With newly developed antisense oligonucleotide (ASO) molecules, early efficacy and toxicity assessment is crucial to prevent costly failures in later stages. But, what are the assays that will give you the best predictions to advance your drug candidate with confidence?

Here you can find 5 key steps you need to consider to successfully develop an ASO in vitro screening assay. Read till the end to discover the extra-advantage of designing a physiologically relevant assay that can shorten your timelines.

Antisense oligonucleotides (ASOs) are a short, synthetic nucleic acid molecule that can bind to specific RNA sequences and can thereby modulate gene expression. This modulation can happen either through inducing degradation of the complimentary sequence, blocking translation or modulating splicing.

This trait can be incredibly valuable in the treatment of genetic disorders and hereditable diseases by either reducing target RNA transcript levels or restoring protein function.

Actually, some ASO therapeutics already made it to the market: Vitravene (Cytomegalovirus (CMV) retinitis), Kynamro (Familial Hypercholesterolemia), Tegsedi (TTR Polyneuropathy) and Waylivra (Familial Chylomicronemia Syndrome).

Nonetheless, safety concerns due to off target effects and limited efficacy are obstacles that need to be overcome, to deliver on the potential of ASOs as an effective therapeutic.

To enhance the likelihood of achieving late-stage success, early adoption of relevant cell-based in vitro screening assays can significantly influence subsequent outcomes.

Delve into 5 essential considerations to design relevant cell-based in vitro assays that empower you to obtain more accurate predictions regarding ASO efficacy and safety.

How to Design a Successful Cell-based In Vitro Assay for
ASO Screening

1. Choosing a Cell Model Relevant to Your Target Disease For the development of an in vitro assay, the basis lies on the selection of the right cell model. In principle, any cell line can be used to develop an assay, but you should look for the one that best fits your research questions and goals.

Each cell model has different (dis)advantages which you must know in advance to be able to get a balance between its physiological relevance, genetic stability, availability, reproducibility, scalability and cost.

One of the most common models used are immortalised cells. The advantage of an immortalised cell is, as the name suggests, the unlimited proliferative capacity.

However, they are not genetically stable and lack the capacity to replicate important cell functions or disease phenotypes. This considerably limits the options of getting accurate predictions for efficacy and toxicity.

Another option is to use primary human cells, which offer a high physiological relevance and enable researchers to mimic a diseased condition in vitro.

The disadvantage of this model is the limited proliferation capacity which hinders their application in primary screenings where large cell batches are required. Primary cells also present inter-donor variability, reducing the robustness of the assay, especially in the context of high-throughput screening.

Overall, primary human cells can be a valuable addition for validation studies where the need for scalability is much lower.

As an alternative, induced pluripotent stem cells (iPSCs) can be a useful tool in many cases, especially when investigating diseases where primary material is difficult to obtain, such as heart and brain related diseases. Availability of primary cells for those tissues is particularly limited, but iPSCs can be differentiated into neural and cardiac cells, as well as any body cell type.

Additionally, iPSCs have unlimited proliferation capacity and genetically stable. They can be patient-specific and recapitulate main disease phenotypes in vitro. To further enhance the relevance of the model and its translatability, different cell types can be cocultured to build more representative environments.

With the right expertise, iPSCs can be differentiated at a large scale producing one single source of cells for the full screening cascade, considerably reducing the variability of your results.

As there is no perfect model, the downside of iPSCs is the required technical expertise to develop robust differentiation procedures, produce large batches and model disease phenotypes in the context of miniaturised assays. Therefore, many researchers partner with expert companies to accelerate pipelines and save resources.