Nucleic acid therapeutics最新文献

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.

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.

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.

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.

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.

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.

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.

Plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (tPA), is a key regulator of fibrinolysis. Elevated levels of PAI-1 are linked to thrombotic disorders and correlate with poor prognosis across various cancers. In this study, we further characterize the RNA aptamer R10-4, previously shown to bind PAI-1 with high affinity and inhibit its antiproteolytic activity. While R10-4's role in modulating fibrinolysis is established, its influence on cancer cell behavior remains unclear. Here, we demonstrate that intracellular transfection of R10-4 in triple negative breast cancer cells significantly impairs migration and invasion without affecting proliferation, mirroring the effects observed with other PAI-1-specific RNA aptamers. Moreover, conditioned media from R10-4 transfected cells suppress endothelial tube formation and exhibit reduced secretion of the pro-angiogenic chemokine (C-C) motif ligand 5 (CCL5). Collectively, these findings reveal that R10-4 restores fibrinolytic balance and disrupts PAI-1-mediated tumor progression, positioning it as a promising multifunctional candidate for therapeutic development.