Table 1 Summary of NAT efficacy models and key controls that need to be considered during NAT efficacy screening
From: Model selection in preclinical nucleic acid therapeutics research
Advantages/Disadvantages | Key controls | |
|---|---|---|
Immortalized cell lines | + Accessibility + Ease of use + Endogenous target/native context factors + Suitable for medium- to high-throughput screening by transfection, gymnosis or electroporation - Target may not be (sufficiently) expressed - Translational relevance is not guaranteed | • Confirmation of target expression (e.g., GTEx searches, short/long-read RNA-seq)29 • Negative control NAT(s) (e.g., non-targeting and mismatch controls)21,123 • Positive control NAT(s) (e.g., to confirm cellular uptake and viability)21 |
Mini-gene assays | + Accessibility + Ease of use + Suitable for medium- to high-throughput screening in immortalized cell lines via transfection - Exogenous target/no native context factors | • Confirmation that the (mis)splicing event of interest is recapitulated52 • Negative control NATs (e.g., non-targeting and mismatch controls)21,123 • Positive control NAT(s) (e.g., to confirm cellular uptake and viability)21 |
Reporter assays | + Accessibility + Ease of use + Suitable for medium- to high-throughput screening in immortalized cell lines via transfection or gymnosis in stable cell lines - Exogenous target/no native context factors - Relies on a proxy (i.e., luciferase enzyme activity) for RNA target levels | • Negative control NATs (e.g., non-targeting and mismatch controls)21,123 • Positive control NAT(s) (e.g., to confirm cellular uptake and viability)21 • Measurements on a second luciferase to account for variation in transfection efficiency and cell viability39 |
Patient-derived cells | + Endogenous target/patient-specific native context factors + Possible to reprogram to specific cell types - Not always accessible for practical and/or ethical reasons - Less user-friendly than immortal cell lines | • Immunocytochemistry/labelled NATs to quantify uptake and distribution69 • Control cell lines (e.g., parental/carrier or isogenic)124 • Cells from multiple patients carrying the same variant (e.g., confirm on-target/functional effects)66 • Multiple iPSC lines/differentiations from the same donor (e.g., to demonstrate reproducibility)124 |
3-D cell models | + Better able to model complex cellular and multicellular processes than 2-D systems + Possible to study hard-to-access cell types/systems + Helps meet the demand to replace animals - Time and cost to generate complex models (e.g., assembloids/multi-tissue systems) - Suitable for low- to medium-throughput screening | • Immunocytochemistry/labelled NATs to quantify uptake and distribution69 • Internal control cell-type markers for culture reproducibility and comparative composition121 • Multiple iPSC lines/differentiations from the same donor (e.g., to demonstrate reproducibility)124 |
Rodent models | + Whole mammalian system + Possible to interrogate biodistribution and toxicology + Availability of established and well-characterized disease models + Surrogate NATs in wild-type rodents enable PK/PD studies/NAT screening - Human gene/locus is not always conserved - Time and cost to generate humanized models - Physiology of rodents may not recapitulate disease/no model may be available - Class-specific toxicology profiles may differ to human - Ethical concerns | • Human wild-type knock-in transgenic line as a comparator for human variant knock-in transgenic line125 • Confirmation of RNA and protein expression (if applicable) and functionality of exogenous humanized (trans)gene96 • Quantification of NAT biodistribution versus efficacy (e.g., PET/radiolabelling, fluorescent-based imaging, mass spectrometry, immunohistochemistry, molecular methods)126 • Diligent experimental design and reporting127 |
Large animal models | + Whole mammalian system/native context factors + Possible to interrogate biodistribution and toxicology + Anatomy and physiology can have more relevance to humans’ than rodents - Cost to generate and maintain models - Ethical concerns | • Human wild-type knock-in transgenic line as a comparator for human variant knock-in transgenic line125 • Confirmation of RNA and protein expression (if applicable) and functionality of exogenous humanized (trans)gene101 |
