Phosphorothioate Antisense Oligonucleotides Research Articles

Abstract 3252: Anti-tumor effect of the combination of a lipid nanoparticle suspension of an AKT-1 antisense oligonucleotide (WGI-0301) with tyrosine kinase inhibitors in an orthotopic murine tumor model

Abstract Background: Archexin is a fully phosphorothioated 20-mer antisense oligonucleotide, complementary to AKT-1 mRNA, which leads to the inhibition of translation and the downstream pathway activities of AKT-1 mRNA. WGI-0301 is a proprietary lipid nanoparticle formulation (QTsomeTM) of Archexin designed to enhance delivery. A phase I clinical study of WGI-0301 as monotherapy in patients with advanced solid tumors is currently ongoing in the U.S. Given the limited efficacy of TKIs compared to immunotherapy and growing incidence resistance, WGI-0301 combined with TKIs may augment the therapeutic response to TKIs by enhancing angiogenic blockade, inhibiting AKT-1, and overcome resistance. Methods: The anti-tumor effect of combining WGI-0301 with Lenvatinib/Sorafenib/Cabozantinib was assessed using a Hep3B-Luciferase orthotopic mouse tumor model. We divided sixty-four female Balb/c nude mice into groups (8 mice per group), including vehicle control, WGI-0301 (8 mg/kg), Lenvatinib (10 mg/kg), Sorafenib (20 mg/kg), Cabozantinib (20 mg/kg), WGI-0301+Lenvatinib (8+10 mg/kg), WGI-0301+Sorafenib (8+20 mg/kg), and WGI-0301+Cabozantinib (8+20 mg/kg). We administered treatments via tail vein injection (vehicle/WGI-0301, QW4 weeks) or oral administration (Lenvatinib/Sorafenib/Cabozantinib, QD28 days). We monitored body weight twice weekly and measured tumor burden using bioluminescence with an IVIS (Lumina III) imaging system twice weekly as well. Treatment was suspended between Day 29 and Day 70 for survival observation. Results: The tumor growth inhibition (TGI) rates of the combination of WGI-0301 with Lenvatinib/Sorafenib/Cabozantinib were 66.05%, 86.81%, and 75.89%, respectively. These values were higher than those of the Lenvatinib/Sorafenib/Cabozantinib single drug group with TGI rate of 50.84%, 55.68%, and 47.84%. The tumor bioluminescence signal intensities of the two drugs combination (56.74*108, 22.28*108, and 40.40*108 photon/s) were slightly lower than that of the Lenvatinib/Sorafenib/Cabozantinib single drug group (81.97*108, 73.95*108, and 86.96*108 photon/s). The median survival time was 35.0, 46.0, 55.5, and 51.5 days in the vehicle, WGI-0301+Lenvatinib, WGI-0301+Sorafenib and WGI-0301+Cabozantinib, respectively. The median survival time of Sorafenib, the two-drug combinations were 55.5, 55.5, 58.0, and 53.5 days, which was significantly prolonged compared to the vehicle in the mice bearing the Hep3B-luc orthotopic tumor model. No severe adverse effects were observed in the drug combination groups. Conclusion: The combination of WGI-0301 with TKIs demonstrated superior anti-tumor efficacy over TKI monotherapy in the absence of substantial increase in toxicity. This finding warrants further robust clinical investigation in patients with advanced HCC. Citation Format: Fengyang Xie, Tao Lin, Chao Wang, Jia Wei, Robert J. Lee, Ben Zhao, Yuxin Angela Men. Anti-tumor effect of the combination of a lipid nanoparticle suspension of an AKT-1 antisense oligonucleotide (WGI-0301) with tyrosine kinase inhibitors in an orthotopic murine tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3252.

Chloroquine and cytosolic galectins affect endosomal escape of antisense oligonucleotides after Stabilin-mediated endocytosis

Non-DNA-binding Stabilin-2/HARE receptors expressed on liver sinusoidal endothelial cells specifically bind to and internalize several classes of phosphorothioate antisense oligonucleotides (PS-ASOs). After Stabilin-mediated uptake, PS-ASOs are trafficked within endosomes (>97%-99%), ultimately resulting in destruction in the lysosome. The ASO entrapment in endosomes lowers therapeutic efficacy, thereby increasing the overall dose for patients. Here, we use confocal microscopy to characterize the intracellular route transverse by PS-ASOs after Stabilin receptor-mediated uptake in stable recombinant Stabilin-1 and -2 cell lines. We found that PS-ASOs as well as the Stabilin-2 receptor transverse the classic path: clathrin-coated vesicle-early endosome-late endosome-lysosome. Chloroquine exposure facilitated endosomal escape of PS-ASOs leading to target knockdown by more than 50% as compared to untreated cells, resulting in increased PS-ASO efficacy. We also characterize cytosolic galectins as novel contributor for PS-ASO escape. Galectins knockdown enhances ASO efficacy by more than 60% by modulating EEA1, Rab5C, and Rab7A mRNA expression, leading to a delay in the endosomal vesicle maturation process. Collectively, our results provide additional insight for increasing PS-ASO efficacy by enhancing endosomal escape, which can further be utilized for other nucleic acid-based modalities.

Controlled sulfur-based engineering confers mouldability to phosphorothioate antisense oligonucleotides.

Phosphorothioates (PS) have proven their effectiveness in the area of therapeutic oligonucleotides with applications spanning from cancer treatment to neurodegenerative disorders. Initially, PS substitution was introduced for the antisense oligonucleotides (PS ASOs) because it confers an increased nuclease resistance meanwhile ameliorates cellular uptake and in-vivo bioavailability. Thus, PS oligonucleotides have been elevated to a fundamental asset in the realm of gene silencing therapeutic methodologies. But, despite their wide use, little is known on the possibly different structural changes PS-substitutions may provoke in DNA·RNA hybrids. Additionally, scarce information and significant controversy exists on the role of phosphorothioate chirality in modulating PS properties. Here, through comprehensive computational investigations and experimental measurements, we shed light on the impact of PS chirality in DNA-based antisense oligonucleotides; how the different phosphorothioate diastereomers impact DNA topology, stability and flexibility to ultimately disclose pro-Sp S and pro-Rp S roles at the catalytic core of DNA Exonuclease and Human Ribonuclease H; two major obstacles in ASOs-based therapies. Altogether, our results provide full-atom and mechanistic insights on the structural aberrations PS-substitutions provoke and explain the origin of nuclease resistance PS-linkages confer to DNA·RNA hybrids; crucial information to improve current ASOs-based therapies.

Advances in the Specificity of Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy Based Structural Characterisation Methods for Synthetic Oligonucleotides

Identity testing is a critical part in the development of a therapeutic synthetic oligonucleotide. Tandem Mass Spectrometry (MS/MS) is commonly used for the analysis of oligonucleotides to obtain structural and sequence information, however there are challenges resulting from chemical modifications introduced to improve their pharmacokinetics and stability. For these structurally complex oligonucleotides, Nuclear Magnetic Resonance (NMR) Spectroscopy has found limited use for characterisation and identity testing, as only partial NMR resonance assignment for oligonucleotides is achieved without isotopic labelling methodologies. Regardless of the choice of method used for oligonucleotide analysis, the specificity is of critical importance. In this work, in-source dissociation mass spectrometry and proton (1H) and carbon (13C) NMR at high temperature were used to analyse danvatirsen, a 16 nucleotide phosphorothioate antisense oligonucleotide, and its closely related switch sequences. Both approaches have shown specificity to distinguish danvatirsen from these similar sequences.

Antisense oligonucleotide therapy for H3.3K27M diffuse midline glioma.

Diffuse midline gliomas (DMGs) are pediatric high-grade brain tumors in the thalamus, midbrain, or pons; the latter subgroup are termed diffuse intrinsic pontine gliomas (DIPG). The brain stem location of these tumors limits the clinical management of DIPG, resulting in poor outcomes for patients. A heterozygous, somatic point mutation in one of two genes coding for the noncanonical histone H3.3 is present in most DIPG tumors. This dominant mutation in the H3-3A gene results in replacement of lysine 27 with methionine (K27M) and causes a global reduction of trimethylation on K27 of all wild-type histone H3 proteins, which is thought to be a driving event in gliomagenesis. In this study, we designed and systematically screened 2'-O-methoxyethyl phosphorothioate antisense oligonucleotides (ASOs) that direct RNase H-mediated knockdown of H3-3A mRNA. We identified a lead ASO that effectively reduced H3-3A mRNA and H3.3K27M protein and restored global H3K27 trimethylation in patient-derived neurospheres. We then tested the lead ASO in two mouse models of DIPG: an immunocompetent mouse model using transduced mutant human H3-3A cDNA and an orthotopic xenograft with patient-derived cells. In both models, ASO treatment restored K27 trimethylation of histone H3 proteins and reduced tumor growth, promoted neural stem cell differentiation into astrocytes, neurons, and oligodendrocytes, and increased survival. These results demonstrate the involvement of the H3.3K27M oncohistone in tumor maintenance, confirm the reversibility of the aberrant epigenetic changes it promotes, and provide preclinical proof of concept for DMG antisense therapy.

Abstract 701: Evaluation of an AKT-1 antisense oligonucleotide in combination with Lenvatinib and Everolimus in Renca-luciferase syngeneic orthotopic murine tumor model

Abstract Background: The combination of 18mg Lenvatinib and 5mg Everolimus per day is used in the clinic for advanced renal cancer patients. However, due to the severe side effects of Lenvatinib, the dose is often reduced to 12mg. Archexin, a fully phosphorothioated 20-mer antisense oligonucleotide, inhibits AKT-1 mRNA translation and inhibits tumor growth. Combination of Archexin with reduced-dosed Lenvatinib and Everolimus may reduce adverse reactions while enhancing therapeutic efficacy. Methods: First, Lenvatinib and Everolimus monotherapies were evaluated for antitumor efficacy in an orthotopic Renca-luciferase syngeneic model. 45 mice were randomly divided into 9 groups (5 in each group), vehicle control, Lenvatinib (5, 10, 20, and 30 mg/kg) and Everolimus (0.2, 0.5, 1, 2, and 4 mg/kg) via daily oral administration for 20 days. Next, we investigated the anti-tumor effect of the two- and three-drug combination. 35 mice were randomly divided into 7 groups (5 in each group), including vehicle control, Lenvatinib/Everolimus combinations (5+0.2, 10+0.2 and 10+0.5 mg/kg) and Archexin/Lenvatinib/Everolimus combinations (60+5+0.2, 60+10+0.2 and 60+10+0.5 mg/kg) via tail vein injection (vehicle/Archexin, Q3D*6 times) or oral administration (Lenvatinib and Everolimus, QD*18 days). Body weight was monitored daily. Tumor burden was measured by bioluminescence through whole-body fluorescence images, performed twice a week. Results: The results showed that Everolimus inhibited tumor growth in a dose-dependent manner. Partial response was observed in mice treated with varying doses of Lenvatinib(TGIs: 57.94%,76.65% and 60.22%), and no significant dose-effect correlation was found. The dose of Lenvatinib and Everolimus that partially inhibited the orthotopic tumor growth had also been identified and selected for subsequent study investigating drug combinations. The drugs combination study showed that three drug combinations (Archexin/Lenvatinib/Everolimus) had superior efficacy compared to the Lenvatinib and Everolimus combination. The fluorescence signal intensities (Lg10) of the three-drug combination were significantly lower than that of the two-drug combination group with P value <0.05. No severe adverse effects were observed in the three drug combinations group. Conclusion: Three drugs combination of Archexin, Lenvatinib and Everolimus, demonstrated superior anti-tumor efficacy compared to the two-drug combination currently used in the clinic. This finding suggests the three-drug combination warrant investigation in the clinical trial in the patients with advanced renal cancer. Citation Format: Fengyang Xie, Jia Wei, Lan Deng, Chao Wang, Ben Zhao, Robert J. Lee, Yuxin Angela Men. Evaluation of an AKT-1 antisense oligonucleotide in combination with Lenvatinib and Everolimus in Renca-luciferase syngeneic orthotopic murine tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 701.

Accessible light-controlled knockdown of cell-free protein synthesis using phosphorothioate-caged antisense oligonucleotides

Controlling cell-free expression of a gene to protein with non-invasive stimuli is vital to the future application of DNA nanodevices and synthetic cells. However, little emphasis has been placed on developing light-controlled ‘off’ switches for cell-free expression. Light-activated antisense oligonucleotides have been developed to induce gene knockdown in living cells; however, they are complicated to synthesise and have not been tested in cell-free systems. Developing simple, accessible methods to produce light-activated antisense oligonucleotides will be crucial for allowing their application in cell-free biology and biotechnology. Here, we report a mild, one-step method for selectively attaching commercially-available photoremovable protecting groups, photocages, onto phosphorothioate linkages of antisense oligonucleotides. Using this photocaging method, upon illumination, the original phosphorothioate antisense oligonucleotide is reformed. Photocaged antisense oligonucleotides, containing mixed phosphorothioate and phosphate backbones, showed a drastic reduction in duplex formation and RNase H activity, which was recovered upon illumination. We then demonstrated that these photocaged antisense oligonucleotides can be used to knock down cell-free protein synthesis using light. This simple and accessible technology will have future applications in light-controlled biological logic gates and regulating the activity of synthetic cells.

Systematic Analysis of Chemical Modifications of Phosphorothioate Antisense Oligonucleotides that Modulate Their Innate Immune Response.

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.

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.

Assessment of the Effect of Organ Impairment on the Pharmacokinetics of 2'-MOE and Phosphorothioate Modified Antisense Oligonucleotides.

The pharmacokinetics (PK) of 2'-O-methoxyethyl and phosphorothioate antisense oligonucleotides (ASOs), with or without N-acetyl galactosamine conjugation, have been well characterized following subcutaneous or intravenous drug administration. However, the effect of organ impairment on ASO PK, primarily hepatic or renal impairment, has not yet been reported. ASOs distribute extensively to the liver and kidneys, where they are metabolized slowly by endo- and exonucleases, with minimal renal excretion as parent drug (<1%-3%). This short review evaluated the effect of organ impairment on ASO PK using 3 case studies: (1) a phase 1 renal impairment study evaluating a N-acetyl galactosamine-conjugated ASO in healthy study participants and study participants with moderate renal impairment, (2) a phase 2 study evaluating an unconjugated ASO in patients with end-stage renal disease; and (3) a phase 3 study evaluating an unconjugated ASO, which included patients with mild hepatic or renal impairment. Results showed that patients with end-stage renal disease had a mild increase (≈34%) in total plasma exposure, whereas mild or moderate renal impairment showed no effect on plasma PK. The effect of hepatic impairment on ASO PK could not be fully evaluated due to lack of data in moderate and severe hepatic impairment study participants. Nonetheless, available data suggest that mild hepatic impairment had no effect on ASO exposure.

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.

Inhomogeneous Diastereomeric Composition of Mongersen Antisense Phosphorothioate Oligonucleotide Preparations and Related Pharmacological Activity Impairment.

Mongersen is a 21-mer antisense oligonucleotide designed to downregulate Mothers against decapentaplegic homolog 7 (SMAD7) expression to treat Crohn's disease. Mongersen was manufactured in numerous batches at different scales during several years of clinical development, which all appeared identical, using common physicochemical analytical techniques, while only phosphorous-31 nuclear magnetic resonance (31P-NMR) in solution showed marked differences. Close-up analysis of 27 mongersen batches revealed marked differences in SMAD7 downregulation in a cell-based assay. Principal component analysis of 31P-NMR profiles showed strong correlation with SMAD7 downregulation and, therefore, with pharmacological efficacy in vitro. Mongersen contains 20 phosphorothioate (PS) linkages, whose chirality (Rp/Sp) was not controlled during manufacturing. A different diastereomeric composition throughout batches would lead to superimposable analytical data, but to distinct 31P-NMR profiles, as indeed we found. We tentatively suggest that this may be the origin of different biological activity. As similar manifolds are expected for other PS-based oligonucleotides, the protocol described here provides a general method to identify PS chirality issues and a chemometric tool to score each preparation for this elusive feature.

Evaluation of Phosphorus and Non-Phosphorus Neutral Oligonucleotide Backbones for Enhancing Therapeutic Index of Gapmer Antisense Oligonucleotides.

The phosphorothioate (PS) linkage in an essential component of therapeutic oligonucleotides. PS in the DNA region of gapmer antisense oligonucleotides (ASOs) supports RNaseH1 activity and enhances nuclease stability. PS also promotes binding to plasma, cell surface, and intracellular proteins, which facilitates tissue distribution, cellular uptake, and endosomal escape of PS ASOs. We recently showed that site-specific replacement of PS in the DNA gap with methoxylpropyl phosphonate (MOP) linkages can enhance the therapeutic index of gapmer ASOs. In this article, we explored 18 phosphorus- and non-phosphorus-based neutral backbone modifications to determine the structure-activity relationship of neutral linkages for enhancing therapeutic index. Replacing MOP with other alkyl phosphonate and phosphotriester linkages enhanced therapeutic index, but these linkages were susceptible to chemical degradation during oligonucleotide deprotection from solid supports following synthesis. Replacing MOP with non-phosphorus linkages resulted in improved chemical stability, but these linkages were introduced into ASOs as nucleotide dimers, which limits their versatility. Overall, linkages such as isopropyl and isobutyl phosphonates and O-isopropyl and O-tetrahydrofuranosyl phosphotriesters, formacetal, and C3-amide showed improved activity in mice relative to MOP. Our data suggest that site-specific incorporation of any neutral backbone linkage can improve therapeutic index, but the size, hydrophobicity, and RNA-binding affinity of the linkage influence ASO activity.

Evaluation of Pharmacokinetics and Metabolism of Phosphorothioate Antisense Oligonucleotide G3139 in Rat by Capillary Electrophoresis with Laser-Induced Fluorescence.

A sensitive and specific capillary electrophoresis with laser-induced fluorescence (CE-LIF) and a simple extraction process was developed to simultaneously detect G3139 and its metabolites as a model of antisense oligonucleotides (ASOs). This method has shown excellent linearity within the tested concentration range for G3139 and its metabolites, with a detection limit of 3.0 pM and a recovery of >84.2%. Based on our developed plasma extraction method, we have evaluated the pharmacokinetics and metabolites from rat plasma after intravenous administration of G3139 at 0.76 mg/kg. The results showed that G3139 and its metabolites were successfully simultaneously detected and analyzed through a single run using CE-LIF with baseline separation until the 30-h test sampling time point. The half-life of G3139 and its metabolites was observed at 31 and 68 h, respectively. This study may provide an effective analytical method for the pharmacokinetic and metabolite evaluation required to develop ASOs to treat a variety of diseases.

Sequence-specific 2'-O-methoxyethyl antisense oligonucleotides activate human platelets through glycoprotein VI, triggering formation of platelet-leukocyte aggregates.

Antisense oligonucleotides (ASO) are DNA-based, disease-modifying drugs. Clinical trials with 2'-O-methoxyethyl (2’MOE) ASO have shown dose- and sequence-specific lowering of platelet counts according to two phenotypes. Phenotype 1 is a moderate (but not clinically severe) drop in platelet count. Phenotype 2 is rare, severe thrombocytopenia. This article focuses on the underlying cause of the more common phenotype 1, investigating the effects of ASO on platelet production and platelet function. Five phosphorothioate ASO were studied: three 2’MOE sequences; 487660 (no effects on platelet count), 104838 (associated with phenotype 1), and 501861 (effects unknown) and two CpG sequences; 120704 and ODN 2395 (known to activate platelets). Human cord bloodderived megakaryocytes were treated with these ASO to study their effects on proplatelet production. Platelet activation (determined by surface P-selectin) and platelet-leukocyte aggregates were analyzed in ASO-treated blood from healthy human volunteers. None of the ASO inhibited proplatelet production by human megakaryocytes. All the ASO were shown to bind to the platelet receptor glycoprotein VI (KD ~0.2-1.5 μM). CpG ASO had the highest affinity to glycoprotein VI, the most potent platelet-activating effects and led to the greatest formation of platelet-leukocyte aggregates. 2’MOE ASO 487660 had no detectable platelet effects, while 2’MOE ASOs 104838 and 501861 triggered moderate platelet activation and SYKdependent formation of platelet-leukocyte aggregates. Donors with higher platelet glycoprotein VI levels had greater ASO-induced platelet activation. Sequence-dependent ASO-induced platelet activation and platelet-leukocyte aggregates may explain phenotype 1 (moderate drops in platelet count). Platelet glycoprotein VI levels could be useful as a screening tool to identify patients at higher risk of ASO-induced platelet side effects.

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.

High-resolution visualization and quantification of nucleic acid-based therapeutics in cells and tissues using Nanoscale secondary ion mass spectrometry (NanoSIMS).

Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial—both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2′-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease.

The Interaction of Phosphorothioate-Containing RNA Targeted Drugs with Proteins Is a Critical Determinant of the Therapeutic Effects of These Agents.

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.

In vitro metabolism of 2'-ribose unmodified and modified phosphorothioate oligonucleotide therapeutics using liquid chromatography mass spectrometry.

Antisense oligonucleotides (ASOs) have been touted as an emerging therapeutic class to treat genetic disorders and infections. The evaluation of metabolic stability of ASOs during biotransformation is critical due to concerns regarding drug safety. Because the effects of the modifications in ASOs on their metabolic stabilities are different from unmodified ASOs, studies that afford an understanding of these effects as well as propose proper methods to determine modified and unmodified ASO metabolites are imperative. An LC-tandem mass spectrometry method offering good selectivity with a high-quality separation using 30 mm N,N-dimethylcyclohexylamine and 100 mm 1,1,1,3,3,3-hexafluoro-2-propanol was utilized to identify each oligonucleotide metabolite. Subsequently, the method was successfully applied to a variety of in vitro systems including endo/exonuclease digestion, mouse liver homogenates, and then liver microsomes, after which the metabolic stability of unmodified versus modified ASOs was compared. Typical patterns of chain-shortened metabolites generated by mainly 3'-exonucleases were observed in phosphodiester and phosphorothioate ASOs, and endonuclease activity was identically observed in gapmers that showed relatively more resistance to nuclease degradation. Overall, the degradation of each ASO occurred more slowly corresponding to the degree of chemical modifications, while 5'-exonuclease activities were only observed in gapmers incubated in mouse liver homogenates. Our findings provide further understanding of the impact of modifications on the metabolic stability of ASOs, which facilitates the development of future ASO therapeutics.

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.