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Nucleic acid-based phosphorothioate containing antisense oligonucleotides (PS-ASOs) have the potential to activate cellular innate immune responses, and the level of activation can vary quite dramatically with sequence. Minimizing the degree of proinflammatory effect is one of the main selection criteria for compounds intended to move into clinical trials. While a recently developed human peripheral blood mononuclear cell (hPBMC)-based assay showed excellent ability to detect innate immune active PS-ASOs, which can then be discarded from the developmental process, this assay is highly resource intensive and easily affected by subject variability. This compelled us to develop a more convenient high-throughput assay. In this study, we describe a new

A human peripheral blood mononuclear cell (PBMC)-based assay was developed to identify antisense oligonucleotide (ASO) with the potential to activate a cellular innate immune response outside of an acceptable level. The development of this assay was initiated when ISIS 353512 targeting the messenger ribonucleic acid for human C-reactive protein (CRP) was tested in a phase I clinical trial, in which healthy human volunteers unexpectedly experienced increases in interleukin-6 (IL-6) and CRP. This level of immune stimulation was not anticipated following rodent and nonhuman primate safety studies in which no evidence of exaggerated proinflammatory effects were observed. The IL-6 increase induced by ISIS 353512 was caused by activation of B cells. The IL-6 induction was inhibited by chloroquine pretreatment of PBMCs and the nature of ASOs suggested that the response is mediated by a Toll-like receptor (TLR), in all likelihood TLR9. While assessing the inter PBMC donor variability, two classes of human PBMC responders to ISIS 353512 were identified (discriminator and nondiscriminators). The discriminator donor PBMCs were shown to produce low level of IL-6 after 24 h in culture, in the absence of ASO treatment. The PBMC assay using discriminator donors was shown to be reproducible, allowing to assess reliably the immune potential of ASOs by comparison to known benchmark ASO controls that were previously shown to be either safe or inflammatory in clinical trials. Clinical Trial registration numbers: NCT00048321 NCT00330330 NCT00519727.

Cellular uptake of antisense oligonucleotides (ASOs) is one of the main determinants of in vivo activity and potency. A significant advancement in improving uptake into cells has come through the conjugation of ASOs to triantenarry N-acetyl-galactosamine (GalNAc3), a ligand for the asialoglycoprotein receptor on hepatocytes. The impact for antisense oligonucleotides, which are already taken up into hepatocytes, is a 10-fold improvement in potency in mice and up to a 30-fold potency improvement in humans, resulting in overall lower effective dose and exposure levels. 2′-Methoxyethyl–modified antisense oligonucleotide conjugated to GalNAc3 (ISIS 702843) is specific for human transmembrane protease serine 6 and is currently in clinical trials for the treatment of β-thalassemia. This report summarizes a chronic toxicity study of ISIS 702843 in nonhuman primates (NHPs), including pharmacokinetic and pharmacology assessments. Suprapharmacologic doses of ISIS 702843 were well tolerated in NHPs after chronic dosing, and the data indicate that the overall safety profile is very similar to that of the unconjugated 2′-(2-methoxyethyl)-D-ribose (2′-MOE) ASOs. Notably, the GalNAc3 moiety did not cause any new toxicities nor exacerbate the known nonspecific class effects of the 2′-MOE ASOs. This observation was confirmed with multiple GalNAc3-MOE conjugates by querying a data base of monkey studies containing both GalNAc3-conjugated and unconjugated 2′-MOE ASOs. SIGNIFICANCE STATEMENT This report documents the potency, pharmacology, and overall tolerability profile of a triantenarry N-acetyl-galactosamine (GalNAc3)-conjugated 2′-(2-methoxyethyl)-D-ribose (2′-MOE) antisense oligonucleotide (ASO) specific to transmembrane protease serine 6 after chronic treatment in the cynomolgus monkey. Collective analysis of 15 independent GalNAc3-conjugated and unconjugated 2′-MOE ASOs shows the consistency in the dose response and character of hepatic and platelet tolerability across sequences that will result in much larger safety margins for the GalNAc3-conjugated 2′-MOE ASOs when compared with the unconjugated 2′-MOE ASOs given the increased potency.

Inotersen, a 2'-O-methoxyethyl (2'-MOE) phosphorothioate antisense oligonucleotide, reduced disease progression and improved quality of life in patients with hereditary transthyretin amyloidosis with polyneuropathy (hATTR-PN) in the NEURO-TTR and NEURO-TTR open-label extension (OLE) trials. However, 300 mg/week inotersen treatment was associated with platelet count reductions in several patients. Mean platelet counts in patients in the NEURO-TTR-inotersen group remained ≥140 × 10

Nucleic acid-based PS-ASOs have the potential to activate cellular innate immune responses, and the level of activation can vary quite dramatically with sequence. Minimizing degree of proinflammatory effect is one of the main selection criteria for compounds intended to move into clinical trials. While a recently developed hPBMC-based assay showed excellent ability to detect innate immune active PS-ASOs, which can then be discarded from the developmental process, this assay is highly resource-intensive and easily affected by subject variability. This compelled us to develop of a more convenient high-throughput assay. Here, we describe a new in vitro assay, utilizing a cultured human Bjab cell line, which was developed and validated to identify PS-ASOs that may cause innate immune activation. The assay was calibrated to replicate results from the hPMBC assay. The Bjab assay was designed to be high throughput and more convenient by using RT-qPCR readout of mRNA of the chemokine Ccl22. The Bjab assay was also shown to be highly reproducible and to provide a large dynamic range in determining the immune potential of PS-ASOs via comparison to known benchmark PS-ASO controls that were previously shown to be either safe or inflammatory in clinical trials. In addition, we demonstrate that Bjab cells can be used to provide mechanistic information on PS-ASO-TLR9 dependent innate immune activation.

Although antisense oligonucleotides (ASOs) are well tolerated preclinically and in the clinic, some sequences of ASOs can trigger an inflammatory response leading to B cell and macrophage activatio...

The 6-month Tg.rasH2 mouse carcinogenicity model provides an acceptable alternative to the 2-year carcinogenicity study in CD-1 mice. However, key questions related to the use of this model for testing antisense oligonucleotides (ASOs) include the similarity in the biologic response between mouse strains and the feasibility of using data from the CD-1 mouse to set doses and dose schedules for a Tg.rasH2 carcinogenicity study. To evaluate the potential strain differences, four distinct 2′- O-(2-methoxyethyl) ASOs were administered to CByB6F1 (wild type), Tg.rasH2 (hemizygous), and CD-1 mice. There were no meaningful differences in clinical signs, body weight, food consumption, or serum chemistry and hematology parameters. Histopathology evaluation indicated little to no difference in the spectrum or magnitude of changes present. The cytokine/chemokine response was also not appreciably different between the strains. This was consistent with the similarity in ASO concentration in the liver between the mouse strains tested. As the class effects of the ASOs were not meaningfully different between CD-1, CByB6F1, or Tg.rasH2 mice, data from nonclinical studies in CD-1 mice can be used for dose selection and expectation of effect in the Tg.rasH2 mouse.

AimsAmyloidogenic transthyretin (ATTR) amyloidosis is a fatal disease characterized by progressive cardiomyopathy and/or polyneuropathy. AKCEA-TTR-LRx (ION-682884) is a ligand-conjugated antisense drug designed for receptor-mediated uptake by hepatocytes, the primary source of circulating transthyretin (TTR). Enhanced delivery of the antisense pharmacophore is expected to increase drug potency and support lower, less frequent dosing in treatment.Methods and resultsAKCEA-TTR-LRx demonstrated an approximate 50-fold and 30-fold increase in potency compared with the unconjugated antisense drug, inotersen, in human hepatocyte cell culture and mice expressing a mutated human genomic TTR sequence, respectively. This increase in potency was supported by a preferential distribution of AKCEA-TTR-LRx to liver hepatocytes in the transgenic hTTR mouse model. A randomized, placebo-controlled, phase 1 study was conducted to evaluate AKCEA-TTR-LRx in healthy volunteers (ClinicalTrials.gov: NCT03728634). Eligible participants were assigned to one of three multiple-dose cohorts (45, 60, and 90mg) or a single-dose cohort (120mg), and then randomized 10:2 (active:placebo) to receive a total of 4 SC doses (Day 1, 29, 57, and 85) in the multiple-dose cohorts or 1 SC dose in the single-dose cohort. The primary endpoint was safety and tolerability; pharmacokinetics and pharmacodynamics were secondary endpoints. All randomized participants completed treatment. No serious adverse events were reported. In the multiple-dose cohorts, AKCEA-TTR-LRx reduced TTR levels from baseline to 2weeks after the last dose of 45, 60, or 90mg by a mean (SD) of -85.7% (8.0), -90.5% (7.4), and -93.8% (3.4), compared with -5.9% (14.0) for pooled placebo (P&nbsp

ISIS 104838, a 2'-O-methoxyethyl (2'-MOE)-modified antisense oligonucleotide (ASO), causes a moderate, reproducible, dose-dependent, but selflimiting decrease in platelet (PLT) counts in monkeys and humans. To determine the etiology of PLT decrease in cynomolgus monkeys, a 12-week repeat dose toxicology study in 5 cynomolgus monkeys given subcutaneous injections of ISIS 104838 (30-60 mg/kg/week). Monkeys were also injected intravenously with 111Indium(In)-oxine-labeled PLTs to investigate PLT sequestration. In response to continued dosing, PLT counts were decreased by 50%-90% by day 30 in all monkeys. PLT decreases were accompanied by 2- to 4.5-fold increases in immunoglobulin M(IgM), which were typified by a 2- to 5-fold increase in antiplatelet factor 4 (antiPF4) IgM and antiPLT IgM, respectively. Monocyte chemotactic protein 1 increased upon dosing of ISIS 104838, concomitant with a 2- to 6-fold increase in monocyte-derived extracellular vesicles (EVs), indicating monocyte activation but not PLT activation. Despite a 2- to 3-fold increase in von Willebrand factor antigen in all monkeys following ASO administration, only 2 monkeys showed a 2- to 4-fold increase in endothelial EVs. Additionally, a ∼60 - 80%% increase in PLT sequestration in liver and spleen was also observed. Collectively, these results suggest the overall increase in total IgM, antiPLT IgM and/or antiPF4 IgM, in concert with monocyte activation contributed to increased PLT sequestration in spleen and liver, leading to decreased PLTs in peripheral blood.

Although antisense oligonucleotides (ASOs) are well tolerated preclinically and in the clinic, some sequences of ASOs can trigger an inflammatory response leading to B cell and macrophage activation in rodents. This prompted our investigation into the contribution of genetic architecture to the ASO-mediated inflammatory response. 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 acute and delayed inflammatory response to a single 75 mg/kg dose of an inflammatory 2′-O-methoxyethyl (2′MOE) modified ASO. The acute response was measured 6 h after ASO administration, via evaluation for increased plasma production of interleukin 6 (IL6), IL10, monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein-1β (MIP-1β). Delayed inflammation was evaluated by spleen weight increases after 96 h. We identified single nucleotide polymorphisms (SNPs) on chromosomes 16 and 17 associated with plasma MIP-1β, IL6, and MCP-1 levels, and one on chromosome 8 associated with increases in spleen weight. Systems genetic analysis utilizing transcriptomic data from HMDP strain macrophages determined that the acute inflammatory SNPs were expression quantitative trait locis (eQTLs) for CCAAT/enhancer-binding protein beta (Cebpb) and salt inducible kinase 1 (Sik1). The delayed inflammatory SNP was an eQTL for Rho guanine nucleotide exchange factor 10 (Arhgef10). In vitro assays in mouse primary cells and human cell lines have confirmed the HMDP finding that lower Sik1 expression increases the acute inflammatory response. Our results demonstrate the utility of using mouse GWA study (GWAS) and the HMDP for detecting genes modulating the inflammatory response to pro-inflammatory ASOs in a pharmacological setting.

The common chemical and biological properties of antisense oligonucleotides provide the opportunity to identify and characterize chemical class effects across species. The chemical class that has proven to be the most versatile and best characterized is the 2'-O-methoxyethyl chimeric antisense oligonucleotides. In this report we present an integrated safety assessment of data obtained from controlled dose-ranging studies in nonhuman primates (macaques) and healthy human volunteers for 12 unique 2'-O-methoxyethyl chimeric antisense oligonucleotides. Safety was assessed by the incidence of safety signals in standardized laboratory tests for kidney and liver function, hematology, and complement activation; as well as by the mean test results as a function of dose level over time. At high doses a number of toxicities were observed in nonhuman primates. However, no class safety effects were identified in healthy human volunteers from this integrated data analysis. Effects on complement in nonhuman primates were not observed in humans. Nonhuman primates predicted safe doses in humans, but over predicted risk of complement activation and effects on platelets. Although limited to a single chemical class, comparisons from this analysis are considered valid and accurate based on the carefully controlled setting for the specified study populations and within the total exposures studied.

The comprehensive structure-activity relationships of triantennary GalNAc conjugated ASOs for enhancing potency via ASGR mediated delivery to hepatocytes is reported. Seventeen GalNAc clusters were assembled from six distinct scaffolds and attached to ASOs. The resulting ASO conjugates were evaluated in ASGR binding assays, in primary hepatocytes, and in mice. Five structurally distinct GalNAc clusters were chosen for more extensive evaluation using ASOs targeting SRB-1, A1AT, FXI, TTR, and ApoC III mRNAs. GalNAc-ASO conjugates exhibited excellent potencies (ED50 0.5-2 mg/kg) for reducing the targeted mRNAs and proteins. This work culminated in the identification of a simplified tris-based GalNAc cluster (THA-GN3), which can be efficiently assembled using readily available starting materials and conjugated to ASOs using a solution phase conjugation strategy. GalNAc-ASO conjugates thus represent a viable approach for enhancing potency of ASO drugs in the clinic without adding significant complexity or cost to existing protocols for manufacturing oligonucleotide drugs.

High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation.

ISIS 481464 is a constrained ethyl (cEt) modified phosphorothioate antisense oligonucleotide (ASO) targeting signal transducer and activator of transcription 3 (STAT3) studied in mice and monkey to support oncology clinical trials. Six-week toxicology studies were performed in mice and cynomolgus monkey (up to 70 and 30 mg/kg/week respectively). Reduction in STAT3 protein up to 90% of control was observed in monkey. Cynomolgus monkey was considered the most relevant species to human with respect to pharmacokinetic properties, but mice are useful in their relative sensitivity to the potential proinflammatory and hepatic effects of oligonucleotides. In monkeys, there was no impact on organ function at doses up to 30 mg/kg/week for 6 weeks. Minimal to slight proximal tubular epithelial cell degeneration and regeneration within the kidney was observed, which had no impact on renal function and showed reversibility at the end of the treatment-free period. Additionally, mild and transient activated partial thromboplastin time elevations and mild increases in complement Bb were observed at the higher doses by intravenous dosing only. In mice, the alterations at 70 mg/kg/week included spleen weight increase up to 1.4-fold relative to control, increases in alanine aminotransferase and aspartate aminotransferase up to 1.8-fold over control, interleukin-10 increases up to 3.7-fold, and monocyte chemoattractant protein-1 increase up to 1.9-fold over control. No significant clinical pathology or histopathology changes were seen in mice at 20 mg/kg/week or less. The toxicity profile of ISIS 481464 is consistent with effects observed with phosphorothioate ASOs containing 2'-O-methoxyethylribose modifications instead of cEt.

Oligonucleotides with a "CpG" motif trigger a proinflammatory response through activation of Toll-like receptor 9 (TLR9) and are being studied to exploit these properties for use as adjuvants and cancer therapies. However, oligonucleotides intended for antisense applications (ASOs) are designed to minimize proinflammatory responses by avoiding CpG motifs and by using chemical modifications [i.e., 2'-methoxyethyl (MOE) sugars and 5-methyl cytosine residues]. Nonetheless, modified ASOs are capable of eliciting a proinflammatory response at high doses, albeit mild compared with CpG oligos. To determine whether this phenomena is TLR-mediated, wild-type, TLR9 knockout, and myeloid differentiation factor 88 (MyD88) knockout mice were treated with a phosphorothioate-modified oligodeoxyribonucleotide CpG optimal oligo (ISIS 12449), and a representative non-CpG 2'-MOE oligonucleotide (ISIS 116847). The non-CpG oligonucleotide had a lower proinflammatory potency relative to ISIS 12449, requiring a >10-fold higher dose in wild-type animals to trigger a proinflammatory response. Furthermore, the inflammatory response to ISIS 12449 at low doses was TLR9 and MyD88-dependent, whereas non-CpG oligonucleotides retained the ability to activate a proinflammatory response in the knockout animals. Animals treated with the non-CpG oligonucleotide exhibited an increased spleen weight, elevated cytokine levels, increased immune cell infiltrates in liver, and an increased level of mRNA for cell surface markers typical of monocyte/macrophage type cells. Bone marrow-derived cells from wild-type and knockout animals treated with non-CpG oligonucleotide responded similarly with the production of MIP-2 and the activation of extracellular signal-regulated kianse1/2. These data implicate a TLR-independent mechanism of activation for non-CpG 2'-MOE oligonucleotides.

Normal blood-cell differentiation is controlled by regulated gene expression and signal transduction. Transcription deregulation due to chromosomal translocation is a common theme in hematopoietic neoplasms. AML1-ETO, which is a fusion protein generated by the 8;21 translocation that is commonly associated with the development of acute myeloid leukemia, fuses the AML1 runx family DNA-binding transcription factor to the ETO corepressor that associates with histone deacetylase complexes. Analyses have demonstrated that AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. Here, we report that the loss of the molecular events of AML1-ETO C-terminal NCoR/SMRT-interacting domain transforms AML1-ETO into a potent leukemogenic protein. Contrary to full-length AML1-ETO, the truncated form promotes in vitro growth and does not obstruct the cell-cycle machinery. These observations suggest a previously uncharacterized mechanism of tumorigenesis, in which secondary mutation(s) in molecular events disrupting the function of a domain of the oncogene promote the development of malignancy.

The t(8;21) is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML). The translocation, which involves the AML1 gene on chromosome 21 and the ETO gene on chromosome 8, generates an AML1-ETO fusion transcription factor. To examine the effect of the AML1-ETO fusion protein on leukemogenesis, we made transgenic mice in which expression of AML1-ETO is under the control of the human MRP8 promoter (hMRP8-AML1-ETO). AML1-ETO is specifically expressed in myeloid cells, including common myeloid progenitors of hMRP8-AML1-ETO transgenic mice. The transgenic mice were healthy during their life spans, suggesting that AML1-ETO alone is not sufficient for leukemogenesis. However, after treatment of newborn hMRP8-AML1-ETO transgenic mice and their wild-type littermates with a strong DNA-alkylating mutagen, N- ethyl- N -nitrosourea, 55% of transgenic mice developed AML and the other 45% of transgenic mice and all of the wild-type littermates developed acute T lymphoblastic leukemia. Our results provide direct evidence that AML1-ETO is critical for causing myeloid leukemia, but one or more additional mutations are required for leukemogenesis. The hMRP8-AML1-ETO-transgenic mice provide an excellent model that can be used to isolate additional genetic events and to further understand the molecular pathogenesis of AML1-ETO-related leukemia.