Sequence- and Structure-Dependent Cytotoxicity of Phosphorothioate and 2'-O-Methyl Modified Single-Stranded Oligonucleotides.

Single-stranded oligonucleotides (SSOs) are a rapidly expanding class of therapeutics that comprises antisense oligonucleotides, microRNAs, and aptamers, with ten clinically approved molecules. Chemical modifications such as the phosphorothioate backbone and the 2'-O-methyl ribose can improve the stability and pharmacokinetic properties of therapeutic SSOs, but they can also lead to toxicity in vitro and in vivo through nonspecific interactions with cellular proteins, gene expression changes, disturbed RNA processing, and changes in nuclear structures and protein distribution. In this study, we screened a mini library of 277 phosphorothioate and 2'-O-methyl-modified SSOs, with or without mRNA complementarity, for cytotoxic properties in two cancer cell lines. Using circular dichroism, nucleic magnetic resonance, and molecular dynamics simulations, we show that phosphorothioate- and 2'-O-methyl-modified SSOs that form stable hairpin structures through Watson-Crick base pairing are more likely to be cytotoxic than those that exist in an extended conformation. In addition, moderate and highly cytotoxic SSOs in our dataset have a higher mean purine composition than pyrimidine. Overall, our study demonstrates a structure-cytotoxicity relationship and indicates that the formation of stable hairpins should be a consideration when designing SSOs toward optimal therapeutic profiles.

Unwinding the SARS-CoV-2 Ribosomal Frameshifting Pseudoknot with LNA and G-Clamp-Modified Phosphorothioate Oligonucleotides Inhibits Viral Replication.

Ribosomal frameshifting (RFS) at the slippery site of SARS-CoV-2 RNA is essential for the biosynthesis of the viral replication machinery. It requires the formation of a pseudoknot (PK) structure near the slippery site and can be inhibited by PK-disrupting oligonucleotide-based antivirals. We obtained and compared three types of such antiviral candidates, namely locked nucleic acids (LNA), LNA-DNA gapmers, and G-clamp-containing phosphorothioates (CPSs) complementary to PK stems. Using optical and electrophoretic methods, we showed that stem 2-targeting oligonucleotide analogs induced PK unfolding at nanomolar concentrations, and this effect was particularly pronounced in the case of LNA. For the leading PK-unfolding LNA and CPS oligonucleotide analogs, we also demonstrated dose-dependent RSF inhibition in dual luciferase assays (DLAs). Finally, we showed that the leading oligonucleotide analogs reduced SARS-CoV-2 replication at subtoxic concentrations in the nanomolar range in two human cell lines. Our findings highlight the promise of PK targeting, illustrate the advantages and limitations of various types of DNA modifications and may promote the future development of oligonucleotide-based antivirals.

SIDT2 Inhibits Phosphorothioate Antisense Oligonucleotide Activity by Regulating Cellular Localization of Lysosomes.

Phosphorothioate (PS)-modified antisense oligonucleotide (ASO) drugs enter cells through endocytic pathways where a majority are entrapped within membrane-bound endosomes and lysosomes, representing a limiting step for antisense activity. While late endosomes have been identified as a major site for productive PS-ASO release, how lysosomes regulate PS-ASO activity beyond macromolecule degradation remains not fully understood. In this study, we reported that SID1 transmembrane family, member 2 (SIDT2), a lysosome transmembrane protein, can robustly regulate PS-ASO activity. We showed that SIDT2 is required for the proper colocalization between PS-ASO and lysosomes, suggesting an important role of SIDT2 in the entrapment of PS-ASOs in lysosomes. Mechanistically, we revealed that SIDT2 regulates lysosome cellular location. Lysosome location is largely determined by its movement along microtubules. Interestingly, we also observed an enrichment of proteins involved in microtubule function among SIDT2-binding proteins, suggesting that SIDT2 regulates lysosome location via its interaction with microtubule-related proteins. Overall, our data suggest that lysosome protein SIDT2 inhibits PS-ASO activity potentially through its interaction with microtubule-related proteins to place lysosomes at perinuclear regions, thus, facilitating PS-ASO's localization to lysosomes for degradation.

Phosphorothioate-linked guanine/cytosine-based stem-loop oligonucleotides induce the extracellular release of mitochondrial DNA from peritoneal B1a cells.

It has been previously demonstrated that phosphorothioate-linked GpC-based stem-loop oligonucleotides (GC-SL ODN) induce the release of mitochondrial DNA (mtDNA) from chronic lymphocytic leukemia (CLL) B cells. Although CLL B cells are believed to originate from CD5+ B cells because of their phenotypic similarities, it remains unclear whether GC-SL ODN can stimulate CD5+ B1 cells to secrete mtDNA. To explore this possibility, we compared the frequency of the mtDNA-producing population among peritoneal cells after GC-SL ODN treatment. We found that mtDNA-releasing cells are enriched for peritoneal CD19+ B cells upon GC-SL ODN challenge. Among peritoneal CD19+ B cells, the CD5+ B1a subpopulation was a primary cellular source of mtDNA secretion in GC-SL ODN-elicited immune responses. GC-SL ODN-stimulated mtDNA release by B1a cells was positively regulated by MyD88 and TRIF signaling pathways. In vivo GC-SL ODN treatment increased lipopolysaccharide-induced activation of innate immune cells such as NK cells, suggesting the immune-enhancing effects of mtDNA secretion. Furthermore, the loop size formed by GC-SL ODNs was a critical factor in inducing mtDNA release by B1a cells. Taken together, our results identified GC-SL ODN as promising biomaterials for enhancing immune responses.

NAT10 and DDX21 Proteins Interact with RNase H1 and Affect the Performance of Phosphorothioate Oligonucleotides.

RNase H1-dependent phosphorothioate oligonucleotides (PS-ASOs) have been developed to treat various diseases through specific degradation of target RNAs. Although many factors or features of RNA and PS-ASOs have been demonstrated to affect antisense activity of PS-ASOs, little is known regarding the roles of RNase H1-associated proteins in PS-ASO performance. In this study, we report that two nucleolar proteins, NAT10 and DDX21, interact with RNase H1 and affect the potency and safety of PS-ASOs. The interactions of these two proteins with RNase H1 were determined using BioID proximity labeling in cells and confirmed biochemically. Reduction of NAT10 and DDX21 decreased PS-ASO activity in cells, and purified NAT10 and DDX21 proteins enhanced RNase H1 cleavage rates, indicating that these two proteins facilitate RNase H1 endoribonuclease activity. Consistently, reduction of these proteins increased the levels of R-loops, and impaired pre-rRNA processing. In addition, reduction of the two proteins increased the cytotoxicity of toxic PS-ASOs, and treatment of toxic PS-ASOs also altered the localization of these proteins. Together, this study shows for the first time that NAT10 and DDX21 interact with RNase H1 protein and enhance its enzymatic activity, contributing to the potency and safety of PS-ASOs.

The Combination of Mesyl-Phosphoramidate Inter-Nucleotide Linkages and 2'-O-Methyl in Selected Positions in the Antisense Oligonucleotide Enhances the Performance of RNaseH1 Active PS-ASOs.

Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2'-O-methyl (2'-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2'-methoxyethyl, 2' constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs. In this study, we evaluated the effects of the combination of MsPA linkages and 2'-OMe nucleotides on PS ASO performance. We found that two MsPA modifications at the 5' end of the gap or in the 3'-wing of a Gap 2'-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap without altering the safety profile. Such effects were observed with multiple sequences in cells and animals. Thus, the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2'-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.

Control of backbone chemistry and chirality boost oligonucleotide splice switching activity.

Although recent regulatory approval of splice-switching oligonucleotides (SSOs) for the treatment of neuromuscular disease such as Duchenne muscular dystrophy has been an advance for the splice-switching field, current SSO chemistries have shown limited clinical benefit due to poor pharmacology. To overcome limitations of existing technologies, we engineered chimeric stereopure oligonucleotides with phosphorothioate (PS) and phosphoryl guanidine-containing (PN) backbones. We demonstrate that these chimeric stereopure oligonucleotides have markedly improved pharmacology and efficacy compared with PS-modified oligonucleotides, preventing premature death and improving median survival from 49 days to at least 280 days in a dystrophic mouse model with an aggressive phenotype. These data demonstrate that chemical optimization alone can profoundly impact oligonucleotide pharmacology and highlight the potential for continued innovation around the oligonucleotide backbone. More specifically, we conclude that chimeric stereopure oligonucleotides are a promising splice-switching modality with potential for the treatment of neuromuscular and other genetic diseases impacting difficult to reach tissues such as the skeletal muscle and heart.

Solid-Phase Separation of Toxic Phosphorothioate Antisense Oligonucleotide-Protein Nucleolar Aggregates Is Cytoprotective.

Phosphorothioate antisense oligonucleotides (PS-ASOs) interact with proteins and can localize to or induce the formation of a variety of subcellular PS-ASO-protein or PS-ASO-ribonucleoprotein aggregates. In this study, we show that these different aggregates that form with varying compositions at various concentrations in the cytosol, nucleus, and nucleolus may undergo phase separations in cells. Some aggregates can form with both nontoxic and toxic PS-ASOs, such as PS bodies, paraspeckles, and nuclear filaments. However, toxic PS-ASOs have been shown to form unique nucleolar aggregates that result in nucleolar dysfunction and apoptosis. These include liquid-like aggregates that we labeled "cloudy nucleoli" and solid-like perinucleolar filaments. Toxic nucleolar aggregates may undergo solid-phase separation and in the solid phase, protein mobility in and out of the aggregates is limited. Other aggregates appear to undergo liquid-phase separation, including paraspeckles and perinucleolar caps, in which protein mobility is negatively correlated with the binding affinity of the proteins to PS-ASOs. However, PS bodies and nuclear filaments are solid-like aggregates. Importantly, in cells that survived treatment with toxic PS-ASOs, solid-like PS-ASO aggregates accumulated, especially Hsc70-containing nucleolus-like structures, in which modest pre-rRNA transcriptional activity was retained and appeared to mitigate the nucleolar toxicity. This is the first demonstration that exogenous drugs, PS-ASOs, can form aggregates that undergo phase separations and that solid-phase separation of toxic PS-ASO-induced nucleolar aggregates is cytoprotective.

Extracellular Nucleotides Affect the Proangiogenic Behavior of Fibroblasts, Keratinocytes, and Endothelial Cells.

Chronic wound healing is currently a severe problem due to its incidence and associated complications. Intensive research is underway on substances that retain their biological activity in the wound microenvironment and stimulate the formation of new blood vessels critical for tissue regeneration. This group includes synthetic compounds with proangiogenic activity. Previously, we identified phosphorothioate analogs of nucleoside 5'-O-monophosphates as multifunctional ligands of P2Y6 and P2Y14 receptors. The effects of a series of unmodified and phosphorothioate nucleotide analogs on the secretion of VEGF from keratinocytes and fibroblasts, as well as their influence on the viability and proliferation of keratinocytes, fibroblasts, and endothelial cells were analyzed. In addition, the expression profiles of genes encoding nucleotide receptors in tested cell models were also investigated. In this study, we defined thymidine 5'-O-monophosphorothioate (TMPS) as a positive regulator of angiogenesis. Preliminary analyses confirmed the proangiogenic potency of TMPS in vivo.

A Fluorescent Assay to Search for Inhibitors of HIV-1 Integrase Interactions with Human Ku70 Protein, and Its Application for Characterization of Oligonucleotide Inhibitors.

The search for compounds that can inhibit the interaction of certain viral proteins with their cellular partners is a promising trend in the development of antiviral drugs. We have previously shown that binding of HIV-1 integrase with human Ku70 protein is essential for viral replication. Here, we present a novel, cheap, and fast assay to search for inhibitors of these proteins' binding based on the usage of genetically encoded fluorescent tags linked to both integrase and Ku70. Using this approach, we have elucidated structure-activity relationships for a set of oligonucleotide conjugates with eosin and shown that their inhibitory activity is primarily achieved through interactions between the conjugate nucleic bases and integrase. Molecular modeling of HIV-1 integrase in complex with the conjugates suggests that they can shield E212/L213 residues in integrase, which are crucial for its efficient binding to Ku70, in a length-dependent manner. Using the developed system, we have found the 11-mer phosphorothioate bearing 3'-end eosin-Y to be the most efficient inhibitor among the tested conjugates.

Phosphorothioate modified oligonucleotide-protein interactions.

Antisense oligonucleotides (ASOs) interact with target RNAs via hybridization to modulate gene expression through different mechanisms. ASO therapeutics are chemically modified and include phosphorothioate (PS) backbone modifications and different ribose and base modifications to improve pharmacological properties. Modified PS ASOs display better binding affinity to the target RNAs and increased binding to proteins. Moreover, PS ASO protein interactions can affect many aspects of their performance, including distribution and tissue delivery, cellular uptake, intracellular trafficking, potency and toxicity. In this review, we summarize recent progress in understanding PS ASO protein interactions, highlighting the proteins with which PS ASOs interact, the influence of PS ASO protein interactions on ASO performance, and the structure activity relationships of PS ASO modification and protein interactions. A detailed understanding of these interactions can aid in the design of safer and more potent ASO drugs, as illustrated by recent findings that altering ASO chemical modifications dramatically improves therapeutic index.

Biological Effects of Morpholin-4-Ium 4 Methoxyphenyl (Morpholino) Phosphinodithioate and Other Phosphorothioate-Based Hydrogen Sulfide Donors.

Significance: Hydrogen sulfide (H2S) is regarded as the third gasotransmitter along with nitric oxide and carbon monoxide. Extensive studies have demonstrated a variety of biological roles for H2S in neurophysiology, cardiovascular disease, endocrine regulation, and other physiological and pathological processes. Recent Advances: Novel H2S donors have proved useful in understanding the biological functions of H2S, with morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate (GYY4137) being one of the most common pharmacological tools used. One advantage of GYY4137 over sulfide salts is its ability to release H2S in a slow and sustained manner akin to endogenous H2S production, rather than the delivery of H2S as a single concentrated burst. Critical Issues: Here, we summarize recent progress made in the characterization of the biological activities and pharmacological effects of GYY4137 in a range of in vitro and in vivo systems. Recent developments in the structural modification of GYY4137 to generate new compounds and their biological effects are also discussed. Future Directions: Slow-releasing H2S donor, GYY4137, and other phosphorothioate-based H2S donors are potent tools to study the biological functions of H2S. Despite recent progress, more work needs to be performed on these new compounds to unravel the mechanisms behind H2S release and pace of its discharge, as well as to define the effects of by-products of donors after H2S liberation. This will not only lead to better in-depth understanding of the biological effects of H2S but will also shed light on the future development of a new class of therapeutic agents with potential to treat a wide range of human diseases.

Phosphorothioate Antisense Oligonucleotides Bind P-Body Proteins and Mediate P-Body Assembly.

Antisense oligonucleotides (ASOs) regulate gene expression by binding to complementary target RNA, and ASOs can be designed to take advantage of a growing array of post RNA binding molecular mechanisms. Intracellular trafficking of ASOs influences their efficacy. We have identified a number of membrane-less structures in the nucleus, nucleolus, and cytoplasm where phosphorothioate-modified ASOs (PS-ASOs) accumulate and have shown that PS-ASOs can induce the formation of new nuclear structures such as PS-bodies and paraspeckle-like structures. In this study, we report that PS-ASOs can localize to cytoplasmic processing bodies (P-bodies) and increase the number of P-bodies in cells. The antisense activity of PS-ASOs was not affected by the absence of essential P-body assembly proteins DDX6 and LSm14A. Moreover, the effects of PS-ASOs on P-body assembly were independent of their antisense activities. The phosphorothioate modification stabilizes the association between ASOs and cellular proteins and is essential for the P-body localization of ASOs. Since PS-ASOs bind to major P-body components, PS-ASOs may serve as scaffolds for P-body formation. Taken together, these results indicate that interactions of PS-ASO with proteins, rather than antisense activities, are essential for the dynamic interplay between PS-ASOs and P-bodies.

mRNA levels can be reduced by antisense oligonucleotides via no-go decay pathway.

Antisense technology can reduce gene expression via the RNase H1 or RISC pathways and can increase gene expression through modulation of splicing or translation. Here, we demonstrate that antisense oligonucleotides (ASOs) can reduce mRNA levels by acting through the no-go decay pathway. Phosphorothioate ASOs fully modified with 2'-O-methoxyethyl decreased mRNA levels when targeted to coding regions of mRNAs in a translation-dependent, RNase H1-independent manner. The ASOs that activated this decay pathway hybridized near the 3' end of the coding regions. Although some ASOs induced nonsense-mediated decay, others reduced mRNA levels through the no-go decay pathway, since depletion of PELO/HBS1L, proteins required for no-go decay pathway activity, decreased the activities of these ASOs. ASO length and chemical modification influenced the efficacy of these reagents. This non-gapmer ASO-induced mRNA reduction was observed for different transcripts and in different cell lines. Thus, our study identifies a new mechanism by which mRNAs can be degraded using ASOs, adding a new antisense approach to modulation of gene expression. It also helps explain why some fully modified ASOs cause RNA target to be reduced despite being unable to serve as substrates for RNase H1.

Lipid Conjugates Enhance Endosomal Release of Antisense Oligonucleotides Into Cells.

Antisense oligonucleotides modified with phosphorothioate linkages (PS-ASOs) can enter cells via endocytic pathways and must escape from membraned organelles to reach target RNAs. We recently found that membrane destabilization induced by different lipid species contributes to PS-ASO release from late endosomes (LEs). In this study, we characterized intracellular uptake, trafficking, and activities of PS-ASOs conjugated with different lipid species. We found that palmitic acid-, tocopherol-, and cholesterol-conjugated PS-ASOs have increased protein binding and enhanced intracellular uptake compared to unconjugated PS-ASOs. Similar to the parental PS-ASO, the lipid-conjugated PS-ASOs traffic from early to LEs without incorporation into lipid droplets. Unlike parental PS-ASOs, the lipid-conjugated PS-ASOs tend to remain associated with plasma or endosomal membranes, and this appears to influence their release from endosomes. The lipid-conjugated PS-ASOs were released more rapidly than parental PS-ASO. These results suggest that lipid conjugation enhances the interactions of PS-ASOs with proteins or membranes, in turn facilitating intracellular trafficking and endosomal release.

Immune cell trafficking and retention in inflammatory bowel disease: mechanistic insights and therapeutic advances.

Intestinal immune cell trafficking has been identified as a central event in the pathogenesis of inflammatory bowel diseases (IBD). Intensive research on different aspects of the immune mechanisms controlling and controlled by T cell trafficking and retention has led to the approval of the anti-α4ß7 antibody vedolizumab, the ongoing development of a number of further anti-trafficking agents (ATAs) such as the anti-ß7 antibody etrolizumab or the anti-MAdCAM-1 antibody ontamalimab and the identification of potential future targets like G-protein coupled receptor 15. However, several aspects of the biology of immune cell trafficking and regarding the mechanism of action of ATAs are still unclear, for example, which impact these compounds have on the trafficking of non-lymphocyte populations like monocytes and how precisely these therapies differ with regard to their effect on immune cell subpopulations. This review will summarise recent advances of basic science in the field of intestinal immune cell trafficking and discuss these findings with regard to different pharmacological approaches from a translational perspective.

Targeted Delivery of miRNA Antagonists to Myeloid Cells In Vitro and In Vivo.

Elevated levels of microRNAs in cancer cells are often associated with oncogenic effects and thus provide potential therapeutic targets. However, the lack of efficient delivery methods for synthetic miRNA inhibitors, antagomiR, or anti-miR oligonucleotides hindered clinical translation of such strategies. We recently developed an approach for targeted delivery of synthetic, 2'-O-methyl-modified antagomiR molecules to normal and malignant myeloid cells and B cells by tethering to the single-stranded, phosphorothioate oligodeoxynucleotides (PSO). The PSO-antagomiR are rapidly internalized through scavenger receptor-mediated endocytosis by human monocytes, dendritic cells, B cells, as well as myeloid leukemia and B-cell lymphoma cells, but not by T cells. Following internalization, the unformulated PSO-antagomiR potently reduces levels of target miRNA and modulates expression of downstream protein targets, both in vitro and in vivo. The simple design of PSO-antagomiR conjugates enable adaptation of this strategy for targeting oncogenic miRNAs in nonmalignant and malignant myeloid cells and B cells.

Evaluating the Impact of Variable Phosphorothioate Content in Tricyclo-DNA Antisense Oligonucleotides in a Duchenne Muscular Dystrophy Mouse Model.

Antisense oligonucleotides (ASOs) hold promise for therapeutic splice switching correction for genetic diseases, in particular for Duchenne muscular dystrophy (DMD), for which ASO-exon skipping represents one of the most advanced therapeutic strategies. We have previously reported the therapeutic potential of tricyclo-DNA (tcDNA) in mouse models of DMD, highlighting the unique pharmaceutical properties and unprecedented uptake in many tissues after systemic delivery, including the heart and central nervous system. TcDNA-ASOs demonstrate an encouraging safety profile and no particular class-related toxicity, however, when administered in high doses for several months, mild renal toxicity is observed secondary to predictable phosphorothioate (PS)-ASO accumulation in kidneys. In this study, we investigate the influence of the relative content of PS linkages in tcDNA-ASOs on exon skipping efficacy. Mdx mice were injected intravenously once weekly for 4 weeks with tcDNA carrying various amounts of PS linkages (0%, 25%, 33%, 50%, 67%, 83%, and 100%). The results indicate that levels of exon-23 skipping and dystrophin rescue increase with the number of PS linkages in most skeletal muscles except in the heart. As expected, plasma coagulation times are shortened with decreasing PS content, and tcDNA-protein binding in serum directly correlates with the number of PS linkages on the tcDNA backbone. Altogether, these data contribute in establishing the appropriate sulfur content within the tcDNA backbone for maximal efficacy and minimal toxicity of the oligonucleotide.

Oligonucleotide⁻Palladacycle Conjugates as Splice-Correcting Agents.

2'-O-Methylribo phosphorothioate oligonucleotides incorporating cyclopalladated benzylamine conjugate groups at their 5'-termini have been prepared and their ability to hybridize with a designated target sequence was assessed by conventional UV melting experiments. The oligonucleotides were further examined in splice-switching experiments in human cervical cancer (HeLa Luc/705), human liver (HuH7_705), and human osteosarcoma (U-2 OS_705) reporter cell lines. Melting temperatures of duplexes formed by the modified oligonucleotides were approximately 5 °C lower than melting temperatures of the respective unmodified duplexes. The cyclopalladated oligonucleotides functioned as splice-correcting agents in the HeLa Luc/705 cell line somewhat more efficiently than their unmodified counterparts. Furthermore, the introduction of this chemical modification did not induce toxicity in cells. These results demonstrate the feasibility of using covalently metalated oligonucleotides as therapeutic agents.

Differential uptake, kinetics and mechanisms of intracellular trafficking of next-generation antisense oligonucleotides across human cancer cell lines.

Antisense oligonucleotides (ASOs) modulate cellular target gene expression through direct binding to complementary RNA. Advances in ASO chemistry have led to the development of phosphorothioate (PS) ASOs with constrained-ethyl modifications (cEt). These next-generation cEt-ASOs can enter cells without transfection reagents. Factors involved in intracellular uptake and trafficking of cEt-ASOs leading to successful target knockdown are highly complex and not yet fully understood. AZD4785 is a potent and selective therapeutic KRAS cEt-ASO currently under clinical development for the treatment of cancer. Therefore, we used this to investigate mechanisms of cEt-ASO trafficking across a panel of cancer cells. We found that the extent of ASO-mediated KRAS mRNA knockdown varied significantly between cells and that this did not correlate with bulk levels of intracellular accumulation. We showed that in cells with good productive uptake, distribution of ASO was perinuclear and in those with poor productive uptake distribution was peripheral. Furthermore, ASO rapidly trafficked to the late endosome/lysosome in poor productive uptake cells compared to those with more robust knockdown. An siRNA screen identified several factors mechanistically involved in productive ASO uptake, including the endosomal GTPase Rab5C. This work provides novel insights into the trafficking of cEt-ASOs and mechanisms that may determine their cellular fate.