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Updated: Aug 19, 2026
Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method
Published on: September 16, 2017
Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA
B H Lloyd1, R V Giles, D G Spiller
1Clatterbridge Cancer Research Trust, J. K. Douglas Research Laboratories, Clatterbridge Hospital, Bebington, Wirral CH63 4JY, UK. bryonyl@ccrt.co.uk
Abstract:
Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFalpha mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.
Insights
Antisense oligonucleotides (ASOs) targeting TNFalpha mRNA were optimized using in vitro selection. This method effectively identified optimal ASO lengths and sequences for gene silencing, reducing mRNA levels by up to 19.8%.
Area of Science:
- Molecular Biology
- Gene Regulation
- Oligonucleotide Therapeutics
Background:
- Antisense oligonucleotides (ASOs) are valuable tools for studying gene function and developing therapeutics.
- Predicting effective ASO binding sites remains a challenge, impacting their utility in target validation and clinical applications.
Purpose of the Study:
- To compare the efficacy of ASOs selected in vitro using random combinatorial libraries against specific target sites in TNFalpha mRNA.
- To investigate the complex relationship between target site accessibility, oligonucleotide length, and ASO efficacy.
- To establish a predictive method for identifying and optimizing ASO activity against any given mRNA sequence.
Main Methods:
- In vitro selection of ASOs using random combinatorial oligonucleotide libraries of varying lengths and complexities.
- Assessment of ASO efficacy on TNFalpha mRNA in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells.
- Application of methylphosphonate:phosphodiester chimaeric oligonucleotides to evaluate mRNA reduction.
Main Results:
- ASO efficacy was complexly related to target site accessibility and oligonucleotide parameters.
- Specific target sites showed a preference for ASOs of a defined optimal length.
- In vitro selected ASO sequences demonstrated efficacy comparable to those observed in PMA-activated U937 cells.
- Chimaeric ASOs reduced TNFalpha mRNA levels by up to 19.8% compared to untreated cells.
Conclusions:
- In vitro selection of ASOs provides a predictive approach for identifying optimal sequences and lengths for gene silencing.
- This method allows for the profiling of any mRNA to identify sites accessible to ASO activity.
- The findings support the use of ASOs as a therapeutic strategy for modulating gene expression.
