Inhibition of EGF Uptake by Nephrotoxic Antisense Drugs In Vitro and Implications for Preclinical Safety Profiling

Abstract

Antisense oligonucleotide (AON) therapeutics offer new avenues to pursue clinically relevant targets inaccessible with other technologies. Advances in improving AON affinity and stability by incorporation of high affinity nucleotides, such as locked nucleic acids (LNA), have sometimes been stifled by safety liabilities related to their accumulation in the kidney tubule. In an attempt to predict and understand the mechanisms of LNA-AON-induced renal tubular toxicity, we established human cell models that recapitulate in vivo behavior of pre-clinically and clinically unfavorable LNA-AON drug candidates. We identified elevation of extracellular epidermal growth factor (EGF) as a robust and sensitive in vitro biomarker of LNA-AON-induced cytotoxicity in human kidney tubule epithelial cells. We report the time-dependent negative regulation of EGF uptake and EGF receptor (EGFR) signaling by toxic but not innocuous LNA-AONs and revealed the importance of EGFR signaling in LNA-AON-mediated decrease in cellular activity. The robust EGF-based in vitro safety profiling of LNA-AON drug candidates presented here, together with a better understanding of the underlying molecular mechanisms, constitutes a significant step toward developing safer antisense therapeutics.

A Sensitive In Vitro Approach to Assess the Hybridization-Dependent Toxic Potential of High Affinity Gapmer Oligonucleotides

“…Overall, our finding of a T m < 55°C threshold level for LNA-ASO toxicity has the potential to improve the design of well-tolerated and efficacious molecules. Moreover, our in vitro screening approach represents a valuable addition to the set of in vitro assays that can identify and deselect ASOs with potential toxicity liabilities, 11 , 14 , 15 further improving the discovery of safer ASO drugs.…”

“…Although our understanding of hybridization-dependent ASO toxicity is increasing, there are only a few reported in silico and/or in vitro approaches to predict this in vivo liability. 6 , 11 , 12 , 13 , 14 , 15 Although in silico methods can identify putative off-target sequences, 11 , 13 setting tolerable thresholds to minimize false positive off-target predictions remains difficult. Thus, in silico approaches can markedly reduce, but not eliminate, the risk of selecting oligonucleotides with hybridization-dependent toxicities.…”

Liver-Targeted Anti-HBV Single-Stranded Oligonucleotides with Locked Nucleic Acid Potently Reduce HBV Gene Expression In Vivo

“…In fact, accumulation in the kidneys has been shown to be associated with nephrotoxicity in clinical settings. 22 , 23 Furthermore, although major distribution to the liver is observed, this distribution is not uniform, and unconjugated SSOs preferentially accumulate in non-parenchymal cells. To enhance the activity of SSOs against a hepatocyte-expressed target, preferential SSO delivery to hepatocytes can be achieved through conjugation to a tri-antennary N-acetylgalactosamine cluster (GalNAc) that directs enhanced uptake via the hepatocyte-specific asialoglycoprotein receptor (ASGPR).…”

Assessing single-stranded oligonucleotide drug-induced effects in vitro reveals key risk factors for thrombocytopenia

“…Oligonucleotide-based therapeutics constitute a promising drug modality to treat diseases in a gene-specific manner. While generally well tolerated, these single-stranded oligonucleotides (ONs) that hybridize with cellular RNA targets sometimes are associated with clinical adverse effects including hepatotoxicity, kidney tubular toxicity or pro-inflammatory effects (injection site reactions and flu-like symptoms) [ 1 – 6 ]. The mechanism of these adverse effects is not fully understood, but mechanisms involving hybridization to off-target RNA sequences [ 2 ] or aptameric binding to proteins [ 7 , 8 ] have been shown to be contributing factors.…”