Background: High von Willebrand factor (VWF) plasma levels are associated with arterial thrombosis. Current antiplatelet therapy to prevent arterial thrombosis increases bleeding risk and often fails to prevent thrombosis. An alternative therapeutic approach would be to lower VWF by allele-selective silencing of VWF. This approach averts complete knockdown of VWF and thus minimizes bleeding risk.
Aims: The aims of this study are to identify small interfering RNAs (siRNAs) that can distinguish between strain-specific differences in murine Vwf to be used in allele-selective knockdown studies in heterozygous mouse models.
Methods: Two commonly used mouse inbred strains, C57BL/6J and 129S1/SvImJ, were selected based on genetic differences between their Vwf genes and comparable plasma VWF levels. siRNAs were designed to target one or two of 11 genetic differences between these mouse strains. In silico analysis predicted 14 siRNAs to be active, meaning that they effectively inhibit either of the strain-specific Vwf alleles. All selected siRNAs were identically chemically modified to increase stability, which included 2’O-Methyl and phosphorothioate backbone modifications. Activity and allele/strain-selectivity of these siRNAs were determined, dose-dependently, in HEK293 cells transiently expressing either C57BL/6J or 129S1/SvImJ Vwf or both.
Results: 7 out of 14 siRNAs effectively inhibited the targeted allele (≥80% at 1 nM siRNA), with minimal inhibition of the untargeted allele. Of the other siRNAs, two showed allele-selective inhibition of approximately 70%, three showed non-selective inhibition of both alleles (at least 60%), and two showed limited inhibitory effect on both alleles. Two lead candidates were chosen based on strong inhibitory activity (>80%), strain-selectivity and ability to target one nucleotide difference.
Conclusions: We have identified strain-selective siRNAs that can distinguish between C57BL/6J and 129S1/SvImJ Vwf based on one or two nucleotide(s) difference between their Vwf genes. The selected lead compounds will be tested in F1 hybrids of cross-bred C57BL/6J and 129S1/SvImJ mice.
To cite this abstract in AMA style:Jongejan YK, Dirven RJ, de Jong A, van Vlijmen BJM, Eikenboom JCJ. Identification of Small Interfering RNAs for Allele-selective Silencing of Murine von Willebrand Factor [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 1). https://abstracts.isth.org/abstract/identification-of-small-interfering-rnas-for-allele-selective-silencing-of-murine-von-willebrand-factor/. Accessed September 24, 2021.
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