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Updated: Mar 15, 2026
A Reporter Based Cellular Assay for Monitoring Splicing Efficiency
Published on: September 15, 2021
Four Novel Splice-Switch Reporter Cell Lines: Distinct Impact of Oligonucleotide Chemistry and Delivery Vector on
Cristina S J Rocha1, Karin E Lundin1, Mark A Behlke2
11 Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital , Huddinge, Sweden .
Abstract:
New advances in oligonucleotide (ON) chemistry emerge continuously, and over the last few years, several aspects of ON delivery have been improved. However, clear knowledge regarding how certain chemistries behave alone, or in combination with various delivery vectors, is limited. Moreover, characterization is frequently limited to a single reporter cell line and, when different cell types are studied, experiments are commonly not carried out under similar conditions, hampering comparative analysis. To address this, we have developed a small "tissue" library of new, stable, pLuc/705 splice-switching reporter cell lines (named HuH7_705, U-2 OS_705, C2C12_705, and Neuro-2a_705). Our data show that, indeed, the cell type used in activity screenings influences the efficiency of ONs of different chemistry (phosphorothioate with locked nucleic acid or 2'-O-methyl with or without N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine). Likewise, the delivery method, Lipofectamine® 2000, PepFect14 nanoparticles, or "naked" uptake, also demonstrates cell-type-dependent outcomes. Taken together, these cell lines can potentially become useful tools for future in vitro evaluation of new nucleic acid-based oligomers as well as delivery compounds for splice-switching approaches and cell-specific therapies.
Insights
New reporter cell lines enable better evaluation of oligonucleotide delivery methods. This research highlights how cell type impacts the effectiveness of different oligonucleotide chemistries and delivery systems for splice-switching therapies.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Delivery
Background:
- Oligonucleotide (ON) chemistry and delivery have advanced, but understanding their performance across different cell types and delivery methods remains limited.
- Current characterization often uses single reporter cell lines or inconsistent conditions, hindering comparative analysis of ON behavior.
Purpose of the Study:
- To develop a standardized set of reporter cell lines for evaluating oligonucleotide activity and delivery across various cell types.
- To assess the influence of cell type and delivery method on the efficacy of different oligonucleotide chemistries for splice-switching applications.
Main Methods:
- Creation of a panel of four stable splice-switching reporter cell lines (HuH7_705, U-2 OS_705, C2C12_705, Neuro-2a_705).
- Testing the activity of various oligonucleotide chemistries (e.g., phosphorothioate with locked nucleic acid, 2 -O-methyl) using different delivery vectors (Lipofectamine® 2000, PepFect14, naked uptake).
Main Results:
- Cell type significantly impacts the efficiency of different oligonucleotide chemistries.
- Delivery methods show cell-type-dependent outcomes, affecting oligonucleotide performance.
- The developed cell lines provide a platform for cell-specific evaluation of ONs.
Conclusions:
- The established reporter cell lines are valuable tools for in vitro assessment of novel nucleic acid-based oligomers.
- These tools facilitate the evaluation of delivery compounds for splice-switching approaches and the development of cell-specific therapies.
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