASO for therapeutic uses due to their resistance to nucleases and known deposition in human physiological systems. On the molecular level, these ASOs are recognized by several scavenger receptors, including scavenger receptors A and B (SR-A, SR-B) and Stabilin receptors, Stab1 and Stab2. The Stabilins bind and internalize a host of endogenous and exogenous molecules with high endocytic rates and low turnover times. To determine their specific role in the biodistribution of PS-ASOs, we intravascularly injected knockout (KO) mice for Stab1, Stab2 or Stab1Stab2 double KO (DKO). Our results show that each receptor contributes to PS-ASO clearance from the blood and accumulation in liver and spleen, locations where these receptors have the highest levels of expression. Moreover, Stab2 is the primary clearance receptor taking up the bulk of the PS-ASO found in blood. Our conclusion is that Stabilins have a profound effect on the catabolism of PS-ASOs, for which internalization and clearance from the tissue need to be taken into consideration in the design of any therapeutic ASO.
Aptamers are single-stranded synthetic oligonucleotides that bind noncovalently to targets with high affinity and selectivity. They are generated through an in vitro selection process known as Systematic Evolution of Ligands by Exponential Enrichment. Aptamers show significant promise in cancer diagnostics and therapeutics due to their specificity, versatility, and tunable biochemical properties. Aptamers can be used to target cellular biomarkers, which are detectable molecules that can indicate specific physiological states of cells, organs, and organisms, making them valuable indicators for diseases. Clinical trials on therapeutic aptamers are providing further insight into their potential use. Leukemia is a hematological malignancy characterized by the uncontrolled proliferation of abnormal blood-forming cells, primarily affecting the bone marrow and peripheral blood. This review will highlight the applications of aptamers in relation to the diagnosis and treatment of leukemia. Novel research on leukemia-related aptamers will be presented along with future directions for aptamer-based diagnostic and therapeutic methods.
Small interfering RNA (siRNA) represents a transformative therapeutic class that enables precise gene silencing through RNA interference (RNAi). N-acetyl galactosamine (GalNAc)-conjugated siRNAs have achieved remarkable clinical success with six FDA-approved therapeutics targeting liver diseases. Following subcutaneous administration, GalNAc-siRNAs rapidly accumulate in hepatocytes via asialoglycoprotein receptor (ASGPR)-mediated endocytosis and are sequestered within endolysosomal compartments, creating an intracellular depot that sustains therapeutic effects for weeks to months. While RNAi-mediated degradation and nuclease metabolism contribute to siRNA clearance, the detection of full-length, active siRNA in circulation long after dosing suggests alternative clearance mechanisms exist. This review examines the pharmacokinetic properties and intracellular trafficking of GalNAc-siRNA, with particular focus on underexplored clearance pathways from hepatocytes. We discuss potential mechanisms including endosomal recycling, efflux via exosomes and lysosomal exocytosis that may facilitate siRNA redistribution from tissues to circulation. Understanding these clearance mechanisms could enable correlation of plasma and tissue drug concentrations, reduce preclinical animal use, inform clinical dosing strategies and guide the design of next-generation siRNA therapeutics with optimized tissue residence time and enhanced therapeutic durability.
Oligonucleotide therapeutics are emerging as a promising modality for targeting disease-associated RNAs. Phosphorothioate (PS)-containing oligonucleotides have gained prominence due to their enhanced stability and pharmacodynamic properties. However, current manufacturing practices afford a mixture of Rp and Sp stereoisomers, and this distribution has been linked to changes in product efficacy. Understanding the sensitivity of analytical methods to changes in this quality attribute has therefore become critically important. Here, we used a suite of analytical techniques-ultraviolet (UV) thermal denaturation, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy-to evaluate the PS diastereomer distribution using Tegsedi, a Food and Drug Administration-approved PS-containing antisense oligonucleotide, and with other synthetic inotersen samples having varied PS diastereomer distributions. While UV and CD techniques showed limited sensitivity, NMR excelled in detecting small changes in the PS diastereomer distribution. The univariate metric of 31P integration was shown to be insufficient for this quality metric evaluation; application of principal component analysis to both 1D 31P and 2D 1H,13C spectra revealed distinct PS changes that arose from the different activators used during manufacturing. This comprehensive evaluation highlights the necessity of advanced analytical techniques in ensuring the quality and consistency of PS-containing oligonucleotide therapeutics.
In the fourth quarter of 2025, a press release announced the approval of the eighth small interfering RNA (siRNA)-based therapeutic. Redemplo (plozasiran), developed by Arrowhead Pharmaceuticals, is the third oligonucleotide-based medicine approved for the treatment of patients with familial chylomicronemia syndrome targeting apolipoprotein C-III. This approval represents the seventh approved siRNA drug based on GalNAc-mediated hepatocyte delivery and introduces the third distinct flavor of this delivery architecture to the clinic.
Adenosine deaminase acting on RNA (ADAR)-mediated RNA editing has emerged as a powerful and precise technology for modifying RNA transcripts, enabling correction of disease-causing mutations without permanent changes to the genome. Recent advances in ADAR protein engineering, guide RNA design, and delivery methods have significantly improved editing efficiency and specificity, overcoming many initial limitations. These developments have expanded the therapeutic potential of ADAR-based editing across a range of conditions, including genetic disorders, cancer, metabolic diseases, and neurodegenerative disorders. Notably, several ADAR-based therapeutics have now entered early clinical trials, marking a critical milestone in translating this technology from bench to bedside. Moreover, its inherent programmability, reversibility, and transient nature make ADAR-mediated RNA editing a highly attractive platform for personalized medicine, enabling tailored interventions based on individual genetic profiles and disease contexts. This review provides a comprehensive comparison of recent innovative advancements in ADAR-based RNA editing technologies, their use in diverse contexts pertinent to human diseases, the key challenges that remain, and future directions for their therapeutic implementation.
Impaired angiogenesis is a common feature of several pathological conditions, including neuromuscular disorders. Such vascular defects not only contribute to disease progression but also may compromise the efficacy of systemically delivered therapies such as antisense oligonucleotides (ASOs) and adeno-associated virus vectors. Enhancing muscle vascularization is therefore an attractive strategy to improve both therapeutic delivery and tissue regeneration. Vascular endothelial growth factor A (VEGF-A) is the principal driver of angiogenesis, but its bioavailability is negatively regulated by VEGFR1/Flt-1, a high-affinity decoy receptor. Here, we investigated a splice-switching ASO (SSO) approach to downregulate Flt-1 expression in murine endothelial cells. We designed ASOs to induce skipping of an out-of-frame exon in the Flt1 transcript, triggering nonsense-mediated decay and reducing protein expression. Screening in C166 endothelial cells identified a lead SSO that efficiently skipped exon 5, resulting in robust Flt-1 downregulation, similar to levels achieved with a control siRNA. Functionally, Flt-1 knockdown enhanced endothelial cell proliferation, survival, and migration upon VEGF-A stimulation. These results provide proof-of-concept for targeting Flt-1 via exon skipping to promote angiogenesis, with potential applications in degenerative or ischemic contexts where vascularization is impaired.
Exon skipping antisense oligonucleotides (AONs) have been extensively studied as a promising method of treating Duchenne muscular dystrophy (DMD), yet the clinical efficacy of the conditionally approved AONs still remains low. Using phosphorothioated locked nucleic acid/2'-fluoro-RNA AONs, we aimed to increase AON efficiency by employing skeletal muscle-targeting conjugate molecules, cholesterol, and docosanoic acid to improve the biodistribution of the therapeutic. While conjugate molecules were able to induce high levels of skipping in an in vitro model, in vivo studies in the hDMDdel52/mdx mouse model caused adverse symptomatic and systemic immune reactions, up to and including death, with little to no appreciable increase in exon skipping. Our study cautions against using these AON conjugates in an animal model due to severe toxicity.
There is a current lack of harmonized regulatory guidance in evaluating the genotoxic potential of oligonucleotide-based therapeutics (ONTs). In particular, guidance has not established the circumstances under which it is acceptable to deviate from the standard test battery. In this study, we analyzed genotoxicity testing strategies and supporting rationales for 91 noncoding ONTs receiving European Scientific Advice between 2004 and 2024. While the standard test battery was performed for the majority of ONTs, reduced test approaches were proposed for 10 products. Furthermore, we examined both the positions of applicants and corresponding European Union (EU) regulatory opinions to identify critical considerations in evaluating genotoxicity. Our findings show that EU regulators see opportunities to deviate from the standard test battery for ONTs if sufficient evidence for class experience can be demonstrated. This was confirmed for several ONTs with well-characterized chemical modifications (ie, phosphorothioate, 2'-methoxyethyl, and 2'-Omethyl), making the standard battery redundant in these cases. Although all reported genotoxicity tests have been uniformly negative, uncertainty remains for future modifications. Ideally, what constitutes sufficient evidence for class experience should be defined in the upcoming International Council for Harmonisation guideline addressing the nonclinical safety evaluation of ONTs (ICH S13), which would allow regulators to accept reduced testing. Together with the industry sharing more knowledge and underlying data that support growing class experience, this development can promote a harmonized approach for future genotoxicity testing of noncoding ONTs.
Advances in backbone modifications are driving the development of nucleic acid therapeutics, yet there is still a need to establish compounds that avoid the potential dose-limiting toxicity of phosphorothioate internucleosidic linkages, particularly outside the central nervous system. We have developed a novel 7',5'-α-bc-DNA (abcDNA) scaffold, and here we benchmark the biophysical and in vivo gene knockdown efficacy of gapmer antisense oligonucleotides (ASOs) containing abcDNA nucleotides. Melting curve analyses show gapmers with abcDNA bases in both wings maintain a good affinity for complementary RNA and demonstrate stable mismatch discrimination, in addition to a high degree of serum biostability. To assess in vivo on-target activity and tolerability, mice were dosed systemically with abcDNA ASOs targeting Malat-1, with or without conjugation to palmitic acid. Multiple tissues were assessed for on-target knockdown efficiency by RT-PCR and in situ hybridization alongside biodistribution analysis by immunohistochemistry. abcDNA ASOs were well tolerated and performed at a comparable level to an equivalent 2'-O-methoxyethylribose gapmer. We also present the first in vivo pharmacokinetic data generated using an enzyme-free nucleic acid nanorobotics method. Together, these data support the utility of abcDNA as a valuable addition to ASO technology that maintains a natural phosphodiester backbone, providing a combination of preferred biostability and on-target affinity with acceptable in vivo tolerability.
In this study, we focused on N-1 impurities in antisense oligonucleotides. We evaluated their binding affinity to the therapeutic target RNA, which had the complementary sequence to the full-length N-mer desired product (DP), and their ability to recruit ribonuclease H (RNase H) using cell-free in vitro assays. The binding affinity of each N-1-mer to the target RNA was extremely low, with binding constants <1: 100 of that of the DP/RNA duplex. However, the degree of destabilization varied significantly depending on the position of the nucleotide defect within the N-1-mer, with differences of up to 5.1 kcal/mol (a 4,000-fold difference in binding constant). This weak binding capability to the target RNA suggests that the presence of the N-1-mer has little effect on DP activity. This study provides essential information for the dissemination of oligonucleotide therapeutics by providing a basis for considering the effect of N-1-mer impurities.
The developmental hazard screening of oligonucleotide therapeutics (ONTs) presents challenges due to their frequent lack of pharmacology in nonclinical species and embryo-fetal exposure is presumed to be limited in vivo. This study demonstrates that direct culture in media containing mipomersen, a 2 '-O-methoxyethyl phosphorothioated antisense oligonucleotide (ASO), results in dose-responsive morphological changes in rat and rabbit whole-embryo culture (WEC). Automated miRNAscope in situ hybridization was used to confirm dose-dependent embryonic exposure and visualize the distribution pattern of mipomersen in the embryo and extraembryonic membranes, suggesting that ONTs may enter the umbilical vasculature and pass into embryo circulation. Neither microinjections nor assisted transfections were required to achieve embryonic exposure. These findings support the utility of WEC as a new approach method (NAM) for developmental hazard identification of ONTs. WEC could complement or partially replace in vivo studies, reducing animal use and required test material amounts, while enabling robust developmental hazard identification for ONTs. This work informs future safety assessment strategies and regulatory guidance for ONTs.
Haploinsufficient autosomal dominant diseases are due to heterozygous mutations that cause inadequate protein expression. Compounds that increase expression of the wild-type allele would be one strategy for treating patients. Synthetic antisense oligonucleotides and double-stranded RNAs have the potential to increase gene expression, making them starting points for drug development. Our goal is to outline strategies for using synthetic nucleic acids to enhance gene expression. We discuss the strengths and limitations of these strategies and the practical challenges behind upregulating the expression of genes as a treatment for haploinsufficient autosomal dominant diseases.
Antisense oligonucleotides (ASOs) represent a promising class of therapeutic agents; yet, their efficacy and/or toxicity profiles are heavily dependent on their tissue distribution and cellular uptake. This study employs nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to elucidate the intracellular distribution of chemically modified ASOs in liver tissue with ultra-high resolution. We demonstrated that fully phosphorothioated ASOs predominantly accumulated in the vesicular structures near nonparenchymal cells, including Kupffer cells. In contrast, partially phosphorothioated ASOs exhibit a uniform distribution throughout the liver. Notably, despite similar overall liver concentrations, ASOs with different chemical modifications exhibited markedly distinct intracellular distribution patterns. These findings highlight the critical importance of subcellular distribution in ASO drug discovery and underscore the utility of NanoSIMS in visualizing the ASO biodistribution. This approach, when combined with electron microscopy, provides invaluable insights into the chemical composition and localization of ASOs within cellular compartments. This study not only advances our understanding of ASO behavior in vivo but also highlights the potential of high-resolution imaging techniques in optimizing ASO delivery strategies. These insights are crucial for enhancing the efficacy and minimizing the adverse effects of ASO-based therapeutics, paving the way for more targeted and effective treatments.
Small interfering RNA (siRNA) therapeutics represent a transformative class of drugs, but their class-specific adverse events (CAE-siRNA) remain incompletely characterized. This study aimed to identify and quantify CAE-siRNA associated with U.S. Food and Drug Administration (FDA)-approved siRNA drugs (patisiran, givosiran, vutrisiran, inclisiran, and lumasiran) using real-world pharmacovigilance data, focusing on potential class-wide effects. A disproportionality analysis was conducted using the FDA Adverse Event Reporting System database (2014–2025Q2) accessed via the MY FAERS platform. The reporting odds ratio (ROR) with 95% confidence interval (CI) was calculated, with signals defined by a lower CI >1 and ≥3 cases. Sensitivity analyses included indication-matched populations (IMPs) and exclusion of concomitant medications. Causality was assessed using Bradford Hill criteria. Among 6200 siRNA-treated patients, 45 CAE-siRNA spanning 10 system organ classes were identified. Pain and pain in extremity, fatigue, and gastrointestinal disorders were the most frequently reported. Notably, patisiran was associated with an elevated risk of back pain (ROR: 2.28, 95% CI: 1.84–2.83), whereas givosiran exhibited significant signals for stress (ROR: 5.29, 95% CI: 3.64–7.70) and weight loss (ROR: 2.35, 95% CI: 1.74–3.16). Of particular concern, inclisiran demonstrated strong hepatic toxicity signals (ROR ranging from 9.11 to 86.06) along with discomfort (ROR: 3.60, 95% CI: 1.34–9.65). Sensitivity analyses confirmed robustness across subgroups. Furthermore, causality assessment supported a likely association between the hepatic toxicity and inclisiran. This study identified clinically relevant CAE-siRNA, particularly hepatic toxicity for inclisiran, supporting enhanced monitoring. While disproportionality analyses are hypothesis generating, these findings underscore the need for targeted pharmacovigilance to optimize the safety of this promising drug class.
Pathogenic variants creating upstream open reading frames (uORFs) in the 5' untranslated region (5'UTR) of the ENG gene can disrupt translation from the main ORF and contribute to hereditary hemorrhagic telangiectasia (HHT). This is the case of the ENG c.-79C>T that introduces a uAUG shown to decrease endoglin expression and associates with HHT. Here, we investigated whether 2'-O-methyl (2'OMe) antisense oligonucleotides (ASOs) could restore protein levels by masking this aberrant uAUG or by targeting predicted secondary structures within the ENG 5'UTR. Several ASOs of varying lengths and backbone chemistries (full phosphodiester or full phosphorothioate) were designed to target the mutant region. Their effects were evaluated in HeLa cells transfected and in HUVECs transduced with wild-type or mutant ENG constructs. Transfection efficiency was verified by MALAT1 knockdown via qPCR, and endoglin protein levels were assessed by Western blot. Despite efficient ASO delivery and optimized experimental conditions, no reproducible increase in endoglin expression was observed upon ASO treatment. These findings highlight the limitations of steric-blocking ASOs targeting 5'UTR variants and underscore the need for deeper mechanistic understanding of uORF-mediated translational regulation.
Antisense oligonucleotides (ASOs) are chemically modified single-stranded oligonucleotides used to modulate the expression or processing of a target RNA transcript. The development of ASOs to treat human disease requires extensive preclinical studies in animal models. A critical component of these studies is determining the concentration of the ASO in tissues and biofluids, which are used to estimate the distribution, half-life, and dose-response relationship. The methods used to quantify ASOs are often constrained by low sensitivities, poor dynamic ranges, and the use of highly specialized equipment. Here, we describe the development of a Splint-Ligation-based quantitative PCR assay to measure the concentration of ASOs in nonhuman primate (NHP) tissues and biofluids. Our results show that the Splint Ligation Assay was highly sensitive across central nervous system (CNS) tissues and biofluids (as low as 100 pM in NHP CNS tissue and 1 pM in NHP plasma), with broad linear dynamic ranges. Overall, our results show that the Splint-Ligation PCR Assay is a reliable, sensitive, and feasible method of ASO quantification.
