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.

Pathogenic variants creating upstream open reading frames (uORFs) in the 5' untranslated region (5'UTR) of the gene can disrupt translation from the main ORF and contribute to hereditary hemorrhagic telangiectasia (HHT). This is the case of the 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 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 constructs. Transfection efficiency was verified by 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) 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 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.

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.

The severe X-linked degenerative neuromuscular disease Duchenne muscular dystrophy (DMD) is caused by the loss of dystrophin through reading frame disruptive mutations in the DMD gene. Dystrophin protein is crucial for the stability of the muscle. Targeting specific exons with antisense oligonucleotides (ASO) will prevent inclusion of the exon during pre-mRNA splicing, which can restore the reading frame, facilitating the production of partially functional dystrophin proteins. For DMD, four ASOs of the phosphorodiamidate morpholino oligomer (PMOs) chemistry are FDA approved. It is anticipated that improved delivery to skeletal muscle and heart will lead to larger therapeutic results. With our research, we sought to identify muscle-homing peptides that can achieve increased delivery of ASOs to muscle or heart when conjugated to PMOs. We applied phage display biopanning mouse models for DMD to identify muscle-homing peptides while simultaneously negatively selecting peptides that home to unwanted organs, such as the kidney and liver. After confirmation of the muscle homing ability , we conjugated selected candidate peptides to PMOs to be tested , where we found that conjugation of one specific muscle homing peptide led to significantly improved delivery to muscle, with a small improvement in exon skipping and dystrophin restoration.

Intrathecally administered RNase H1-active gapmer antisense oligonucleotides (ASOs) are promising therapeutics for brain diseases where lowering the expression of one target gene is expected to be therapeutically beneficial. Such ASOs are active, to varying degrees, across most or all cell types in the cortex and cerebellum of mouse and non-human primate (NHP) brain regions with substantial drug accumulation. Intrathecally delivered ASOs, however, exhibit a gradient of exposure across the brain, with more limited drug accumulation and weaker target engagement in deep brain regions of NHP. Here, we profiled the activity of a tool, ASO, against in three deep brain regions of NHP: thalamus, caudate, and putamen. All neuronal subtypes exhibited knockdown similar to, or deeper than, the bulk tissue. Among non-neuronal cells, knockdown was deepest in microglia and weakest in endothelial stalk. Overall, we observed broad target engagement across all cell types detected, supporting the relevance of intrathecal ASOs to diseases with deep brain involvement.

Mini/midigene splicing assays are often used to evaluate splicing modulation therapy, for example, employing antisense oligonucleotides (AONs). Twenty-five AONs targeting the splicing defect caused by a recurrent variant in (c.768G>T) were tested using a midigene containing a part of intron 5, exon 6, and a part of intron 6 of the gene. Surprisingly, almost all AONs showed high efficacy, complicating candidate selection. We hypothesized that the lack of genomic context may lead to a very accessible transcript for AONs. Indeed, the use of an maxigene that contains a part of intron 5, exon 6, parts of intron 6, and the genomic region between exons 7 and 11 allowed a clear distinction between efficacious and less efficacious AONs, corroborating the results we recently observed in patient-derived retinal cells. These underscore the necessity of a proper genetic context included in constructs used in splicing assays to assess the potential of splicing modulation therapy.

Clustered regularly interspaced short palindromic repeats-based editing is inefficient at over two-thirds of genetic targets. A primary cause is ribonucleic acid (RNA) misfolding that can occur between the spacer and scaffold regions of the gRNA, which hinders the formation of functional Cas9 ribonucleoprotein (RNP) complexes. Here, we uncover hundreds of highly efficient gRNA variant scaffolds for (Sa)Cas9 utilizing an innovative binding and ligand activation driven enrichment (BLADE) methodology, which leverages asymmetrical product dissociation over rounds of evolution. SaBLADE-derived gRNA scaffolds contain 7%-42% of nucleotide variation relative to wild type. gRNA variants are able to improve gene editing efficiency at all targets tested, and they achieve their highest levels of editing improvement (>400%) at the most challenging DNA target sites for the wild-type SaCas9 gRNA. This arsenal of SaBLADE-derived gRNA variants showcases the power and flexibility of combinatorial chemistry and directed evolution to enable efficient gene editing at challenging, or previously intractable, genomic sites.

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 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.

The 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 , a transgenic human knock-in mouse (hu) 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 hu mice. Overall, these data demonstrate the hu 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.

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.

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.

BK polyomavirus, a virus that latently resides in tubular epithelial cells of human kidneys, represents a major clinical problem for immunocompromised patients undergoing kidney transplantation. No proven effective specific antiviral therapies other than reduction of immunosuppressants exist. We exploited splice-modulating antisense oligonucleotides (ASOs) to block expression of the viral "gatekeeper protein" T antigen to specifically inhibit viral replication. Candidate oligonucleotides were screened for inhibitory activity in BK virus-infected cells, resulting in up to 97% reduction of viral T antigen mRNA and virus-encapsulating protein. The most promising ASO candidates were validated in BK virus-infected human kidney epithelial cells, showing sequence-specific activity that was exacerbated by chemical modifications. Administration of the candidate oligonucleotide in mice revealed long-lasting uptake in proximal tubules of the kidney, where BK virus resides. A final optimization round yielded an oligonucleotide displaying superior activity. Studies in transformed cells revealed a discernible shift in T antigen splicing not observed with a scrambled control, indicating that targeting of the donor splice site was responsible for the observed effects. Here, we demonstrate proof-of-principle for a direct-acting, splice-modulating, universal ASO that distributes to sites where BK viral replication occurs, warranting further development of this novel therapeutic to combat BK virus replication in kidney transplant patients.

We present a general method for in-cellulo delivery of 2'--methyl (2'-OMe) RNA oligonucleotides (oligos) to mitochondria for antisense applications, with potential for implementation in other mitochondrial DNA (mtDNA)-targeted therapies. Exosomes, which are nanoscale, naturally occurring extracellular vesicles (EVs), have been employed for biotechnology applications in oligonucleotide delivery in recent years. We discovered that exosomes from fetal bovine serum (FBS) can be used as a simple and biologically compatible delivery agent of 2'-OMe RNA antisense oligonucleotides to cellular mitochondria, leading to target protein knockdown. While most RNA interference and antisense mechanisms occur in the cytoplasm or nucleus, the need for mitochondrial targeting has become increasingly apparent. Mitochondrial disease describes a variety of currently incurable syndromes that especially affect organs requiring significant energy including the muscles, heart, and brain. Many of these syndromes result from mutations in mtDNA, which codes for the 13 proteins of the oxidative phosphorylation system and are thus often implicated in inherited metabolic disorders.

Antisense oligonucleotides (ASOs) are short, synthetic nucleic acids designed to specifically bind to complementary sequences of RNA. They have become powerful tools in research and medicine due to their ability to modulate gene expression through RNase H-mediated target reduction as well as splice modulation. Molecular dynamics simulations and molecular modeling play critical roles in the study, design, and optimization of ASOs. These computational techniques provide detailed insights into the structure, behavior, and interactions of ASOs at the molecular level. Here, we present a summary of the applications of computational chemistry tools in the study of ASOs and discuss the strengths and disadvantages of each approach.

The efficacy of nucleic acid therapeutics (NATs) such as antisense oligonucleotides (ASOs) and small interfering RNAs relies on multiple stages of extra- and intracellular trafficking. Assessing uptake and efficacy often relies on fluorescent tagging of the NAT for imaging, although the exogenous tag undoubtedly influences the kinetics of intracellular transport and does not represent the compound used clinically. Therefore, better methods to assess the cellular and tissue distribution of NATs are needed. Here, we have validated new panels of antibody reagents that target clinically relevant nucleic acid modifications for visualizing ASOs both and . Using the ModDetect™ library of antibodies, we have tested ASOs for intracellular localization by immunocytochemistry and for biodistribution in mouse tissues by immunohistochemistry. Antibodies specific for the commonly used phosphorothioate (PS) or 2'--methoxyethyl (2'-MOE) modifications successfully detected gapmer ASOs, facilitating colocalization studies with endosomal markers in 2D and 3D cell models. In addition, we assessed colocalization of anti-PS signals with fluorescently tagged ASOs. Our data demonstrate the utility of these reagents for the NAT field, where modified nucleic acids can be detected irrespective of the nucleotide sequence, rendering the system amenable for multiple clinical and preclinical workflows and quantitative immunoassays.

Amido-bridged nucleic acid (AmNA) and a 2'-O,4'-C-spirocyclopropylene bridged nucleic acid (scpBNA) are bridged nucleic acid analogs with high binding affinity toward complementary strands along with high nuclease resistance. AmNA and scpBNA have been developed to overcome phosphorothioate modified gapmer hepatotoxicity, while the mechanism of reducing hepatotoxicity still remains unknown. Here, we found that antisense oligonucleotides (ASOs) with combination of AmNA, scpBNA, and phosphodiester (PO) bonds could significantly reduce hepatotoxicity in mice. Histopathological findings of the periportal spaces of the liver were observed only in the locked nucleic acid and AmNA-scpBNA groups, but not in the AmNA-scpBNA-PO group. Furthermore, bioinformatics and histopathological analysis revealed that the reduced hepatotoxicity might be related to mitochondrial abnormalities, such as decreased expression levels of Atp5o and Sdhb genes. Taken together, the results of this study demonstrated that AmNA, scpBNA, and PO modification are able to reduce hepatotoxicity for improving the potential of ASOs.

Nonclinical safety screening of small interfering RNAs (siRNAs) conjugated to a trivalent -acetylgalactosamine (GalNAc) ligand is typically carried out in rats at exaggerated exposures in a repeat-dose regimen. We have previously shown that at these suprapharmacological doses, hepatotoxicity observed with a subset of GalNAc-siRNAs is largely driven by undesired RNA-induced silencing complex (RISC)-mediated antisense strand seed-based off-target activity, similar to microRNA-like regulation. However, the RISC component requirements for off-target activity of siRNAs have not been evaluated. Here, we evaluate the roles of major RISC components, AGO and TNRC6 (or GW182) proteins, in driving on- and off-target activity of GalNAc-siRNAs in hepatocytes, and . We demonstrate that knocking down AGO2, but not AGO1 or AGO4, is protective against GalNAc-siRNA-driven off-target activity and hepatotoxicity. As expected, knocking down AGO2, but not AGO1 or AGO4, reduces the on-target activity of GalNAc-siRNA. Similarly, knocking down TNRC6 paralogs, TNRC6A or TNRC6B, but not TNRC6C, is protective against off-target activity and hepatotoxicity while having minimal impact on the on-target activity of GalNAc-siRNA. These data indicate that while AGO2 is the only RISC component required for the on-target activity of GalNAc-siRNAs, the undesired off-target activity and hepatotoxicity of a subset of GalNAc-siRNAs are mediated via the RISC composed predominantly of AGO2 and TNRC6 paralogs TNRC6A and/or TNRC6B.

Chemically modified short interfering RNAs (siRNAs) unequivocally represent a groundbreaking class of drugs. The deliberate chemical modification of the natural structure has been pivotal to their resounding success. Specific modifications at certain positions bolster their potency, safety, stability, and specificity. In clinical research, 2'--methyl and 2'-fluoro are the most used modifications. The effects of a wide range of chemical changes in fully modified siRNAs have not been thoroughly evaluated for tolerability. In this study, we utilized two sequences in a fully modified siRNA to systematically assess the tolerability of single nucleotide backbone and sugar modifications, including deoxyribonucleic acid, 2'--(2-methoxyethyl), locked nucleic acid, unlocked nucleic acid, mismatches, butane diol substitution, and butane diol insertion. We synthesized 522 siRNA variants and evaluated their efficacy . Our findings demonstrate that individual tolerability is significantly influenced by the modification's sequence, pattern, and position, with limited universal principles identifiable from this dataset. The efficacy results are probably driven by the thermodynamic balance defined by a combination of parameters. The framework presented here will serve as a reference dataset to facilitate the expansion of chemical diversity in therapeutic siRNAs.

A 2023 workshop brought together stakeholders involved in the development and safety assessment of oligonucleotide (ONT) therapeutics. The purpose was to discuss potential strategies and opportunities for enhancing developmental and reproductive toxicity (DART) assessment of ONTs. The workshop was timely, bringing together regulators, industry representatives, consultants, and contract research organization partners interested in the ongoing development of internationally harmonized guidance for nonclinical safety assessment of ONTs. Given DART's importance in nonclinical safety assessment and the unique attributes of ONTs, the forum discussed case studies, consensus approaches, and areas needing further development to optimize DART strategies. This report covers the workshop proceedings, highlighting methods to achieve a robust DART assessment for ONTs. It includes case studies that described strategies for dose level selection, dosing frequency, species selection, and alternative animal model approaches. Topics also cover surrogate ONT use, exposure of the placenta and embryo/fetal compartment, and weight of evidence approaches. A goal of these workshop proceedings is to describe example approaches to hopefully inform the DART strategy expectations in the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidance currently under development for nonclinical safety assessment of ONTs.