Activity Of Antisense Oligonucleotides Research Articles

Regulation of human microglial gene expression and function via RNAase-H active antisense oligonucleotides in vivo in Alzheimer’s disease

BackgroundMicroglia play important roles in maintaining brain homeostasis and neurodegeneration. The discovery of genetic variants in genes predominately or exclusively expressed in myeloid cells, such as Apolipoprotein E (APOE) and triggering receptor expressed on myeloid cells 2 (TREM2), as the strongest risk factors for Alzheimer’s disease (AD) highlights the importance of microglial biology in the brain. The sequence, structure and function of several microglial proteins are poorly conserved across species, which has hampered the development of strategies aiming to modulate the expression of specific microglial genes. One way to target APOE and TREM2 is to modulate their expression using antisense oligonucleotides (ASOs).MethodsIn this study, we identified, produced, and tested novel, selective and potent ASOs for human APOE and TREM2. We used a combination of in vitro iPSC-microglia models, as well as microglial xenotransplanted mice to provide proof of activity in human microglial in vivo.ResultsWe proved their efficacy in human iPSC microglia in vitro, as well as their pharmacological activity in vivo in a xenografted microglia model. We demonstrate ASOs targeting human microglia can modify their transcriptional profile and their response to amyloid-β plaques in vivo in a model of AD.ConclusionsThis study is the first proof-of-concept that human microglial can be modulated using ASOs in a dose-dependent manner to manipulate microglia phenotypes and response to neurodegeneration in vivo.

A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides.

Antisense oligonucleotide (ASO) therapy is a novel therapeutic approach in which ASO specifically binds target mRNA, resulting in mRNA degradation; however, cellular uptake of ASOs remains critically low, warranting improvement. Transient receptor potential canonical (TRPC) channels regulate Ca2+ influx and are activated upon stimulation by phospholipase C-generated diacylglycerol. Herein, we report that a novel TRPC3/C6/C7 activator, L687, can induce cellular ASO uptake. L687-induced ASO uptake was enhanced in a dose- and incubation-time-dependent manner. L687 enhanced the knockdown activity of various ASOs both in vitro and in vivo. Notably, suppression of TRPC3/C6 by specific siRNAs reduced ASO uptake in A549 cells. Application of BAPTA-AM, a Ca2+ chelator, and SKF96365, a TRPC3/C6 inhibitor, suppressed Ca2+ influx via TRPC3/C6, resulting in reduced ASO uptake, thereby suggesting that Ca2+ influx via TRPC3/C6 is critical for L687-mediated increased ASO uptake. L687 also induced dextran uptake, indicating that L687 increased endocytosis. Adding ASO to L687 resulted in endosome accumulation; however, the endosomal membrane disruptor UNC7938 facilitated endosomal escape and enhanced knockdown activity. We discovered a new function for TRPC activators regarding ASO trafficking in target cells. Our findings provide an opportunity to formulate an innovative drug delivery system for the therapeutic development of ASO.

Prenatal delivery of a therapeutic antisense oligonucleotide achieves broad biodistribution in the brain and ameliorates Angelman syndrome phenotype in mice

Angelman syndrome (AS), an early-onset neurodevelopmental disorder characterized by abnormal gait, intellectual disabilities, and seizures, occurs when the maternal allele of the UBE3A gene is disrupted, since the paternal allele is silenced in neurons by the UBE3A antisense (UBE3A-AS) transcript. Given the importance of early treatment, we hypothesized that prenatal delivery of an antisense oligonucleotide (ASO) would downregulate the murine Ube3a-AS, resulting in increased UBE3A protein and functional rescue. Using a mouse model with a Ube3a-YFP allele that reports on-target ASO activity, we found that in utero, intracranial (IC) injection of the ASO resulted in dose-dependent activation of paternal Ube3a, with broad biodistribution. Accordingly, in utero injection of the ASO in a mouse model of AS also resulted in successful restoration of UBE3A and phenotypic improvements in treated mice on the accelerating rotarod and fear conditioning. Strikingly, even intra-amniotic (IA) injection resulted in systemic biodistribution and high levels of UBE3A reactivation throughout the brain. These findings offer a novel strategy for early treatment of AS using an ASO, with two potential routes of administration in the prenatal window. Beyond AS, successful delivery of a therapeutic ASO into neurons has implications for a clinically feasible prenatal treatment for numerous neurodevelopmental disorders.

Lipid nanoparticle delivery limits antisense oligonucleotide activity and cellular distribution in the brain after intracerebroventricular injection

Antisense oligonucleotide (ASO) therapeutics are being investigated for a broad range of neurological diseases. While ASOs have been effective in the clinic, improving productive ASO internalization into target cells remains a key area of focus in the field. Here, we investigated how the delivery of ASO-loaded lipid nanoparticles (LNPs) affects ASO activity, subcellular trafficking, and distribution in the brain. We show that ASO-LNPs increase ASO activity up to 100-fold in cultured primary brain cells as compared to non-encapsulated ASO. However, in contrast to the widespread ASO uptake and activity observed following free ASO delivery invivo, LNP-delivered ASOs did not downregulate mRNA levels throughout the brain after intracerebroventricular injection. This lack of activity was likely due to ASO accumulation in cells lining the ventricles and blood vessels. Furthermore, we reveal a formulation-dependent activation of the immune system post dosing, suggesting that LNP encapsulation cannot mask cellular ASO backbone-mediated toxicities. Together, these data provide insights into how LNP encapsulation affects ASO distribution as well as activity in the brain, and a foundation that enables future optimization of brain-targeting ASO-LNPs.

Synthesis of multivalent fatty acid-conjugated antisense oligonucleotides: Cell internalization, physical properties, and in vitro and in vivo activities

Herein, we describe the design and synthesis of multi-conjugatable fatty acid monomer phosphoramidites and their conjugation to antisense oligonucleotides (ASOs). Multivalent long-chain fatty acid conjugation improved the cellular uptake of ASOs but decreased in vitro activity due to alterations in physical properties and cellular localization. In addition, multivalently fatty acid-conjugated ASOs showed different organ specificity compared with that of unconjugated ASO in in vivo experiment. Although optimization of the linker structure between the fatty acid moiety and the ASO may be required, divalent long-chain fatty acid conjugation provides a new approach to increase endocytosis, thereby potentially improving the activity of therapeutic ASOs.

Synthesis and biophysical properties of tetravalent PEG-conjugated antisense oligonucleotide

This study was aimed at developing a novel platform for tetravalent conjugation of 4-arm polyethylene glycol (PEG) with an antisense oligonucleotide (ASO). The ASO technology has several limitations, such as low cellular uptake, poor nuclease stability, and short half-life. PEG-conjugated ASOs may result in an improvement in the pharmacokinetic behavior of the drug. Moreover, PEGylation can reduce enzymatic degradation and renal excretion of the conjugates, thereby, increasing its blood stability and retention time. In this study, we successfully synthesized PEG-ASO conjugate consisting of 4-arm-PEG and four molecules of ASO (4-arm-PEG-tetra ASO). Its hybridization ability with complementary RNA, enzymatic stability, and in vitro gene silencing ability were evaluated. No significant difference in hybridization ability was observed between 4-arm-PEG-tetra ASO and the parent ASO. In addition, gene silencing activity of the 4-arm-PEG-tetra ASO was observed in vitro. However, the in vitro activity of the 4-arm-PEG-tetra ASO was slightly reduced as that of the parent ASO. Moreover, the 4-arm-PEG-tetra ASO showed appreciable stability in cellular extract, suggesting that it hybridizes with mRNA in its intact form, without being cleaved in the cell, and exhibits ASO activity.

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.

Generation of Human iPSC-Derived Myotubes to Investigate RNA-Based Therapies In Vitro.

Alternative pre-mRNA splicing can be cell-type specific and results in the generation of different protein isoforms from a single gene. Deregulation of canonical pre-mRNA splicing by disease-associated variants can result in genetic disorders. Antisense oligonucleotides (AONs) offer an attractive solution to modulate endogenous gene expression through alteration of pre-mRNA splicing events. Relevant in vitro models are crucial for appropriate evaluation of splicing modifying drugs. In this chapter, we describe how to investigate the splicing modulating activity of AONs in an in vitro skeletal muscle model, applied to Pompe disease. We also provide a detailed description of methods to visualize and analyze gene expression in differentiated skeletal muscle cells for the analysis of muscle differentiation and splicing outcome. The methodology described here is relevant to develop treatment options using AONs for other genetic muscle diseases as well, including Duchenne muscular dystrophy, myotonic dystrophy, and facioscapulohumeral muscular dystrophy.

α-Synuclein antisense oligonucleotides as a disease-modifying therapy for Parkinson's disease.

Parkinson’s disease (PD) is a prevalent neurodegenerative disease with no approved disease-modifying therapies. Multiplications, mutations, and single nucleotide polymorphisms in the SNCA gene, encoding α-synuclein (aSyn) protein, either cause or increase risk for PD. Intracellular accumulations of aSyn are pathological hallmarks of PD. Taken together, reduction of aSyn production may provide a disease-modifying therapy for PD. We show that antisense oligonucleotides (ASOs) reduce production of aSyn in rodent preformed fibril (PFF) models of PD. Reduced aSyn production leads to prevention and removal of established aSyn pathology and prevents dopaminergic cell dysfunction. In addition, we address the translational potential of the approach through characterization of human SNCA-targeting ASOs that efficiently suppress the human SNCA transcript in vivo. We demonstrate broad activity and distribution of the human SNCA ASOs throughout the nonhuman primate brain and a corresponding decrease in aSyn cerebral spinal fluid (CSF) levels. Taken together, these data suggest that, by inhibiting production of aSyn, it may be possible to reverse established pathology; thus, these data support the development of SNCA ASOs as a potential disease-modifying therapy for PD and related synucleinopathies.

Mechanisms Regulating Myeloma Cell Responsiveness to Antisense Oligonucleotides Targeting IRF4

Functional genomics studies from our group and others, including CRISPR screens, have documented that interferon regulatory factor 4 (IRF4) is a critical transcription factor (TF) for multiple myeloma (MM) cells in preclinical in vitro and in vivo models; and one of the top most pronounced and recurrent dependencies for MM cells vs. other neoplasias. IRF4 lacks known ligand-binding pocket(s) amenable to selective inhibition by small-molecule pharmacological agents. IRF4 is thus considered, as many TFs, as "undruggable". Recent progress in antisense oligonucleotides (ASOs) and their in vivo properties motivated us to systematically evaluate the response of MM cells to anti-IRF4 ASOs, to obtain direct insights into their potential therapeutic applications in MM and also as functional probes into IRF4 biology. We observed that a panel of anti-IRF4 ASOs (but not control ASO) exhibited dose- and time-dependent activity against genotypically diverse MM cell lines (sub-uM IC50s for most MM lines vs. >10uM against non-MM cells lacking IRF4, e.g. HS5 bone marrow stromal cells [BMSCs]). Major in vitro anti-MM activity could be observed within 3-5 days of treatment; and was preceded by significant decrease in intracellular IRF4 protein levels. Importantly, 24-hour exposure to IRF4 ASO (followed by washout and subsequent culture in ASO-free media for up to 6 days) can induce similar anti-MM activity as continuous in vitro exposure over 6 days. Combinations of IRF4-ASO with several established (e.g. proteasome inhibitor, thalidomide derivative, glucocorticoids) or investigational (e.g. venetoclax) agents for MM result led to enhanced or even synergistic effects in a panel of 6 MM cell lines. For some of these MM cell lines, their in vitro response to IRF4 ASO had statistically significant attenuation in co-cultures with BMSCs: this effect was partially recapitulated in MM cell monocultures supplemented by conditioned media from BMSCs or (to a lesser extent) rhIL6 treatment. To obtain insights into cell-autonomous and nonautonomous mechanisms regulating MM cell responses to IRF4 ASOs, we examined the transcriptional profiles of MM.1S cells exposed to ASOs; and also performed genome-scale CRISPR studies to define genes whose editing (loss-of-function, LOF) or activation (gain-of-function, GOF) alter the MM cell response to the IRF4 ASOs. The transcriptional signature of MM cell treatment with IRF4 ASO (vs. control ASO) comprised distinct clusters of downregulated genes with preferential expression in normal or malignant plasma cells vs. other lineages; CRISPR-validated roles as dependencies for MM cells in vitro (preferentially essential for MM vs. non-MM tumors; or pan-essential); and/or proximity to large areas of chromatin accessibility (defined by H3K27Ac genome-wide ChIP-Seq or ATAC-Seq). Importantly, several genes downregulated by IRF4 ASO treatment are upregulated in MM cells cocultured with BMSCs, possibly explaining at least in part the impact of co-cultures on anti-MM activity of ASO. In our integrated genome-scale CRISPR studies (and validation of many genes with individual sgRNAs), MM cell response to IRF4 ASOs was attenuated by LOF of endonuclease RNase H1 (RNASEH1; which degrades the target RNA when it establishes intracellular hybrids with the ASOs); LOF of diverse genes involved in ASO endocytosis (e.g. IGF2R, PICALM, SH3GL1, RAB5C) or regulation of chondroitin/heparan sulfate (SLC35B2, B3GAT3, B4GALT7, ALG5 and ALG6); GOF of IRF4 itself (likely reflecting a "stoichiometric" effect, e.g. induction of higher IRF4 mRNA levels may require higher concentrations of ASO to maintain anti-MM effect); and GOF of exocytosis-related genes(e.g. CLU, QPCT). Importantly, LOF or GOF of individual genes typically associated with high-risk MM was not associated with decreased MM cell response to the IRF4 ASOs: therefore, retaining intracellular accumulation of IRF4 ASO and productive knockdown of IRF4 mRNA remains a primary driver of anti-MM activity of ASOs even in the context of biologically aggressive MM cells with "high-risk" features. Our results provide comprehensive integrated assessment of the molecular and functional landscapes associated with dysregulation of IRF4; have direct implications for our mechanistic understanding of the role of IRF4 in MM biology; and provide a framework for IRF4 targeting by ASOs or potentially other therapeutic approaches. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Luo:Ionis Pharmaceuticals, Inc.: Current Employment. Kim:Ionis Pharmaceuticals, Inc.: Current Employment. MacLeod:Ionis Pharmaceuticals, Inc.: Current Employment. Mitsiades:Arch Oncology: Research Funding; Sanofi: Research Funding; FIMECS: Consultancy, Honoraria; Karyopharm: Research Funding; Abbvie: Research Funding; Takeda: Other: employment of a relative; TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; EMD Serono: Research Funding; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria.

Targeted Delivery of Antisense Oligonucleotides Using Neurotensin Peptides.

Despite recent advances, targeted delivery of therapeutic oligonucleotide to extra-hepatic tissues continues to be a challenging endeavor and efficient ligand-receptor systems need to be identified. To determine the feasibility of using neurotensin to improve the productive uptake of antisense oligonucleotides (ASO), we synthesized neurotensin-ASO conjugates and evaluated their cellular uptake and activity in cells and in mice. We performed a comprehensive structure-activity relationship study of the conjugates and determined the influence of ASO charge, ASO length, peptide charge, linker chemistry and ligand identity on receptor binding and internalization. We identified a modified neurotensin peptide capable of improving the cellular uptake and activity of gapmer ASOs in sortilin expressing cells (sixfold) and in spinal cord in mice (twofold). Neurotensin conjugation also improved the potency of morpholino ASO designed to correct splicing of survival motor neuron pre-mRNA in the cortex and striatum after intracerebroventricular injection. Neurotensin-mediated targeted delivery represents a possible approach for enhancing the potency of ASOs with diverse nucleic acid modifications.

Mechanisms of palmitic acid-conjugated antisense oligonucleotide distribution in mice

Conjugation of antisense oligonucleotide (ASO) with a variety of distinct lipophilic moieties like fatty acids and cholesterol increases ASO accumulation and activity in multiple tissues. While lipid conjugation increases tissue exposure in mice and reduces excretion of ASO in urine, histological review of skeletal and cardiac muscle indicates that the increased tissue accumulation of lipid conjugated ASO is isolated to the interstitium. Administration of palmitic acid-conjugated ASO (Palm-ASO) in mice results in a rapid and substantial accumulation in the interstitium of muscle tissue followed by relatively rapid clearance and only slight increases in intracellular accumulation in myocytes. We propose a model whereby increased affinity for lipid particles, albumin, and other plasma proteins by lipid-conjugation facilitates ASO transport across endothelial barriers into tissue interstitium. However, this increased affinity for lipid particles and plasma proteins also facilitates the transport of ASO from the interstitium to the lymph and back into circulation. The cumulative effect is only a slight (∼2-fold) increase in tissue accumulation and similar increase in ASO activity. To support this proposal, we demonstrate that the activity of lipid conjugated ASO was reduced in two mouse models with defects in endothelial transport of macromolecules: caveolin-1 knockout (Cav1−/−) and FcRn knockout (FcRn−/−).

Enhanced Potency of GalNAc-Conjugated Antisense Oligonucleotides in Hepatocellular Cancer Models.

Antisense oligonucleotides (ASOs) are a novel therapeutic approach to target difficult-to-drug protein classes by targeting their corresponding mRNAs. Significantly enhanced ASO activity has been achieved by the targeted delivery of ASOs to selected tissues. One example is the targeted delivery of ASOs to hepatocytes, achieved with N-acetylgalactosamine (GalNAc) conjugation to ASO, which results in selective uptake by asialoglycoprotein receptor (ASGR). Here we have evaluated the potential of GalNAc-conjugated ASOs as a therapeutic approach to targeting difficult-to-drug pathways in hepatocellular carcinoma (HCC). The activity of GalNAc-conjugated ASOs was superior to that of the unconjugated parental ASO in ASGR (+) human HCC cells in vitro, but not in ASGR (−) cells. Both human- and mouse-derived HCC displayed reduced levels of ASGR, however, despite this, GalNAc-conjugated ASOs showed a 5- to 10-fold increase in potency in tumors. Systemically administered GalNAc-conjugated ASOs demonstrated both enhanced antisense activity and antitumor activity in the diethylnitrosamine-induced HCC tumor model. Finally, GalNAc conjugation enhanced ASO activity in human circulating tumor cells from HCC patients, demonstrating the potential of this approach in primary human HCC tumor cells. Taken together, these results provide a strong rationale for a potential therapeutic use of GalNAc-conjugated ASOs for the treatment of HCC.

Mouse genome-wide association studies and systems genetics uncover the genetic architecture associated with hepatic pharmacokinetic and pharmacodynamic properties of a constrained ethyl antisense oligonucleotide targeting Malat1.

Antisense oligonucleotides (ASOs) have demonstrated variation of efficacy in patient populations. This has prompted our investigation into the contribution of genetic architecture to ASO pharmacokinetics (PK) and pharmacodynamics (PD). Genome wide association (GWA) and transcriptomic analysis in a hybrid mouse diversity panel (HMDP) were used to identify and validate novel genes involved in the uptake and efficacy of a single dose of a Malat1 constrained ethyl (cEt) modified ASO. The GWA of the HMDP identified two significant associations on chromosomes 4 and 10 with hepatic Malat1 ASO concentrations. Stabilin 2 (Stab2) and vesicle associated membrane protein 3 (Vamp3) were identified by cis-eQTL analysis. HMDP strains with lower Stab2 expression and Stab2 KO mice displayed significantly lower PK than strains with higher Stab2 expression and the wild type (WT) animals respectively, confirming the role of Stab2 in regulating hepatic Malat1 ASO uptake. GWA examining ASO efficacy uncovered three loci associated with Malat1 potency: Small Subunit Processome Component (Utp11l) on chromosome 4, Rho associated coiled-coil containing protein kinase 2 (Rock2) and Aci-reductone dioxygenase (Adi1) on chromosome 12. Our results demonstrate the utility of mouse GWAS using the HMDP in detecting genes capable of impacting the uptake of ASOs, and identifies genes critical for the activity of ASOs in vivo.

Sub‐organ Fractionation of Hepatic Cells after Antisense Oligonucleotide Treatment in Mice

Nonalcoholic Steatohepatitis (NASH) causes chronic liver disease and leads to advanced fibrosis and cirrhosis. Current research is unclear which liver cell population is key for treatment of the disease. Antisense oligonucleotide (ASO) drugs are a likely therapy for NASH due to their preferential distribution to the liver. Moreover, conjugates can be attached to the ASO and enable targeted cell specific drug delivery to the liver. Conjugations to the ASO also facilitate enhanced potency in targeted cell types by utilizing different uptake mechanisms. Unconjugated ASOs preferentially accumulate in the non‐parenchymal (NP) cells of the liver including liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC) and hepatic stellate cells (HSC) before reaching the hepatocytes. Conversely triantennary N‐acetyl galactosamine (GalNAc)‐conjugation of ASOs results in a shift of ASO to preferentially accumulate in the hepatocytes over the NP cells (Prakash et al. NAR, 42: 8796, 2014). A recent study was designed to investigate the ASO activity in hepatocytes, LSEC, KC, and HSC from mice fed a standard chow diet compared to mice fed a Western diet. The liver pathology of the mice fed a Western diet at the time of ASO treatment was indicative of a NASH diseased state (steatosis, steatohepatitis and fibrosis). Mice were injected subcutaneously, at the indicated doses, with unconjugated or GalNAc‐conjugated ASO for 6 weeks. Following treatment, mouse livers were perfused, the hepatocytes were isolated by Percoll gradient and the NP cells by Optiprep (Lodixanol) gradient. NP cells were further isolated into enriched populations by fluorescence‐activated cell sorting (FACS). Whole liver and isolated cell populations were evaluated for RNA reduction after treatment. Drug concentration in each cell type was also measured using an ELISA based assay. As expected, there was target mRNA reduction with unconjugated and conjugated ASOs in all hepatocyte cell fractions. There was also targeted mRNA reduction in all NP cell populations in both healthy and diseased mice. However, the HSC cell population contained the least amount of ASO resulting in the lowest level of targeted reduction. These results suggest that GalNAc‐conjugated ASO can decrease targeted RNA not only in hepatocyte cells, but also in LSEC, KC and HSC cells despite a lower dose and a sever diseased state. This finding illustrates the ability of GalNAc‐conjugated ASO to enhance drug potency yet still allow drug delivery to all liver cell types. Therefore, ASO treatment is an attractive therapy for liver diseases, such as NASH, that involve multiple cell types contributing to disease progression.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract B197: Selective depletion of YAP1 with next-generation antisense oligonucleotides leads to immune cell infiltration and tumor regression in mouse models of HCC

Abstract The Hippo pathway plays important roles in maintaining tissue homeostasis. When dysregulated, however, it contributes to the development of a variety of cancers including hepatocellular carcinoma (HCC). YAP1 is the final regulator of the pathway and its causal role in promoting HCC growth has been demonstrated previously in multiple animal models. YAP1 is therefore considered an attractive drug target for HCC but as a transcription coactivator is difficult to inhibit by conventional approaches. We investigated how selective downregulation of YAP1 would affect the growth of established HCC using optimized Gen2.5 antisense oligonucleotides (ASOs) for mouse YAP1. Here, we describe the antitumor activity of YAP1 ASOs in 2 mouse models of HCC, which appears to be mediated by both tumor autonomous and immune modulatory functions of YAP1. In YAP1-activated Salvador KO mice with established HCC, systemic treatment of YAP1 ASO for 2 months at 75 mg/kg/week resulted in a marked reduction (>90%) in tumor YAP1 levels and regression of HCC tumors, which is in contrast to the tumors in the control ASO-treated animals that significantly increased in size. Proliferation of tumor cells was drastically reduced in YAP1 ASO-treated animals as demonstrated by low Ki-67 staining along with a decrease in Cyr61, a downstream YAP1 target. YAP1 ASOs were also tested in the diethylnitrosamine-induced HCC model. Although the model has been widely used in HCC research, lack of reliable biomarkers and high variability in tumor take between animals make it less ideal for an efficacy study. To overcome these limitations, we established a subcutaneous model by in vivo passaging tumors excised from the liver in either nude or immune-competent C57BL/6 mice. Activation of YAP1 in this model was demonstrated by strong nuclear localization of YAP1 protein in tumors. While the effect of YAP1 ASO on tumor was primarily delaying its growth in nude mice, the same YAP1 ASO induced tumor regression when DEN tumors were grown in immune competent mice, suggesting an immune component for activity. Strong infiltration of myeloid lineage cells was observed in tumors of YAP1 ASO-treated animals at the end of the 6 week study, suggesting potential activation of immune cells as a result of YAP1 depletion. Significant infiltration of T cells and myeloid lineage cells into YAP1 ASO-treated tumors was also observed at an early time point as demonstrated by increases in CD3 and F4/80 signals by IHC. Immune cell infiltration into tumors was more pronounced in the combination of ASOs for YAP1 and its paralog mouse TAZ than YAP1 ASO alone; however, the combination group exhibited notable signs of inflammation on the skin and in the liver. Importantly, when tumor-bearing animals were taken off the treatment after tumor regression was observed with YAP1 ASO, the antitumor response was sustained for more than 6 weeks with 2 out of 8 animals remaining tumor free in YAP1 ASO group. Further, safety of ASO-mediated YAP1 inhibition was confirmed in both normal mice with no notable toxicity and tumor-bearing mice with improvement in liver transaminases despite near complete depletion of YAP1 in the liver. Collectively, these results provide evidence for immune activation as a result of YAP1 downregulation and also suggest a potential therapeutic use of YAP1 ASO in the treatment of HCC with YAP1 activation. Citation Format: Youngsoo Kim, Joanna Schmidt, Minji Jo, Jing Yi, Tianyuan Zhou, Xiaolin Luo, Sun Young Yim, Ju-Seog Lee, Randy L. Johnson, Brett P. Monia, Gordon Mills, A. Robert MacLeod. Selective depletion of YAP1 with next-generation antisense oligonucleotides leads to immune cell infiltration and tumor regression in mouse models of HCC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B197.

Asialoglycoprotein receptor 1 mediates productive uptake of N-acetylgalactosamine-conjugated and unconjugated phosphorothioate antisense oligonucleotides into liver hepatocytes.

Antisense oligonucleotide (ASO) therapeutics show tremendous promise for the treatment of previously intractable human diseases but to exert their effects on cellular RNA processing they must first cross the plasma membrane by endocytosis. The conjugation of ASOs to a receptor ligand can dramatically increase their entry into certain cells and tissues, as demonstrated by the implementation of N-acetylgalactosamine (GalNAc)-conjugated ASOs for Asialoglycoprotein Receptor (ASGR)-mediated uptake into liver hepatocytes. We compared the internalization and activity of GalNAc-conjugated ASOs and their parents in endogenous ASGR-expressing cells and were able to recapitulate hepatocyte ASO uptake and activity in cells engineered to heterologously express the receptor. We found that the minor receptor subunit, ASGR2, is not required for effective in vitro or in vivo uptake of GalNAc-conjugated ASO and that the major subunit, ASGR1, plays a small but significant role in the uptake of unconjugated phosphorothioate ASOs into hepatocytes. Moreover, our data demonstrates there is a large excess capacity of liver ASGR for the effective uptake of GalNAc–ASO conjugates, suggesting broad opportunities to exploit receptors with relatively moderate levels of expression.

Antisense oligonucleotides selectively suppress target RNA in nociceptive neurons of the pain system and can ameliorate mechanical pain.

There is an urgent need for better treatments for chronic pain, which affects more than 1 billion people worldwide. Antisense oligonucleotides (ASOs) have proven successful in treating children with spinal muscular atrophy, a severe infantile neurological disorder, and several ASOs are currently being tested in clinical trials for various neurological disorders. Here, we characterize the pharmacodynamic activity of ASOs in spinal cord and dorsal root ganglia (DRG), key tissues for pain signaling. We demonstrate that activity of ASOs lasts up to 2 months after a single intrathecal bolus dose. Interestingly, comparison of subcutaneous, intracerebroventricular, and intrathecal administration shows that DRGs are targetable by systemic and central delivery of ASOs, while target reduction in the spinal cord is achieved only after direct central delivery. Upon detailed characterization of ASO activity in individual cell populations in DRG, we observe robust target suppression in all neuronal populations, thereby establishing that ASOs are effective in the cell populations involved in pain propagation. Furthermore, we confirm that ASOs are selective and do not modulate basal pain sensation. We also demonstrate that ASOs targeting the sodium channel Nav1.7 induce sustained analgesia up to 4 weeks. Taken together, our findings support the idea that ASOs possess the required pharmacodynamic properties, along with a long duration of action beneficial for treating pain.

High Boron-loaded DNA-Oligomers as Potential Boron Neutron Capture Therapy and Antisense Oligonucleotide Dual-Action Anticancer Agents.

Boron cluster-modified therapeutic nucleic acids with improved properties are of interest in gene therapy and in cancer boron neutron capture therapy (BNCT). High metallacarborane-loaded antisense oligonucleotides (ASOs) targeting epidermal growth factor receptor (EGFR) were synthesized through post-synthetic Cu (I)-assisted “click” conjugation of alkyne-modified DNA-oligonucleotides with a boron cluster alkyl azide component. The obtained oligomers exhibited increased lipophilicity compared to their non-modified precursors, while their binding affinity to complementary DNA and RNA strands was slightly decreased. Multiple metallacarborane residues present in the oligonucleotide chain, each containing 18 B-H groups, enabled the use of IR spectroscopy as a convenient analytical method for these oligomers based on the diagnostic B-H signal at 2400–2650 cm−1. The silencing activity of boron cluster-modified ASOs used at higher concentrations was similar to that of unmodified oligonucleotides. The screened ASOs, when used in low concentrations (up to 50 μM), exhibited pro-oxidative properties by inducing ROS production and an increase in mitochondrial activities in HeLa cells. In contrast, when used at higher concentrations, the ASOs exhibited anti-oxidative properties by lowering ROS species levels. In the HeLa cells (tested in the MTT assay) treated (without lipofectamine) or transfected with the screened compounds, the mitochondrial activity remained equal to the control level or only slightly changed (±30%). These findings may be useful in the design of dual-action boron cluster-modified therapeutic nucleic acids with combined antisense and anti-oxidant properties.

RNase H1-Dependent Antisense Oligonucleotides Are Robustly Active in Directing RNA Cleavage in Both the Cytoplasm and the Nucleus

RNase H1-dependent antisense oligonucleotides (ASOs) are active in reducing levels of both cytoplasmic mRNAs and nuclear retained RNAs. Although ASO activity in the nucleus has been well demonstrated, the cytoplasmic activity of ASOs is less clear. Using kinetic and subcellular fractionation studies, we evaluated ASO activity in the cytoplasm. Upon transfection, ASOs targeting exonic regions rapidly reduced cytoplasmically enriched mRNAs, whereas an intron-targeting ASO that only degrades the nuclear pre-mRNA reduced mRNA levels at a slower rate, similar to normal mRNA decay. Importantly, some exon-targeting ASOs can rapidly and vigorously reduce mRNA levels without decreasing pre-mRNA levels, suggesting that pre-existing cytoplasmic mRNAs can be cleaved by RNase H1-ASO treatment. In addition, we expressed a cytoplasm-localized mutant 7SL RNA that contains a partial U16 small nucleolar RNA (snoRNA) sequence. Treatment with an ASO simultaneously reduced both the nuclear U16 snoRNA and the cytoplasmic 7SL mutant RNA as early as 30 min after transfection in an RNase H1-dependent manner. Both the 5′ and 3′ cleavage products of the 7SL mutant RNA were accumulated in the cytoplasm. Together, these results demonstrate that RNase H1-dependent ASOs are robustly active in both the cytoplasm and nucleus.