Nucleic acid therapeutics最新文献

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.

The PNPLA3 single nucleotide polymorphism, rs738409, is the strongest known genetic risk factor for metabolic dysfunction-associated steatotic liver disease; thus, targeting the minor allele with a GalNAc-conjugated siRNA is an attractive strategy to treat patients carrying the genetic variant. To enable translational safety assessment of a GalNAc-conjugated siRNA that specifically targets the rs738409 sequence of PNPLA3, a transgenic human PNPLA3^I148M knock-in mouse (huPNPLA3^I148M) was utilized. This model showed no significant genotype-related phenotypic differences to wild-type mice in a phenotype characterization study when maintained on standard rodent chow. Additionally, a repeat-dose toxicology study using a GalNAc-conjugated siRNA specific for rs738409 resulted in comparable findings between genotypes (i.e., liver enzyme and histopathology changes), indicating the findings were due to the siRNA therapeutic and not a result of target knockdown in huPNPLA3^I148M mice. Overall, these data demonstrate the huPNPLA3^I148M mouse is suitable for repeat-dose toxicology studies, suggesting this approach could be applied to other siRNA programs lacking a pharmacologically relevant nonclinical species to support translational safety assessments during drug development.

Cryopreservation is a routine step in the manufacturing process of adoptive cell therapies (ACT), providing critical logistic flexibility. RNA interference (RNAi)-based therapies are increasingly being explored as enhancers or modulators of ACT. However, the impact of cryopreservation on cells treated with RNAi-based therapies has not been investigated before. In this study, we addressed this knowledge gap by examining silencing efficacy in small interfering RNA (siRNA)-treated cells that undergo cryopreservation. Our findings demonstrate that silencing in cryopreserved cells is comparable to that in cells maintained continuously in culture. Moreover, we found that the duration of siRNA exposure plays a significant role in cells that later undergo cryopreservation, with extended exposure improving silencing efficiency. However, this effect diminishes at higher siRNA concentrations. Additionally, we showed that siRNA treatment is feasible at low temperatures (2°C-8°C), and siRNA-treated cells can be cryopreserved for extended periods (at least 1 month) without loss of efficacy. Our work establishes the feasibility of integrating siRNA treatments into current manufacturing processes for ACT.

Posttranscriptional regulation is crucial for siRNA design, as decay rates in cell lines influence perceived siRNA potency. This study profiles transcripts with 'fast' and 'slow' half-lives in HeLa and SH-SY5Y cells, commonly used in drug discovery. We calculated half-lives for 1,815 HeLa and 5,376 SH-SY5Y transcripts, finding comparable half-lives between cell lines, though HeLa cells generally had longer half-lives. Comparing mRNA and protein half-lives, 'fast' decay transcripts encoded proteins with shorter half-lives, while 'slow' decay transcripts encoded stable proteins. We linked mRNA decay rates to siRNA activity by comparing HeLa data to a previous siRNA screen, discovering that faster decay transcripts had lower knockdown. Surprisingly, stable transcripts, more amenable to knockdown, were over-represented by membrane protein-coding transcripts. Despite their stability, these transcripts had low-to-moderate expression, regardless of miRNA regulation. We explored cis- and trans- features affecting mRNA stability and expression, suggesting that low RNA binding protein (RBP) binding, combined with specific stabilizing RBP regulation, contributes to the stability of these membrane protein-coding transcripts. This study highlights the importance of understanding transcript features, mRNA decay and its potential impact on siRNA efficacy, particularly for transcripts encoding membrane proteins.

The emergence of nucleic acid (NA) therapeutics, including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), which are usually delivered directly, and messenger RNAs (mRNAs), which are typically encapsulated in lipid nanoparticles (LNPs), marks a transformative era in precision medicine. While these therapies offer precise approaches for gene regulation or expression, they can trigger unwanted innate and/or adaptive immune responses that can either have no significant impact or adversely affect treatment efficacy and/or patient safety. Consequently, therapies where an adaptive immune response is desired, such mRNA/LNP-based vaccines against infectious diseases or cancer are out of scope of this article. In the present work, the Innovation and Quality Consortium Nucleic Acids Immunogenicity Working Group examines how the various components of NA-based therapies might contribute to their immunogenic potential and describes risk mitigation strategies through product design adaptations during early development stages. In addition, a comprehensive immunogenicity risk assessment framework is described, allowing to effectively define a tailored clinical testing strategy for different NA modalities with varying immunogenicity (IG) consequences. A streamlined monitoring strategy is recommended when minimal impact is expected, whereas extensive testing is suggested when safety concerns arise. Overall, these recommendations ensure that safe and effective NA-based therapies reach patients with an appropriate assessment of the IG potential.

Antisense oligonucleotides (ASOs) designed to recruit RNase H1 (gapmer ASOs) have been used successfully to downregulate the expression of therapeutic targets. Gapmer ASOs can be identified that selectively reduce the expression of transcripts containing the perfectly complementary intended ASO target site without affecting the expression of unintended transcripts (selective ASOs). However, ASOs can also be identified that reduce the expression of unintended transcripts with target sites that are not perfectly complementary to the ASO (nonselective ASOs). Currently, the understanding of in silico rules for predicting off-targets is suboptimal. In order to determine the selectivity of gapmer ASOs, we therefore developed an experimental workflow called concentration-response digital gene expression (CR-DGE). In CR-DGE, ASO treatment is performed at increasing concentrations, and the effect on the transcriptome is measured using 3'Tag-Seq. Expression data are then analyzed to identify genes with concentration-responsive knockdown. We demonstrate that CR-DGE identifies gapmer ASO concentration-responsive genes with high reproducibility and greater sensitivity than conventional single-concentration assays. Applying CR-DGE to a panel of gapmer ASOs identifies ASOs with a range of selectivity. These results demonstrate that CR-DGE can be used effectively to assess the selectivity of gapmer ASOs, offering a valuable tool for research and therapeutic development.

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.

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) 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.