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KIF1A-associated neurological disorder (KAND) is a neurodegenerative and often lethal ultrarare disease with a wide phenotypic spectrum associated with largely heterozygous de novo missense variants in KIF1A. Antisense oligonucleotide treatments represent a promising approach for personalized treatments in ultrarare diseases. Here we report the case of one patient with a severe form of KAND characterized by refractory spells of behavioral arrest and carrying a p.Pro305Leu variant in KIF1A, who was treated with intrathecal injections of an allele-specific antisense oligonucleotide specifically designed to degrade the mRNA from the pathogenic allele. The first intrathecal administration was complicated by an epidural cerebrospinal fluid collection, which resolved spontaneously. Otherwise, the antisense oligonucleotide was safe and well tolerated over the 9-month treatment. Most outcome measures, including severity of the spells of behavioral arrest, number of falls and quality of life, improved. There was little change in the 6-min Walk Test distance, but qualitative changes in gait resulting in meaningful reductions in falls and increasing independence were observed. Cognitive performance was stable and did not degenerate over time. Our findings provide preliminary insights on the safety and efficacy of an allele-specific antisense oligonucleotide as a possible treatment for KAND., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The triantennary N -acetylgalactosamine (GalNAc 3 ) cluster has demonstrated the utility of receptor-mediated uptake of ligand-conjugated antisense drugs targeting RNA expressed by hepatocytes. GalNAc 3 -conjugated 2'- O -methoxyethyl (2'MOE) modified antisense oligonucleotides (ASOs) have demonstrated a higher potency than the unconjugated form to support lower doses for an equivalent pharmacological effect. We utilized the Ionis integrated safety database to compare four GalNAc 3 -conjugated and four same-sequence unconjugated 2'MOE ASOs. This assessment evaluated data from eight randomized placebo-controlled dose-ranging phase 1 studies involving 195 healthy volunteers (79 GalNAc 3 ASO, 24 placebo; 71 ASO, 21 placebo). No safety signals were identified by the incidence of abnormal threshold values in clinical laboratory tests for either ASO group. However, there was a significant increase in mean alanine transaminase levels compared with placebo in the upper dose range of the unconjugated 2'MOE ASO group. The mean percentage of subcutaneous injections leading to local cutaneous reaction was 30-fold lower in the GalNAc 3 -conjugated ASO group compared with the unconjugated ASO group (0.9% vs. 28.6%), with no incidence of flu-like reactions (0.0% vs. 0.7%). Three subjects (4.2%) in the unconjugated ASO group discontinued dosing. An improvement in the overall safety and tolerability profile of GalNAc 3 -conjugated 2'MOE ASOs is evident in this comparison of short-term clinical data in healthy volunteers.

Single-stranded phosphorothioate oligonucleotides (PS-oligos) can activate TLR9, leading to an innate immune response. This can occur with PS-oligos containing unmethylated CpG sites, the canonical motif, or PS-oligos that do not contain those motifs (non-CpG). Structural evidence shows that TLR9 contains two PS-oligo binding sites, and recent data suggest that synergistic cooperative activation of TLR9 can be achieved by adding two separate PS-oligos to cells, each engaging with a separate site on TLR9 to enhance TLR9 activation as a pair. Here, we demonstrate and characterize this cooperativity phenomenon using PS-oligos in human cell lines, and we introduce several novel PS-oligo pairs (CpG and non-CpG pairs) that show cooperative activation. Indeed, we find that cooperative PS-oligos likely bind at different sites on TLR9. Interestingly, we find that PS-oligos that generate little TLR9 activation on their own can prime TLR9 to be activated by other PS-oligos. Finally, we determine that previous models of TLR9 activation cannot be used to fully explain data from systems using human TLR9 and PS-oligos. Overall, we reveal new details of TLR9 activation, but we also find that more work needs to be done to determine where certain PS-oligos are binding to TLR9., Competing Interests: All authors are employees and shareholders of Ionis Pharmaceuticals, Inc., (© 2023 The Authors.)

While rare, some gapmer phosphorothioate (PS) antisense oligonucleotides (ASOs) can induce a noncanonical TLR9-dependent innate immune response. In this study, we performed systematic analyses of the roles of PS ASO backbone chemistry, 2' modifications, and sequence in PS ASO induced TLR9 signaling. We found that each of these factors can contribute to altering PS ASO induced TLR9 signaling, and in some cases the effects are quite dramatic. We also found that the positioning (5' vs. 3') of a particular backbone or 2' modification within a PS ASO can affect its TLR9 signaling. Interestingly, medicinal chemical strategies that decrease TLR9 signaling for one sequence can have opposing effects on another sequence. Our results demonstrate that TLR9 signaling is highly PS ASO sequence dependent, the mechanism of which remains unknown. Despite this, we determined that placement of two mesyl phosphoramidate linkages within the PS ASO gap is the most promising strategy to mitigate PS ASO dependent TLR9 activation to enhance the therapeutic index and, therefore, further streamline PS ASO drug development.

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.

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.

Non-CpG PS-ASOs can activate the innate immune system, leading to undesired outcomes. This response can vary-in part-as a function of 2'modifications and sequence. Here we investigated the molecular steps involved in the varied effects of PS-ASOs on the innate immune system. We found that pro-inflammatory PS-ASOs require TLR9 signaling based on the experimental systems used. However, the innate immunity of PS-ASOs does not correlate with their binding affinity with TLR9. Furthermore, the innate immune responses of pro-inflammatory PS-ASOs were reduced by coincubation with non-inflammatory PS-ASOs, suggesting that both pro-inflammatory and non-inflammatory PS-ASOs can interact with TLR9. We show that the kinetics of the PS-ASO innate immune responses can vary, which we speculate may be due to the existence of alternative PS-ASO binding sites on TLR9, leading to full, partial, or no activation of the pathway. In addition, we found that several extracellular proteins, including HMGB1, S100A8 and HRG, enhance the innate immune responses of PS-ASOs. Reduction of the binding affinity by reducing the PS content of PS-ASOs decreased innate immune responses, suggesting that PS-ASO-protein complexes may be sensed by TLR9. These findings thus provide critical information concerning how PS-ASOs can interact with and activate TLR9., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

For more than three decades, Ionis Pharmaceutics has pursued the challenging mission of creating a new platform for drug discovery. To overcome the numerous challenges faced required the integration of innovation across many scientific areas, despite many disappointments and failures. The approaches implemented to create and maintain a scientific environment to achieve the mission demanded the rigorous practice of science over three decades. The approaches taken are discussed in this perspective., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Phosphorothioate modified antisense oligonucleotides (PS-ASOs) can reduce gene expression through hybridization to target RNAs and subsequent cleavage by RNase H1. Target reduction through this mechanism is influenced by numerous features of the RNA, which modulate PS-ASO binding affinities to the RNA target, and how the PS-ASO-RNA hybrid is recognized by RNase H1 for RNA cleavage. Endogenous RNAs are frequently chemically modified, which can regulate intra- and intermolecular interactions of the RNA. The effects of PS-ASO modifications on antisense activity have been well studied; however, much less is known regarding the effects of RNA modifications on PS-ASO hybridization and RNase H1 cleavage activity. Here, we determine the effects of three different RNA modifications on PS-ASO binding and antisense activity in recombinant and cell-based systems. Some RNA modifications can reduce PS-ASO hybridization, the cleavage activity of RNase H1, or both, while other modifications had minimal effects on PS-ASO function. In addition to these direct effects, RNA modifications can also change the RNA structure, which may affect PS-ASO accessibility in a cellular context. Our results elucidate the effects of three prevalent RNA modifications on PS-ASO-mediated RNase H1 cleavage activity, and such findings will help improve PS-ASO target site selection., Competing Interests: All authors are current or previous employees of Ionis Pharmaceuticals., (© 2022 Ionis Pharmaceuticals.)

Thanks to the advent of genomic sequencing and numerous personalized medicine initiatives in various medical centers, it is now known that there are many patients who have heretofore never been diagnosed who have mutations that are unique to them and them only and others that may be members of an extremely rare mutation (<30 patients in the world). Although each mutation may be unique it is now estimated that there are millions of these unique or vanishingly small patient groups. Patients with diseases caused by ultra-rare mutations present challenges to the health care system that are as unique as their mutation. n-Lorem was founded to take advantage of the antisense technology that we created at Ionis to discover and develop personalized antisense oligonucleotides (ASOs) one patient at a time and provide those experimental ASO treatments for free for life. In our first 18 months of operation, we have demonstrated this goal is achievable and worked with the FDA to develop guidance for ASO treatment of patients with ultra-rare diseases. In this article, I define the problem, discuss the ASO solution, and our progress at n-Lorem to date. I then focus on important steps that we have taken to assure that these complex risk/benefit judgments are made with high quality and that each patient receives the highest quality ASO possible. I then describe the processes we have created to assure that the opportunity to learn from each patient and our aggregate experience are maximized and shared with all stakeholders.

Patients with ultrarare diseases present unique challenges to the health care systems of developed economies that demand novel approaches, beginning with achieving a diagnosis and concluding with long-term treatment. The challenges derive from numbers. On the one hand, the rarity of the disease phenotypes means that the vast majority of ultrarare patients are never diagnosed, and for the fortunate few who are diagnosed, the journey to a genetic diagnosis is long and perilous. On the other hand, as more human genomes are sequenced, the number of these patients identified is growing logarithmically. Once patients are diagnosed, personalized medicines must be rapidly developed and delivered. Here I define the problems and propose a nonprofit model to meet the needs of some of these patients., (Copyright © 2021. Published by Elsevier Ltd.)

The healthcare systems in the developed economies were established primarily to address the more prevalent diseases and have been customized to support the provision of therapeutics to rare patients. However, with the ever-broader implementation of genomic sequencing, it is clear that there are significantly more disease-causing mutations in the human genome than realized and that many mutations are much rarer than current definitions of rare disease populations. Given this, I propose parsing patient populations and defining patient populations more precisely. Nano-rare patients are defined as patients having disease-causing mutations that are unique to a single patient or having a known worldwide prevalence of less than 30. These patient populations present unique challenges to healthcare systems that demand the development of novel models for delivery of therapeutics and novel, more efficient drug discovery technologies, such as antisense technology. The challenges presented by nano-rare patients, a novel non-profit model as a means of providing experimental treatments rather than the traditional commercial model, and progress in establishing a non-profit solution are discussed., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Phosphorothioate (PS) modified antisense oligonucleotide (ASO) drugs that act on cellular RNAs must enter cells and be released from endocytic organelles to elicit antisense activity. It has been shown that PS-ASOs are mainly released by late endosomes. However, it is unclear how endosome movement in cells contributes to PS-ASO activity. Here, we show that PS-ASOs in early endosomes display Brownian type motion and migrate only short distances, whereas PS-ASOs in late endosomes (LEs) move linearly along microtubules with substantial distances. In cells with normal microtubules and LE movement, PS-ASO-loaded LEs tend to congregate perinuclearly. Disruption of perinuclear positioning of LEs by reduction of dynein 1 decreased PS-ASO activity, without affecting PS-ASO cellular uptake. Similarly, disruption of perinuclear positioning of PS-ASO-LE foci by reduction of ER tethering proteins RNF26, SQSTM1 and UBE2J1, or by overexpression of P50 all decreased PS-ASO activity. However, enhancing perinuclear positioning through reduction of USP15 or over-expression of RNF26 modestly increased PS-ASO activity, indicating that LE perinuclear positioning is required for ensuring efficient PS-ASO release. Together, these observations suggest that LE movement along microtubules and perinuclear positioning affect PS-ASO productive release., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Phosphorothioate (PS) modified antisense oligonucleotide (ASO) drugs can trigger RNase H1 cleavage of cellular target RNAs to modulate gene expression. Internalized PS-ASOs must be released from membraned endosomal organelles, a rate limiting step that is not well understood. Recently we found that M6PR transport between Golgi and late endosomes facilitates productive release of PS-ASOs, raising the possibility that Golgi-mediated transport may play important roles in PS-ASO activity. Here we further evaluated the involvement of Golgi in PS-ASO activity by examining additional Golgi proteins. Reduction of certain Golgi proteins, including Golgi-58K, GCC1 and TGN46, decreased PS-ASO activity, without substantial effects on Golgi integrity. Upon PS-ASO cellular uptake, Golgi-58K was recruited to late endosomes where it colocalized with PS-ASOs. Reduction of Golgi-58K caused slower PS-ASO release from late endosomes, decreased GCC2 late endosome relocalization, and led to slower retrograde transport of M6PR from late endosomes to trans-Golgi. Late endosome relocalization of Golgi-58K requires Hsc70, and is most likely mediated by PS-ASO-protein interactions. Together, these results suggest a novel function of Golgi-58K in mediating Golgi-endosome transport and indicate that the Golgi apparatus plays an important role in endosomal release of PS-ASO, ensuring antisense activity., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Phosphorothioate-modified antisense oligonucleotide (PS-ASO) drugs are commonly used to modulate gene expression through RNase H1-mediated cleavage of target RNAs. Upon internalization through endocytic pathways into cells, PS-ASOs must be released from membraned endosomal organelles to act on target RNAs, a limiting step of PS-ASO activity. Here we report that Hsc70 protein mediates productive release of PS-ASOs from endosomes. Hsc70 protein was enriched in endosome fractions shortly after PS-ASO incubation with cells. Reduction of Hsc70 significantly decreased the activities of PS-ASOs in reducing target RNAs. PS-ASO uptake and transport from early endosomes to late endosomes (LEs) were not affected upon Hsc70 reduction; however, endosomal release of PS-ASOs was impaired. Reduction of Hsc70 led to more scattered mannose-6-phosphate receptor (M6PR) localization at LEs in the cytoplasm, in contrast to the perinuclear localization at trans -Golgi network (TGN) in control cells, suggesting that retrograde transport of M6PR from LEs to TGN was affected. Consistently, reduction of Hsc70 increased colocalization of M6PR and PS-ASOs at LEs, and also delayed M6PR antibody transport from LE to TGN. Together, these results suggest that Hsc70 protein is involved in M6PR vesicle escape from LEs and may thus enhance PS-ASO release from LEs.

When coined, the term "antisense" included oligonucleotides of any structure, with any chemical modification and designed to work through any post-RNA hybridization mechanism. However, in practice the term "antisense" has been used to describe single stranded oligonucleotides (ss ASOs) designed to hybridize to RNAswhile the term "siRNA" has come to mean double stranded oligonucleotides designed to activate Ago2. However, the two approaches share many common features. The medicinal chemistry developed for ASOs greatly facilitated the development of siRNA technology and remains the chemical basis for both approaches. Many of challenges faced and solutions achieved share many common features. In fact, because ss ASOs can be designed to activate Ago2, the two approaches intersect at this remarkably important protein. There are also meaningful differences. The pharmacokinetic properties are quite different and thus potential routes of delivery differ. ASOs may be designedto use a variety of post-RNA binding mechanismswhile siRNAs depend solely on the robust activity of Ago2. However, siRNAs and ASOs are both used for therapeutic purposes and both must be and can be understood in a pharmacological context. Thus, the goals of this review are to put ASOs in pharmacological context and compare their behavior as pharmacological agents to the those of siRNAs., (Copyright © 2020. Published by Elsevier Inc.)

Antisense technology is now beginning to deliver on its promise to treat diseases by targeting RNA. Nine single-stranded antisense oligonucleotide (ASO) drugs representing four chemical classes, two mechanisms of action and four routes of administration have been approved for commercial use, including the first RNA-targeted drug to be a major commercial success, nusinersen. Although all the approved drugs are for use in patients with rare diseases, many of the ASOs in late- and middle-stage clinical development are intended to treat patients with very common diseases. ASOs in development are showing substantial improvements in potency and performance based on advances in medicinal chemistry, understanding of molecular mechanisms and targeted delivery. Moreover, the ASOs in development include additional mechanisms of action and routes of administration such as aerosol and oral formulations. Here, we describe the key technological advances that have enabled this progress and discuss recent clinical trials that illustrate the impact of these advances on the performance of ASOs in a wide range of therapeutic applications. We also consider strategic issues such as target selection and provide perspectives on the future of the field.

Site-specific incorporation of 2'-modifications and neutral linkages in the deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined the effect of introducing 2',5'-linked RNA in the deoxynucleotide gap region on toxicity and potency of PS ASOs. Our results demonstrate that incorporation of 2',5'-linked RNA in the gap region dramatically improved hepatotoxicity profile of PS-ASOs without compromising potency and provide a novel alternate chemical approach for improving therapeutic index of ASO drugs., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

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.

We recently found that toxic PS-ASOs can cause P54nrb and PSF nucleolar mislocalization in an RNase H1-dependent manner. To better understand the underlying mechanisms of these observations, here we utilize different biochemical approaches to demonstrate that PS-ASO binding can alter the conformations of the bound proteins, as illustrated using recombinant RNase H1, P54nrb, PSF proteins and various isolated domains. While, in general, binding of PS-ASOs or ASO/RNA duplexes stabilizes the conformations of these proteins, PS-ASO binding may also cause the unfolding of RNase H1, including both the hybrid binding domain and the catalytic domain. The extent of conformational change correlates with the binding affinity of PS-ASOs to the proteins. Consequently, PS-ASO binding to RNase H1 induces the interaction of RNase H1 with P54nrb or PSF in a 2'-modification and sequence dependent manner, and toxic PS-ASOs tend to induce more interactions than non-toxic PS-ASOs. PS-ASO binding also enhances the interaction between P54nrb and PSF. However, the interaction between RNase H1 and P32 protein can be disrupted upon binding of PS-ASOs. Together, these results suggest that stronger binding of PS-ASOs can cause greater conformational changes of the bound proteins, subsequently affecting protein-protein interactions. These observations thus provide deeper understanding of the molecular basis of PS-ASO-induced protein mislocalization or degradation observed in cells and advance our understanding of why some PS-ASOs are cytotoxic., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We recently showed that site-specific incorporation of 2'-modifications or neutral linkages in the oligo-deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined if introducing substitution at the 5'-position of deoxynucleotide monomers in the gap can also enhance therapeutic index. Introducing R- or S-configured 5'-Me DNA at positions 3 and 4 in the oligodeoxynucleotide gap enhanced the therapeutic profile of the modified ASOs suggesting a different positional preference as compared to the 2'-OMe gap modification strategy. The generality of these observations was demonstrated by evaluating R-5'-Me and R-5'-Ethyl DNA modifications in multiple ASOs targeting HDAC2, FXI and Dynamin2 mRNA in the liver. The current work adds to a growing body of evidence that small structural changes can modulate the therapeutic properties of PS ASOs and ushers a new era of chemical optimization with a focus on enhancing the therapeutic profile as opposed to nuclease stability, RNA-affinity and pharmacokinetic properties. The 5'-methyl DNA modified ASOs exhibited excellent safety and antisense activity in mice highlighting the therapeutic potential of this class of nucleic acid analogs for next generation ASO designs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Antisense technology is beginning to deliver on the broad promise of the technology. Ten RNA-targeted drugs including eight single-strand antisense drugs (ASOs) and two double-strand ASOs (siRNAs) have now been approved for commercial use, and the ASOs in phase 2/3 trials are innovative, delivered by multiple routes of administration and focused on both rare and common diseases. In fact, two ASOs are used in cardiovascular outcome studies and several others in very large trials. Interest in the technology continues to grow, and the field has been subject to a significant number of reviews. In this review, we focus on the molecular events that result in the effects observed and use recent clinical results involving several different ASOs to exemplify specific molecular mechanisms and specific issues. We conclude with the prospective on the technology., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, we demonstrate that 5S ribosomal RNA (rRNA), a highly structured and protein-bound RNA, is quite difficult to reduce with antisense oligonucleotides (ASOs). However, we found a single accessible site that was targetable with a high-affinity complementary ASO. The ASO appeared to bind to the site, recruit RNaseH1, and cause degradation of the 5S RNA. Intriguingly, we also observed that the same ASO induced an accumulation of pre-5S RNA, which may contribute to reduced levels of mature 5S rRNA. As expected, ASO mediated reduction of 5S RNA, and modest inhibition of processing of pre-5S RNA resulted in nucleolar toxicity. However, the toxicity induced was minimal compared with actinomycin D, consistent with its modest effects on pre-5S rRNA. Mechanistically, we show that the accumulation of pre-5S rRNA required ASO hybridization to the cognate rRNA sequence but was independent of RNaseH1 activity. We found that Ro60 and La, proteins known to bind misprocessed RNAs, likely sequester the ASO-pre-5S rRNA species and block RNaseH1 activity, thus identifying another example of competitive mechanisms mediated by proteins that compete with RNaseH1 for binding to ASO-RNA heteroduplexes.

Antisense oligonucleotide (ASO) drugs that trigger RNase H1 cleavage of target RNAs have been developed to treat various diseases. Basic pharmacological principles suggest that the development of tolerance is a common response to pharmacological interventions. In this manuscript, for the first time we report a molecular mechanism of tolerance that occurs with some ASOs. Two observations stimulated our interest: some RNA targets are difficult to reduce with RNase H1 activating ASOs and some ASOs display a shorter duration of activity than the prolonged target reduction typically observed. We found that certain ASOs targeting the coding region of some mRNAs that initially reduce target mRNAs can surprisingly increase the levels of the corresponding pre-mRNAs. The increase in pre-mRNA is delayed and due to enhanced transcription and likely also slower processing. This process requires that the ASOs bind in the coding region and reduce the target mRNA by RNase H1 while the mRNA resides in the ribosomes. The pre-mRNA increase is dependent on UPF3A and independent of the NMD pathway or the XRN1-CNOT pathway. The response is consistent in multiple cell lines and independent of the methods used to introduce ASOs into cells., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recent progress in understanding phosphorothioate antisense oligonucleotide (PS-ASO) interactions with proteins has revealed that proteins play deterministic roles in the absorption, distribution, cellular uptake, subcellular distribution, molecular mechanisms of action, and toxicity of PS-ASOs. Similarly, such interactions can alter the fates of many intracellular proteins. These and other advances have opened new avenues for the medicinal chemistry of PS-ASOs and research on all elements of the molecular pharmacology of these molecules. These advances have recently been reviewed. In this Perspective article, we summarize some of those learnings, the general principles that have emerged, and a few of the exciting new questions that can now be addressed.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The activity of PS-ASOs is strongly influenced by association with both inter- and intracellular proteins. The sequence, chemical nature, and structure of the ASO can have profound influences on the interaction of PS-ASOs with specific proteins. A more thorough understanding of how these pharmacological agents interact with various proteins and how chemical modifications, sequence, and structure influence interactions with proteins is needed to inform future ASO design efforts. To better understand the chemistry of PS-ASO interactions, we have focused on human positive cofactor 4 (PC4). Although several studies have investigated the in vitro binding properties of PC4 with endogenous nucleic acids, little is known about the chemistry of interaction of PS-ASOs with this protein. Here we examine in detail the impact of ASO backbone chemistry, 2'-modifications, and buffer environment on the binding affinity of PC4. In addition, using site-directed mutagenesis, we identify those amino acids that are specifically required for ASO binding interactions, and by substitution of abasic nucleotides we identify the positions on the ASO that most strongly influence affinity for PC4. Finally, to confirm that the interactions observed in vitro are biologically relevant, we use a recently developed complementation reporter system to evaluate the kinetics and subcellular localization of the interaction of ASO and PC4 in live cells.

The phosphorothioate backbone modification (PS) is one of the most widely used chemical modifications for enhancing the drug-like properties of nucleic acid-based drugs, including antisense oligonucleotides (ASOs). PS-modified nucleic acid therapeutics show improved metabolic stability from nuclease-mediated degradation and exhibit enhanced interactions with plasma, cell-surface, and intracellular proteins, which facilitates their tissue distribution and cellular uptake in animals. However, little is known about the structural basis of the interactions of PS nucleic acids with proteins. Here, we report a crystal structure of the DNA-binding domain of a model ASO-binding protein PC4, in complex with a full PS 2'-OMe DNA gapmer ASO. To our knowledge this is the first structure of a complex between a protein and fully PS nucleic acid. Each PC4 dimer comprises two DNA-binding interfaces. In the structure one interface binds the 5'-terminal 2'-OMe PS flank of the ASO, while the other interface binds the regular PS DNA central part in the opposite polarity. As a result, the ASO forms a hairpin-like structure. ASO binding also induces the formation of a dimer of dimers of PC4, which is stabilized by base pairing between homologous regions of the ASOs bound by each dimer of PC4. The protein interacts with the PS nucleic acid through a network of electrostatic and hydrophobic interactions, which provides insights into the origins for the enhanced affinity of PS for proteins. The importance of these contacts was further confirmed in a NanoBRET binding assay using a Nano luciferase tagged PC4 acting as the BRET donor, to a fluorescently conjugated ASO acting as the BRET acceptor. Overall, our results provide insights into the molecular forces that govern the interactions of PS ASOs with cellular proteins and provide a potential model for how these interactions can template protein-protein interactions causative of cellular toxicity.

Therapeutic oligonucleotides are often modified using the phosphorothioate (PS) backbone modification which enhances stability from nuclease mediated degradation. However, substituting oxygen in the phosphodiester backbone with sulfur introduce chirality into the backbone such that a full PS 16-mer oligonucleotide is comprised of 215 distinct stereoisomers. As a result, the role of PS chirality on the performance of antisense oligonucleotides (ASOs) has been a subject of debate for over two decades. We carried out a systematic analysis to determine if controlling PS chirality in the DNA gap region can enhance the potency and safety of gapmer ASOs modified with high-affinity constrained Ethyl (cEt) nucleotides in the flanks. As part of this effort, we examined the effect of systematically controlling PS chirality on RNase H1 cleavage patterns, protein mislocalization phenotypes, activity and toxicity in cells and in mice. We found that while controlling PS chirality can dramatically modulate interactions with RNase H1 as evidenced by changes in RNA cleavage patterns, these were insufficient to improve the overall therapeutic profile. We also found that controlling PS chirality of only two PS linkages in the DNA gap was sufficient to modulate RNase H1 cleavage patterns and combining these designs with simple modifications such as 2'-OMe to the DNA gap resulted in dramatic improvements in therapeutic index. However, we were unable to demonstrate improved potency relative to the stereorandom parent ASO or improved safety over the 2'-OMe gap-modified stereorandom parent ASO. Overall, our work shows that while controlling PS chirality can modulate RNase H1 cleavage patterns, ASO sequence and design are the primary drivers which determine the pharmacological and toxicological properties of gapmer ASOs., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Release of phosphorothioate antisense oligonucleotides (PS-ASOs) from late endosomes (LEs) is a rate-limiting step and a poorly defined process for productive intracellular ASO drug delivery. Here, we examined the role of Golgi-endosome transport, specifically M6PR shuttling mediated by GCC2, in PS-ASO trafficking and activity. We found that reduction in cellular levels of GCC2 or M6PR impaired PS-ASO release from endosomes and decreased PS-ASO activity in human cells. GCC2 relocated to LEs upon PS-ASO treatment, and M6PR also co-localized with PS-ASOs in LEs or on LE membranes. These proteins act through the same pathway to influence PS-ASO activity, with GCC2 action preceding that of M6PR. Our data indicate that M6PR binds PS-ASOs and facilitates their vesicular escape. The co-localization of M6PR and of GCC2 with ASOs is influenced by the PS modifications, which have been shown to enhance the affinity of ASOs for proteins, suggesting that localization of these proteins to LEs is mediated by ASO-protein interactions. Reduction of M6PR levels also decreased PS-ASO activity in mouse cells and in livers of mice treated subcutaneously with PS-ASO, indicating a conserved mechanism. Together, these results demonstrate that the transport machinery between LE and Golgi facilitates PS-ASO release., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

The rapid RNase H1-dependent mislocalization of heterodimer proteins P54nrb and PSF to nucleoli is an early event in the pathway that explains the effects of most toxic phosphorothioate ASOs (PS-ASOs). Using a recently developed NanoLuciferace (NLuc)-based structural complementation reporter system which allows us to observe ASO/protein interactions in real time in live cells, we have determined that safe and toxic PS-ASOs associate with these proteins with kinetics and impact on subcellular localization that differ. Toxic PS-ASOs interact in a complex that includes RNase H1, P54nrb and PSF; but RNase H1/P54nrb complexes were observed in only the cells treated with toxic, but not safe PS-ASOs. In addition, experiments performed in vitro suggest that RNA is also a required component of the complex. The protein-protein interaction between P54nrb and RNase H1 requires the spacer region of RNAse H1, while the P54nrb core domains are required for association with RNase H1. In addition, we have determined that PS-ASOs bind P54nrb via RRM1 and RRM2, while they bind RNase H1 primarily via the hybrid binding domain, however catalytic domain interactions also contribute to overall affinity. These ASO-protein interactions are highly influenced by the chemistry of the PS-ASO binding environment, however little correlation between affinity for specific proteins and PS-ASO toxicity was observed., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5'-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The molecular mechanisms of toxicity of chemically modified phosphorothioate antisense oligonucleotides (PS-ASOs) are not fully understood. Here, we report that toxic gapmer PS-ASOs containing modifications such as constrained ethyl (cEt), locked nucleic acid (LNA) and 2'-O-methoxyethyl (2'-MOE) bind many cellular proteins with high avidity, altering their function, localization and stability. We show that RNase H1-dependent delocalization of paraspeckle proteins to nucleoli is an early event in PS-ASO toxicity, followed by nucleolar stress, p53 activation and apoptotic cell death. Introduction of a single 2'-O-methyl (2'-OMe) modification at gap position 2 reduced protein-binding, substantially decreasing hepatotoxicity and improving the therapeutic index with minimal impairment of antisense activity. We validated the ability of this modification to generally mitigate PS-ASO toxicity with more than 300 sequences. Our findings will guide the design of PS-ASOs with optimal therapeutic profiles.

Advances in medicinal chemistry have produced new chemical classes of antisense oligonucleotides (ASOs) with enhanced therapeutic properties. Conjugation of the triantennary N-acetylgalactosamine (GalNAc 3 ) moiety to the extensively characterized phosphorothioate (PS)-modified 2'-O-methoxyethyl (2'MOE) ASO exemplifies such an advance. This structure-activity optimized moiety effects receptor-mediated uptake of the ASO prodrug through the asialoglycoprotein receptor 1 to support selective targeting of RNAs expressed by hepatocytes. In this study we report the integrated assessment of data available from randomized placebo-controlled dose-ranging studies of this chemical class of ASOs administered systemically to healthy human volunteers. First, we compare the pharmacokinetic and pharmacodynamic profiles of a subset of the GalNAc 3 -conjugated PS-modified 2'MOE ASOs to the parent PS-modified 2'MOE ASOs for which plasma analytes are available. We then evaluate the safety profile of the full set of GalNAc 3 -conjugated PS-modified 2'MOE ASO conjugates by the incidence of signals in standardized laboratory tests and by the mean laboratory test results as a function of dose level over time. With hepatocyte targeted delivery, the ED 50 for the GalNAc 3 -conjugated PS-modified 2'MOE ASO subset ranges from 4 to 10 mg/week, up to 30-fold more potent than the parent PS-modified 2'MOE ASO. No GalNAc 3 -conjugated PS-modified 2'MOE ASO class effects were identified from the assessment of the integrated laboratory test data across all doses tested with either single or multidose regimens. The increase in potency supports an increase in the safety margin for this new chemical class of ASOs now under broad investigation in the clinic. Although the total exposure is limited in the initial phase 1 trials, ongoing and future investigations in patient populations will support evaluation of the effects of long-term exposure.

Chemically modified antisense oligonucleotides with phosphorothioate linkages (PS-ASOs) mediate site-specific cleavage of RNA by RNase H1 and are broadly used as research and therapeutic tools. PS-ASOs can enter cells via endocytic pathways and escape from membrane-enclosed endocytic organelles to reach target RNAs. We recently found that lysobisphosphatidic acid is required for release of PS-ASOs from late endosomes. Here, we evaluated the effects of other lipids on PS-ASO intracellular trafficking and activities. We show that free fatty acids, ceramide, and cholesterol increase PS-ASO activities. Free fatty acids induced formation of lipid droplets without changing the intracellular localization of PS-ASOs in early or late endosomes. Ceramide and cholesterol did not obviously induce the formation of lipid droplets, but cholesterol caused enlargement of endosome size and volume. Although none of those lipids appeared to influence PS-ASO internalization or intracellular trafficking processes, all led to an increase in leakiness of late endosomes. Thus, the membrane destabilization induced by these lipids likely contributes to PS-ASO release from late endosomes, which, in turn, increases PS-ASO activity., (Copyright © 2018. Published by Elsevier Inc.)

RNase H1-dependent, phosphorothioate-modified antisense oligonucleotides (PS-ASOs) can enter cells through endocytic pathways and need to be released from the membrane-enclosed organelles, a limiting step for antisense activity. Accumulating evidence has suggested that productive PS-ASO release mainly occurs from late endosomes (LEs). However, how PS-ASOs escape from LEs is not well understood. Here, we report that upon PS-ASO incubation, COPII vesicles, normally involved in ER-Golgi transport, can re-locate to PS-ASO-containing LEs. Reduction of COPII coat proteins significantly decreased PS-ASO activity, without affecting the levels of PS-ASO uptake and early-to-late endosome transport, but caused slower PS-ASO release from LEs. COPII co-localization with PS-ASOs at LEs does not require de novo assembly of COPII at ER. Interestingly, reduction of STX5 and P115, proteins involved in tethering and fusion of COPII vesicles with Golgi membranes, impaired COPII re-localization to LEs and decreased PS-ASO activity. STX5 can re-locate to LEs upon PS-ASO incubation, can bind PS-ASOs, and the binding appears to be required for this pathway. Our study reveals a novel release pathway in which PS-ASO incubation causes LE re-localization of STX5, which mediates the recruitment of COPII vesicles to LEs to facilitate endosomal PS-ASO release, and identifies another key PS-ASO binding protein.