Hybrid Mouse Diversity Panel Identifies Genetic Architecture Associated with the Acute Antisense Oligonucleotide-Mediated Inflammatory Response to a 2'-O-Methoxyethyl Antisense Oligonucleotide.

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-1ß (MIP-1ß). 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-1ß, 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.

Comparison of the Class Effects of Antisense Oligonucleotides in CByB6F1-Tg(HRAS)2Jic and CD-1 Mice.

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.

The Distinct and Cooperative Roles of Toll-Like Receptor 9 and Receptor for Advanced Glycation End Products in Modulating In Vivo Inflammatory Responses to Select CpG and Non-CpG Oligonucleotides.

Antisense oligonucleotides (ASOs) are widely accepted therapeutic agents that suppress RNA transcription. While the majority of ASOs are well tolerated in vivo, few sequences trigger inflammatory responses in absence of conventional CpG motifs. In this study, we identified non-CpG oligodeoxy-nucleotide (ODN) capable of triggering an inflammatory response resulting in B cell and macrophage activation in a MyD88- and TLR9-dependent manner. In addition, we found the receptor for advance glycation end product (RAGE) receptor to be involved in the initiation of inflammatory response to suboptimal concentrations of both CpG- and non-CpG-containing ODNs. In contrast, dosing RAGE KO mice with high doses of CpG or non-CpG ODNs lead to a stronger inflammatory response than observed in wild-type mice. Together, our data provide a previously uncharacterized in vivo mechanism contingent on ODN-administered dose, where TLR9 governs the primary response and RAGE plays a distinct and cooperative function in providing a pivotal role in balancing the immune response.

Comprehensive Structure-Activity Relationship of Triantennary N-Acetylgalactosamine Conjugated Antisense Oligonucleotides for Targeted Delivery to Hepatocytes.

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.

Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts.

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.