In Vitro Toxicity Screening Assay for Oligonucleotides
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Solution Overview
Problem
Current methods for predicting the in vivo toxicity of oligonucleotides, such as antisense oligonucleotides, are inadequate, as in vitro cell-based assays are not predictive of in vivo toxicity, and existing in silico methods are limited in their ability to identify off-target effects and are not universally applicable across different oligonucleotide designs.
Innovation Solution
An in vitro toxicity assay using primary mammalian hepatocytes or hepatocytes derived from induced pluripotent stem cells, where oligonucleotides are administered and their toxicity is assessed by measuring biomarkers like LDH release and ATP levels, providing a predictive model for in vivo hepatotoxicity that is applicable regardless of oligonucleotide sequence, chemistry, or design.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If in vitro cell-based assays are used to predict oligonucleotide toxicity, then the use of animals in drug discovery is reduced, but the assays are not predictive of in vivo toxicity
Solution Approach 1:
The patent changes the cell type parameter from conventional cell lines to primary hepatocytes, and modifies the assay parameters by measuring specific biomarkers (ALT, AST, LDH, ATP) to achieve predictive accuracy for in vivo toxicity while maintaining reduced animal use
Solution Approach 2:
The patent creates an in vitro model that copies the liver's toxicological response by using primary hepatocytes and measuring biomarkers that mirror in vivo liver injury, thereby predicting human and animal toxicity without requiring actual in vivo experiments
2Reliability
If in silico bioinformatics algorithms are used to predict in vivo toxicity, then predictive accuracy improves, but the methods are limited to specific chemistry and designs
Solution Approach 1:
The patent develops a universal in vitro assay system using primary hepatocytes that can evaluate any oligonucleotide chemistry or design, making the methodology broadly applicable across different drug candidates without being restricted to specific molecular types
3Reliability
If in vivo toxicity screening is performed in rodents, then predictive data for human toxicity may be obtained, but animal welfare concerns and resource demands increase
Solution Approach 1:
The patent introduces primary hepatocytes as an intermediary system that bridges the gap between simple in vitro assays and complex in vivo models, providing human-relevant toxicity data without requiring animal experimentation
Data Source
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AI summary
The invention relates to methods for predicting the in vivo toxicity of oligonucleotides, such as antisense oligonucleotides using in vitro cell based assays based on gymnotically administering oligonucleotides to primary mammalian hepatocytes and subsequently measuring the levels of toxicity biomarkers such as the release of LDH into the cell culture media and/or intracellular ATP.