Antisense Oligonucleotide Targeting Research Articles

Antisense Oligonucleotide Therapy for Calmodulinopathy.

Calmodulinopathies are rare inherited arrhythmia syndromes caused by dominant heterozygous variants in CALM1, CALM2, or CALM3, which each encode the identical CaM (calmodulin) protein. We hypothesized that antisense oligonucleotide (ASO)-mediated depletion of an affected calmodulin gene would ameliorate disease manifestations, whereas the other 2 calmodulin genes would preserve CaM level and function. We tested this hypothesis using human induced pluripotent stem cell-derived cardiomyocyte and mouse models of CALM1 pathogenic variants. Human CALM1F142L/+ induced pluripotent stem cell-derived cardiomyocytes exhibited prolonged action potentials, modeling congenital long QT syndrome. CALM1 knockout or CALM1-depleting ASOs did not alter CaM protein level and normalized repolarization duration of CALM1F142L/+ induced pluripotent stem cell-derived cardiomyocytes. Similarly, an ASO targeting murine Calm1 depleted Calm1 transcript without affecting CaM protein level. This ASO alleviated drug-induced bidirectional ventricular tachycardia in CalmN98S/+ mice without a deleterious effect on cardiac electrical or contractile function. These results provide proof of concept that ASOs targeting individual calmodulin genes are potentially effective and safe therapies for calmodulinopathies.

Targeting the transferrin receptor to transport antisense oligonucleotides across the mammalian blood-brain barrier.

Antisense oligonucleotides (ASOs) are promising therapeutics for treating various neurological disorders. However, ASOs are unable to readily cross the mammalian blood-brain barrier (BBB) and therefore need to be delivered intrathecally to the central nervous system (CNS). Here, we engineered a human transferrin receptor 1 (TfR1) binding molecule, the oligonucleotide transport vehicle (OTV), to transport a tool ASO across the BBB in human TfR knockin (TfRmu/hu KI) mice and nonhuman primates. Intravenous injection and systemic delivery of OTV to TfRmu/hu KI mice resulted in sustained knockdown of the ASO target RNA, Malat1, across multiple mouse CNS regions and cell types, including endothelial cells, neurons, astrocytes, microglia, and oligodendrocytes. In addition, systemic delivery of OTV enabled Malat1 RNA knockdown in mouse quadriceps and cardiac muscles, which are difficult to target with oligonucleotides alone. Systemically delivered OTV enabled a more uniform ASO biodistribution profile in the CNS of TfRmu/hu KI mice and greater knockdown of Malat1 RNA compared with a bivalent, high-affinity TfR antibody. In cynomolgus macaques, an OTV directed against MALAT1 displayed robust ASO delivery to the primate CNS and enabled more uniform biodistribution and RNA target knockdown compared with intrathecal dosing of the same unconjugated ASO. Our data support systemically delivered OTV as a potential platform for delivering therapeutic ASOs across the BBB.

Lowering an ER stress-regulated long noncoding RNA protects mice from diabetes and isolated pancreatic islets from cell death

We investigated the role of the endoplasmic reticulum (ER) stress-regulated long noncoding-RNA (lncRNA) lncMGC in pancreatic islets and the pathology of type 1 diabetes (T1D), as well as the potential of lncMGC-based therapeutics. In vivo, blood glucose levels (BGL) and HbA1c were significantly lower in lncMGC-knockout (KO)-streptozotocin (STZ)-treated diabetic mice compared to wild-type-STZ. Antisense oligonucleotides (GapmeR) targeting lncMGC significantly attenuated insulitis and BGL in T1D NOD mice compared to GapmeR-negative control (NC). GapmeR-injected T1D Akita mice showed significantly lower BGL compared to Akita-NC mice. hlncMGC-GapmeR lowered BGL in partially humanized lncMGC (hlncMGC)-STZ mice compared to NC-injected mice. CHOP (ER stress regulating transcription factor) and lncMGC were upregulated in islets from diabetic mice but not in lncMGC-KO and GapmeR-injected diabetic mice, suggesting ER stress involvement. In vitro, hlncMGC-GapmeR increased viability of isolated islets from human donors and hlncMGC mice and protected them from cytokine induced apoptosis. Anti-ER stress and anti-apoptotic genes were upregulated, but pro-apoptotic genes were down-regulated in lncMGC KO mice islets and GapmeR-treated human islets. Taken together, these results show that a GapmeR targeting lncMGC is effective in ameliorating diabetes in mice and also preserves human and mouse islet viability, implicating clinical translation potential.

In-vitro Efficacy and In-vivo Biodistribution Studies of an Antisense Oligonucleotide Targeting Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation (S37.010)

In-vitro Efficacy and In-vivo Biodistribution Studies of an Antisense Oligonucleotide Targeting Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation (S37.010)

Chondrocyte-Targeted Delivery System of Sortase A-Engineered Extracellular Vesicles Silencing MMP13 for Osteoarthritis Therapy.

Targeted drug delivery and the reduction of off-target effects are crucial for the promising clinical application of nucleic acid drugs. To address this challenge, a new approach for treating osteoarthritis (OA) that accurately delivers antisense oligonucleotides (ASO) targeting matrix metalloproteinase-13 (ASO-MMP13) to chondrocytes, is developed. Small extracellular vesicles (exos) are ligated with chondrocyte affinity peptide (CAP) using Sortase A and subsequently incubated with cholesterol-modified ASO-MMP13 to construct a chondrocyte-targeted drug delivery exo (CAP-exoASO). Compared with exos without CAP (ExoASO), CAP-exoASOs attenuate IL-1β-induced chondrocyte damage and prolong the retention time of ASO-MMP13 in the joint without distribution in major organs following intra-articular injection. Notably, CAP-exoASOs decrease MMP13 expression (P < 0.001) and upregulate COL2A1 expression (P = 0.006), resulting in reorganization of the cartilage matrix and alleviation of progression in the OA model. Furthermore, the Osteoarthritis Research Society International (OARSI) score of articular cartilage tissues treated with CAP-exoASO is comparable with that of healthy rats (P = 0.148). A mechanistic study demonstrates that CAP-exoASO may reduce inflammation by suppressing the IL-17 and TNF signaling pathways. Based on the targeted delivery effect, CAP-exoASOs successfully accomplish cartilage repair and have considerable potential for development as a promising therapeutic modality for satisfactory OA therapy.

Olezarsen for Hypertriglyceridemia in Patients at High Cardiovascular Risk.

Reducing the levels of triglycerides and triglyceride-rich lipoproteins remains an unmet clinical need. Olezarsen is an antisense oligonucleotide targeting messenger RNA for apolipoprotein C-III (APOC3), a genetically validated target for triglyceride lowering. In this phase 2b, randomized, controlled trial, we assigned adults either with moderate hypertriglyceridemia (triglyceride level, 150 to 499 mg per deciliter) and elevated cardiovascular risk or with severe hypertriglyceridemia (triglyceride level, ≥500 mg per deciliter) in a 1:1 ratio to either a 50-mg or 80-mg cohort. Patients were then assigned in a 3:1 ratio to receive monthly subcutaneous olezarsen or matching placebo within each cohort. The primary outcome was the percent change in the triglyceride level from baseline to 6 months, reported as the difference between each olezarsen group and placebo. Key secondary outcomes were changes in levels of APOC3, apolipoprotein B, non-high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol. A total of 154 patients underwent randomization at 24 sites in North America. The median age of the patients was 62 years, and the median triglyceride level was 241.5 mg per deciliter. The 50-mg and 80-mg doses of olezarsen reduced triglyceride levels by 49.3 percentage points and 53.1 percentage points, respectively, as compared with placebo (P<0.001 for both comparisons). As compared with placebo, each dose of olezarsen also significantly reduced the levels of APOC3, apolipoprotein B, and non-HDL cholesterol, with no significant change in the LDL cholesterol level. The risks of adverse events and serious adverse events were similar in the three groups. Clinically meaningful hepatic, renal, or platelet abnormalities were uncommon, with similar risks in the three groups. In patients with predominantly moderate hypertriglyceridemia at elevated cardiovascular risk, olezarsen significantly reduced levels of triglycerides, apolipoprotein B, and non-HDL cholesterol, with no major safety concerns identified. (Funded by Ionis Pharmaceuticals; Bridge-TIMI 73a ClinicalTrials.gov number, NCT05355402.).

Structural dynamics of therapeutic nucleic acids with phosphorothioate backbone modifications.

Antisense oligonucleotides (ASOs) offer ground-breaking possibilities for selective pharmacological intervention for any gene product-related disease. Therapeutic ASOs contain extensive chemical modifications that improve stability to enzymatic cleavage and modulate binding affinity relative to natural RNA/DNA. Molecular dynamics (MD) simulation can provide valuable insights into how such modifications affect ASO conformational sampling and target binding. However, force field parameters for chemically modified nucleic acids (NAs) are still underdeveloped. To bridge this gap, we developed parameters to allow simulations of ASOs with the widely applied phosphorothioate (PS) backbone modification, and validated these in extensive all-atom MD simulations of relevant PS-modified NA systems representing B-DNA, RNA, and DNA/RNA hybrid duplex structures. Compared to the corresponding natural NAs, single PS substitutions had marginal effects on the ordered DNA/RNA duplex, whereas substantial effects of phosphorothioation were observed in single-stranded RNA and B-DNA, corroborated by the experimentally derived structure data. We find that PS-modified NAs shift between high and low twist states, which could affect target recognition and protein interactions for phosphorothioated oligonucleotides. Furthermore, conformational sampling was markedly altered in the PS-modified ssRNA system compared to that of the natural oligonucleotide, indicating sequence-dependent effects on conformational preference that may in turn influence duplex formation.

Retroelements in targeted antitumor therapy (review)

Background: Pathological expression of retroelements is determined in most malignant tumors, which reflects the pathogenesis of tumors, since activated retrotransposons cause genomic instability due to a cascade of mutual regulatory influence events between transposons, tumor suppressors and oncogenes. These relationships are reflected in the expression profiles of certain long non-coding RNAs and miRNAs in specific tumor tissues, since retroelements are key sources of non-coding RNAs. Since retroelements serve as drivers for the initiation and progression of carcinogenesis, it is promising to use drugs aimed at inhibiting their expression. The aim of the study: To determine the available methods of influencing retroelements in antitumor therapy, to evaluate the ways of their further improvement for the effective treatment of malignant neoplasms. Materials and methods: The databases Scopus, WoS, PubMed were used to analyze the role of retroelements in the development of tumors, methods of influencing them in the treatment of neoplasms. Results: Reverse transcriptase inhibitors have proven to be effective. Silencing of retrotransposons by acting on histone modifiers can lead to a weakening of the antitumor immune response; therefore, this approach requires RNA guides that direct the formation of heterochromatin in the area where strictly specific retroelements are located. A promising method is viral mimicry aimed at activating the interferon and T-cell response to artificially activated endogenous retroviruses. For this purpose, inhibitors of histone methyltransferases, DNA methyltransferases, and histone deacetylases are used. Their joint use is most effective, especially with PD-1 checkpoint blockers. The arrangement of retroelements in gene introns has become the basis for the development of spliceosomal targeted therapy, which also initiates an antitumor immune response to double-stranded RNA transcripts. We suggest the possibility of using antisense oligonucleotides targeting retroelements in anticancer therapy. Such approach is used to treat age-related macular degeneration, Fukuyama muscular dystrophy, amyotrophic lateral sclerosis. Antisense oligonucleotides targeting microRNAs, proto-oncogenes, and oncosuppressors are used in anticancer therapy and are closely related to retroelements, which could become promising targets for antisense oligonucleotides. Conclusion: In antitumor therapy, it is possible to use two diametrically opposed strategies for influencing retroelements: inhibition and activation. The most promising way is a combination of these methods with specific silencing of retrotransposons using guides (complementary RNAs) and stimulation of the expression of elements not involved in carcinogenesis, but activating the immune response.

CircHIPK3 regulates fatty acid metabolism through miR-637/FASN axis to promote esophageal squamous cell carcinoma

The oncogenic role of circRNA in cancers including esophageal cancer (EC) has been well studied. However, whether and how circRNAs are involved in cancer cell metabolic processes remains largely unknown. Here, we reported that circRNA, circHIPK3, is highly expressed in ESCC cell lines and tissues. Knockdown of circHIPK3 significantly restrained cell proliferation, colony formation, migration, and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, circHIPK3 was found to act as a ceRNA by sponging miR-637 to regulate FASN expression and fatty acid metabolism in ESCC cells. Anti-sense oligonucleotide (ASO) targeting circHIPK3 substantially inhibited ESCC both in vitro and in vivo. Therefore, these results uncover a modulatory axis constituting of circHIPK3/miR-637/FASN may be a potential biomarker and therapeutic target for ESCC in the clinic.

Use of Therapeutic RNAs to Accelerate Wound Healing in Diabetic Rabbit Wounds.

Introduction: Diabetes mellitus (DM) affects over 422 million people globally. Patients with DM are subject to a myriad of complications, of which diabetic foot ulcers (DFUs) are the most common with ∼25% chance of developing these wounds throughout their lifetime. Innovation: Currently there are no therapeutic RNAs approved for use in DFUs. Use of dressings containing novel layer-by-layer (LbL)-formulated therapeutic RNAs that inhibit PHD2 and miR-210 can significantly improve diabetic wound healing. These dressings provide sustained release of therapeutic RNAs to the wounds locally without systemic side effects. Clinical Problem Addressed: Diabetic foot wounds are difficult to heal and often result in significant patient morbidity and mortality. Materials and Methods: We used the diabetic neuroischemic rabbit model of impaired wound healing. Diabetes was induced in the rabbits with alloxan, and neuroischemia was induced by ligating the central neurovascular bundle of each ear. Four 6-mm full-thickness wounds were created on each ear. A LbL technique was used to conformally coat the wound dressings with chemically modified RNAs, including an antisense oligonucleotide (antimiR) targeting microRNA-210 (miR-210), an short synthetic hairpin RNA (sshRNA) targeting PHD2, or both. Results: Wound healing was improved by the antimiR-210 but not the PHD2-sshRNA. Specific knockdown of miR-210 in tissue as measured by RT-qPCR was ∼8 Ct greater than nonspecific controls, and this apparent level of knockdown (>99%) suggests that delivery to the tissue is highly efficient at the administered dose. Discussion: Healing of ischemic/neuropathic wounds in diabetic rabbits was accelerated upon inhibition of miR-210 by LbL delivery to the wound bed. miR-210 inhibition was achieved using a chemically modified antisense RNA.

Targeting NPM1 Epigenetically Promotes Postinfarction Cardiac Repair by Reprogramming Reparative Macrophage Metabolism.

Reparative macrophages play a crucial role in limiting excessive fibrosis and promoting cardiac repair after myocardial infarction (MI), highlighting the significance of enhancing their reparative phenotype for wound healing. Metabolic adaptation orchestrates the phenotypic transition of macrophages; however, the precise mechanisms governing metabolic reprogramming of cardiac reparative macrophages remain poorly understood. In this study, we investigated the role of NPM1 (nucleophosmin 1) in the metabolic and phenotypic shift of cardiac macrophages in the context of MI and explored the therapeutic effect of targeting NPM1 for ischemic tissue repair. Peripheral blood mononuclear cells were obtained from healthy individuals and patients with MI to explore NPM1 expression and its correlation with prognostic indicators. Through RNA sequencing, metabolite profiling, histology, and phenotype analyses, we investigated the role of NPM1 in postinfarct cardiac repair using macrophage-specific NPM1 knockout mice. Epigenetic experiments were conducted to study the mechanisms underlying metabolic reprogramming and phenotype transition of NPM1-deficient cardiac macrophages. The therapeutic efficacy of antisense oligonucleotide and inhibitor targeting NPM1 was then assessed in wild-type mice with MI. NPM1 expression was upregulated in the peripheral blood mononuclear cells from patients with MI that closely correlated with adverse prognostic indicators of MI. Macrophage-specific NPM1 deletion reduced infarct size, promoted angiogenesis, and suppressed tissue fibrosis, in turn improving cardiac function and protecting against adverse cardiac remodeling after MI. Furthermore, NPM1 deficiency boosted the reparative function of cardiac macrophages by shifting macrophage metabolism from the inflammatory glycolytic system to oxygen-driven mitochondrial energy production. The oligomeric NPM1 recruited histone demethylase KDM5b to the promoter of Tsc1 (TSC complex subunit 1), the mTOR (mechanistic target of rapamycin kinase) complex inhibitor, reduced histone H3K4me3 modification, and inhibited TSC1 expression, which then facilitated mTOR-related inflammatory glycolysis and antagonized the reparative function of cardiac macrophages. The in vivo administration of antisense oligonucleotide targeting NPM1 or oligomerization inhibitor NSC348884 substantially ameliorated tissue injury and enhanced cardiac recovery in mice after MI. Our findings uncover the key role of epigenetic factor NPM1 in impeding postinfarction cardiac repair by remodeling metabolism pattern and impairing the reparative function of cardiac macrophages. NPM1 may serve as a promising prognostic biomarker and a valuable therapeutic target for heart failure after MI.

Advances in Dyslipidaemia Treatments: Focusing on ApoC3 and ANGPTL3 Inhibitors.

Apolipoprotein C3 (apoC3) and angiopoietin-like protein 3 (ANGPTL3) inhibit lipolysis by lipoprotein lipase and may influence the secretion and uptake of various lipoproteins. Genetic studies show that depletion of these proteins is associated with improved lipid profiles and reduced cardiovascular events so it was anticipated that drugs which mimic the effects of loss-of-function mutations would be useful lipid treatments. ANGPTL3 inhibitors were initially developed as a treatment for severe hypertriglyceridaemia including familial chylomicronaemia syndrome (FCS), which is usually not adequately controlled with currently available drugs. However, it was found ANGPTL3 inhibitors were also effective in reducing low-density lipoprotein cholesterol (LDL-C) and they were studied in patients with homozygous familial hypercholesterolaemia (FH). Evinacumab targets ANGPTL3 and reduced LDL-C by about 50% in patients with homozygous FH and it has been approved for that indication. The antisense oligonucleotide (ASO) vupanorsen targeting ANGPTL3 was less effective in reducing LDL-C in patients with moderate hypertriglyceridaemia and its development has been discontinued but the small interfering RNA (siRNA) ARO-ANG3 is being investigated in Phase 2 studies. ApoC3 can be inhibited by the ASO volanesorsen, which reduced triglycerides by >70% in patients with FCS and it was approved for FCS in Europe but not in the United States because of concerns about thrombocytopaenia. Olezarsen is an N-acetylgalactosamine-conjugated ASO targeting apoC3 which appears as effective as volanesorsen without the risk of thrombocytopaenia and is undergoing Phase 3 trials. ARO-APOC3 is an siRNA targeting apoC3 that is currently being investigated in Phase 3 studies.

An anti-PD-1 antisense oligonucleotide promotes the expression of soluble PD-1 by blocking the interaction between SRSF3 and an exonic splicing enhancer of PD-1 exon 3

PD-1 is a key immune checkpoint molecule. Anti-PD-1 immunotherapy is encouraging in cancer treatment. However, it still needs to be improved. PD-1 has at least five isoforms generated by alternative splicing. An isoform without exon 3 encoding soluble PD-1 (sPD-1) can activate anti-tumor immunity by inhibiting the interaction between cellular surface full-length PD-1 (flPD-1) and PD-L1. However, the regulatory mechanism of exon 3 splicing remains largely unknown. Here, we screened the exon 3 sequence by mutation and searched corresponding splicing factors by SpliceAid database and pulldown assay. The alternative splicing of PD-1 exon 3 was analyzed by RT-PCR. The expression levels of flPD-1 and sPD-1 were analyzed by Western blot, flow cytometry, and ELISA. We discovered that an exonic splicing enhancer (ESE) of exon 3 is essential for its inclusion. Moreover, SRSF3 can bind to this ESE and enhance exon 3 inclusion and flPD-1 expression. We designed and screened out an antisense oligonucleotide (ASO) targeting PD-1 to block the interaction between SRSF3 and ESE, and significantly increase exon 3 skipping and sPD-1 expression, which was verified in various tumor cells in addition to oral cancer cells. Altogether, our results uncovered the regulatory mechanism of human PD-1 exon 3 splicing and sPD-1 expression and further designed a novel anti-PD-1 ASO, which are useful for developing a new method of anti-cancer immunotherapy.

Abstract A174: Antitumor activity of lipid nanoparticle-delivered anti-miR-21

Abstract Background: MicroRNAs (miRNAs) are small non-coding RNA molecules that play crucial roles in various biological processes, such as growth, development, aging, and apoptosis. Several studies have shown that miRNAs, including miR-21, can function as either oncogenes or tumor suppressor genes, influencing tumor initiation and progression. Aberrant expression of miR-21 has been observed in different cancer cells, indicating its significant involvement as an oncogenic miRNA in the development of various types of tumors. Antisense oligonucleotides (ASOs) have been identified as effective tools for downregulating target genes by either triggering RNase H endonuclease activity to cleave the ASO-target gene heteroduplex or by interfering with splicing and translation regulation of the target RNA sequence. ASOs have the potential to target not only mRNA but also oligonucleotides like miRNAs or small interfering RNAs (siRNAs), making them a promising approach for miRNA inhibition. However, the efficient delivery of freely circulating ASOs to the tumor microenvironment poses a challenge due to their high density of negative charges, impeding cell uptake. To address this issue, a novel lipid nanoparticle (LNP) platform called QTsome has been developed, which combines quaternary amine cationic lipids and tertiary amine ionizable lipids to enhance drug delivery. The utilization of a mixture of cationic and ionizable lipids in QTsome allows for optimal pH-dependent drug loading and release characteristics. In this study, our objective was to employ the QTsome system to deliver an ASO targeting miR-21 (AM-21) for potential tumor therapy. Methods: A total of 8E6 A549 cells were inoculated into mice, and when the tumor volume reached 100mm3, the mice were randomly divided into seven groups based on tumor volume, with each group consisting of five mice.On the day of grouping, drug administration was initiated with the following doses: 1mg/kg, every 3 days (Q3D); 2mg/kg, Q3D; 3mg/kg, Q3D; 4mg/kg, Q3D; 4mg/kg, every 4 days (Q4D); 4mg/kg, every 5 days (Q5D); 4mg/kg, every 7 days (Q7D). The drugs were administered a total of 5 times. Additionally, the A549/MC38 model was constructed using the same method. The antitumor activity of the drug and its therapeutic effect in combination with Erlotinib were evaluated in both the A549 CDX and MC38 syngeneic mouse models through intravenous injection, with a dose of 4mg/kg, Q3D. Results: The study revealed that the optimal dose for achieving the maximum antitumor effect was 4mg/kg, administered 3 times per day. In the A549 model, the tumor growth inhibition rates were 71.91%, 60.93%, and 80.44% for the AM-21/QTsome group, Erlotinib group, and the combination of AM-21/QTsome and Erlotinib group, respectively. In the MC38 mouse model, the tumor inhibition rate of AM-21/QTsome was 33.29%. Conclusion: These results emphasize the potential of miR-21 as a therapeutic target for cancer treatment, demonstrating that AM-21/QTsome holds great promise as a groundbreaking drug in this field. Citation Format: Jing Li, Qiaofang Ma, Xinyue Zhang, Xiaojie Chen, Jialiang Lin, Jun Bai, Fei Su, Xiaobin Zhao, Robert J. Lee, Yongsheng Yang. Antitumor activity of lipid nanoparticle-delivered anti-miR-21 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A174.

Abstract 18861: Therapeutic Targeting of Rpl13a -Small Nucleolar RNAs Mitigates Atherosclerosis in Apoe -/- Mice

Specific non-coding, small nucleolar (sno) RNAs encoded within introns of the Rpl13a gene elevate cellular levels of reactive oxygen species (ROS), and ROS are known to aggravate atherosclerosis (athero). We found that genetic deficiency of Rpl13a -snoRNAs reduces brachiocephalic artery athero in Apoe -/- mice by 50%. We therefore tested the hypothesis that athero could be reduced by acute pharmacologic intervention targeting Rpl13a -snoRNAs in Apoe -/- mice. To that end, we injected male and female Apoe -/- mice subcutaneously with 48 mg/kg antisense oligonucleotides (ASOs) targeting (a) the four Rpl13a -snoRNAs (ASO-snoRNA), or (b) green fluorescent protein (ASO-control). We tested two distinct strategies: ( i ) preventive, in which ASO injections and Western diet were started in 10-wk-old mice, with 4 weekly and 5 bi-weekly ASO treatments over 14 wk, then sacrifice; and ( ii ) “therapeutic,” in which ASO injections were started in 24-wk-old mice that had been fed Western diet for 14 wk and then were continued on Western diet with 6 weekly ASO treatments and then sacrificed. ASO injections were administered as indicated until 1 wk prior to sacrifice. Compared with saline-injected mice, ASO-control and ASO-snoRNA showed no hepatotoxicity (as assessed by serum ALT and AST). Compared with ASO-control, ASO-snoRNA administration for 6 wk reduced Rpl13a -snoRNA levels in the aorta and spleen by 54-80% for snoRNAs U32a , U33 , and U34 (RT-qPCR). In the “therapeutic” cohort, brachiocephalic artery cross-sectional athero was reduced by 38% in ASO-snoRNA-treated as compared with ASO-control-treated mice (n=7/group, p &lt;0.03). Additionally, male ASO-snoRNA-treated mice exhibited a 98.1% decrease in circulating IL1-β transcripts (n=6-8/group, p&lt;0.003). In the “preventive” cohort, aortic athero was reduced by 40% (13±5 vs 7±3 % aortic lesion area, n= 11-13/group, p &lt;0.02) in ASO-snoRNA-treated as compared with ASO-control-treated mice. We conclude that therapeutic targeting of Rpl13a -snoRNAs with ASOs mitigates athero, both during the early and later stages of atherogenesis.

Antisense oligonucleotide-based targeting of Tau-tubulin kinase 1 prevents hippocampal accumulation of phosphorylated tau in PS19 tauopathy mice

Tau tubulin kinase-1 (TTBK1), a neuron-specific tau kinase, is highly expressed in the entorhinal cortex and hippocampal regions, where early tau pathology evolves in Alzheimer’s disease (AD). The protein expression level of TTBK1 is elevated in the cortex brain tissues with AD patients compared to the control subjects. We therefore hypothesized that antisense oligonucleotide (ASO) based targeting Ttbk1 could prevent the accumulation of phosphorylated tau, thereby delaying the development of tau pathology in AD. Here we show that in vivo administration of ASO targeting mouse Ttbk1 (ASO-Ttbk1) specifically suppressed the expression of Ttbk1 without affecting Ttbk2 expression in the temporal cortex of PS19 tau transgenic mice. Central administration of ASO-Ttbk1 in PS19 mice significantly reduced the expression level of representative phosphor-tau epitopes relevant to AD at 8 weeks post-dose, including pT231, pT181, and pS396 in the sarkosyl soluble and insoluble fractions isolated from hippocampal tissues as determined by ELISA and pS422 in soluble fractions as determined by western blotting. Immunofluorescence demonstrated that ASO-Ttbk1 significantly reduced pS422 phosphorylated tau intensity in mossy fibers region of the dentate gyrus in PS19 mice. RNA-sequence analysis of the temporal cortex tissue revealed significant enrichment of interferon-gamma and complement pathways and increased expression of antigen presenting molecules (Cd86, Cd74, and H2-Aa) in PS19 mice treated with ASO-Ttbk1, suggesting its potential effect on microglial phenotype although neurotoxic effect was absent. These data suggest that TTBK1 is an attractive therapeutic target to suppress TTBK1 without compromising TTBK2 expression and pathological tau phosphorylation in the early stages of AD.

A patient-derived organoid-based study identified an ASO targeting SNORD14E for endometrial cancer through reducing aberrant FOXM1 Expression and β-catenin nuclear accumulation

BackgroundMost of the endometrial cancer (EC) patients are diagnosis in early stage with a good prognosis while the patients with locally advanced recurrent or metastatic result in a poor prognosis. Adjuvant therapy could benefit the prognosis of patients with high-risk factors. Unfortunately, the molecular classification of great prognostic value has not yet reached an agreement and need to be further refined. The present study aims to identify new targets that have prognostic value in EC based on the method of EC patient-derived organ-like organs (PDOs), and further investigate their efficacy and mechanism.MethodsThe Cancer Genome Atlas (TCGA) database was used to determine SNORD14E expression. The effects of SNORD14E were investigated using CCK8, Transwell, wound-healing assays, and a xenograft model experiment; apoptosis was measured by flow cytometry. Antisense oligonucleotide (ASO) targeting SNORD14E was designed and patient-derived organoids (PDO) models in EC patients was established. A xenograft mouse and PDO model were employed to evaluate the effects of ASO targeting SNORD14E. RNA-seq, Nm-seq, and RNA immunoprecipitation (RIP) experiments were employed to confirm the alternative splicing (AS) and modification induced by SNORD14E. A minigene reporter gene assay was conducted to confirm AS and splicing factors on a variable exon. Actinomycin-d (Act-D) and Reverse Transcription at Low deoxy-ribonucleoside triphosphate concentrations followed by PCR (RTL-P) were utilized to confirm the effects of 2′-O methylation modification on FOXM1.ResultsWe found that SNORD14E was overexpressed in EC tissues and patients with high expressed SNORD14E were distributed in the TCGA biomolecular classification subgroups without difference. Further, SNORD14E could reduce disease-free survival (DFS) and recurrence free survival (RFS) of EC patients. SNORD14E promoted proliferation, migration, and invasion and inhibited the apoptosis of EC cells in vitro. ASOs targeting SNORD14E inhibited cell proliferation, migration, invasion while promoted cell apoptosis. ASOs targeting SNORD14E inhibited tumor growth in the xenograft mouse model. TCGA-UCEC database showed that the proportion of patients with high expression of SNORD14E in middle-high risk and high-risk patients recommended by EMSO-ESGO-ESTRO guidelines for adjuvant therapy is more than 50%. Next, we enrolled 8 cases of high-risk and high-risk EC patients according to EMSO-ESGO-ESTRO guidelines and successfully constructed EC-PDOs. ASOs targeting SNORD14E inhibited the EC-PDO growth. Mechanistically, SNORD14E could recognize the mRNA of FOXM1 and recruit SRSF1 to promote the shearing of the variable exon VIIa of FOXM1, resulting in the overexpression of the FOXM1 malignant subtypes FOXM1b and FOXM1c. In addition, SNORD14E modified FOXM1 mRNA with 2`-O-methylation, which prolonged the half-life of FOXM1 mRNA. The nucleus accumulation of β-catenin caused by aberrant FOXM1 expression led to EC progression.ConclusionsASO targeting SNORD14E can be an effective treatment for EC.

Updates in Drug Treatment of Severe Hypertriglyceridemia.

To provide an insight into the new pharmacological options for the treatment of severe hypertriglyceridemia (sHTG). sHTG is difficult to treat. The majority of the traditional pharmacological agents available have limited success in both robustly decreasing triglyceride levels and/or in reducing the incidence of acute pancreatitis (AP), the most severe complication of sHTG. Therapeutic options with novel mechanisms of action have been developed, such as antisense oligonucleotides (ASO) and small interfering RNA (siRNA) targeting APOC3 and ANGPTL3. The review discusses also 2 abandoned drugs for sHTG treatment, evinacumab and vupanorsen. The ASO targeting APOC3, volanesorsen, is approved for use in patients with familial chylomicronemia syndrome (FCS) in Europe. Olezarsen, an N-acetylgalactosamine (GalNAc)-conjugated ASO with the same target, seems to have a better safety and efficacy profile. siRNA targeting APOC3 and ANGPTL3, namely ARO-APOC3 and ARO-ANG3, are also promising for the treatment of sHTG. However, the ultimate clinical goal of any sHTG treatment, the decrease in the risk of AP, has not been definitively achieved till now by any pharmacotherapy, either approved or in development.

A Systemically Administered Unconjugated Antisense Oligonucleotide Targeting DUX4 Improves Muscular Injury and Motor Function in FSHD Model Mice.

Facioscapulohumeral muscular dystrophy (FSHD), one of the most common muscular dystrophies, is caused by an abnormal expression of the DUX4 gene in skeletal muscles, resulting in muscle weakness. In this study, we investigated MT-DUX4-ASO, a novel gapmer antisense oligonucleotide (ASO). MT-DUX4-ASO decreased the expression of DUX4 and its target genes in FSHD patient-derived myoblasts. For the first time, we demonstrated that a systemically administered ASO, even without a ligand for drug delivery, could significantly improve muscle injury and motor function in the ACTA1-MCM/FLExDUX4 (DUX4-TG) mouse model of FSHD. Tamoxifen (TMX) injection transiently induces skeletal-muscle-specific DUX4 expression in DUX4-TG mice, while the skeletal muscles of TMX-untreated DUX4-TG mice have leaky DUX4 expression in a small subset of myofibers similar to those of FSHD patients. Subcutaneous 10 mg/kg of MT-DUX4-ASO at two-week intervals significantly suppressed muscular DUX4 target gene expression, histological muscle injury, and blood muscle injury marker elevation in TMX-untreated DUX4-TG mice. Notably, MT-DUX4-ASO at 10 mg/kg every other week significantly prevented the TMX-induced declines in treadmill test running speed and muscle force in DUX4-TG mice. Thus, the systemically administered unconjugated MT-DUX4-ASO suppressed disease progression in DUX4-TG mice, extending the potential of unconjugated ASOs as a promising FSHD treatment strategy.

Preclinical evaluation of JAK2 specific investigational oligonucleotide for the treatment of MDS/PV.

122 Background: Myeloproliferative disorders (MPD) are clonal hematopoietic stem cell malignancies with cytokine independency or hypersensitivity. Polycythemia vera (PV), an acquired MPD characterized by increased blood cell mass and hematocrit and leukocyte count, is associated with incidental myelofibrosis (MF). MDS is characterized by cytopenia and the presence of morphological dysplasia of precursor and mature bone marrow blood cells. PV and MDS leave patients at risk for progression to acute myeloid leukemia. Abnormal cytokine signaling due to an aberrant JAK2-STAT pathway has a vital role in PV and MDS pathogenesis. JAK2 mutations can result in hematologic malignancies, where hyperactive signaling of the JAK2-STAT pathway promotes tumor cell proliferation, invasion, and angiogenesis. Increased JAK2 kinase activity is observed in hematologic malignancies; somatic JAK2 V617F gain-of-function mutations are found in at least 95% of PV patients and is implicated in MDS cases. We hypothesize that preventing JAK2 transcription by antisense oligonucleotide (ASO) mediated exon masking of the JAK2 intron-exon junction will result in reduced JAK2 mRNA, and thus JAK2 protein, by providing nonsense mediated decay (NMD) in the reading frame. This may alleviate disease manifestations of hematologic malignancies like PV and MDS. Methods: This study utilized a HEL cell line harboring the V617F JAK2 gain-of-function mutation. We designed a series 19mer ASO targeting JAK2 exon-intron junctions ( JAK2 intron-exon junction providing NMD in the reading frame, steric, non-RNAse H1) and tested these at a range of concentrations. The ASOs were designed with a phosphorothioate 2’-O-methoxyethyl backbone and prioritized based on in silico binding affinity and limited off-target binding. HEL cells underwent ASO treatment (1µM) and 72-hour incubation. Results: We observed significant JAK2 protein decrease (~50%) in ASO-treated samples compared to untreated samples. JAK2 qPCR results confirmed 40-60% of target transcript. STAT5 phosphorylation status further confirmed this effect, and we report a 35% pSTAT5 reduction. Furthermore, this ASO showed limited off-target effects in silico. siRNA and CRISPR knockout lysate were used as controls. Our preclinical data support this ASO as a highly specific JAK2 agent, affecting direct levels of JAK2 as well as downstream STAT signaling. Conclusions: Around 50-60% of primary MF patients harbor the JAK2 V617F gain-of-function mutation. ASOs offer the capability to directly target JAK2 mutations with high precision and effectively reduce JAK2 protein production, without off-target kinase effects. The ability to reduce JAK2 protein may alleviate the disease burden that patients with hematologic malignancies face, resulting in a higher quality of life through prevention and treatment.