Abstract
Interactions of chemically modified nucleic acid therapeutics with plasma proteins play an important role in facilitating distribution from the injection site to peripheral tissues by reducing renal clearance. Despite the importance of these interactions, analytical methods that can characterize binding constants with individual plasma proteins in a reliable and high throughput manner are not easily available. We developed a fluorescence polarization (FP) based assay and measured binding constants for the 25 most abundant human plasma proteins with phosphorothioate (PS) modified antisense oligonucleotides (ASOs). We evaluated the influence of sequence, sugar modifications, and PS content on ASO interactions with several abundant human plasma proteins and determined the effect of salt and pH on these interactions. PS ASOs were found to associate predominantly with albumin and histidine-rich glycoprotein (HRG) in mouse and human plasma by size-exclusion chromatography. In contrast, PS ASOs associate predominantly with HRG in monkey plasma because of higher concentrations of this protein in monkeys. Finally, plasma proteins capable of binding PS ASOs in human plasma were confirmed by employing affinity chromatography and proteomics. Our results indicate distinct differences in contributions from the PS backbone, nucleobase composition and oligonucleotide flexibility to protein binding.
Highlights
The phosphorothioate (PS) backbone modification, where one of the non-bridging oxygen atoms of the phosphodiester (PO) linkage is replaced with sulfur, represents one of the most widely used chemical modifications in nucleic acid therapeutics [1]
We recently reported that PS antisense oligonucleotides (ASOs) show 2fold improved activity in ␣-2-macroglobulin knockout mice suggesting that interactions with specific plasma proteins can shuttle ASOs into non-productive cellular compartments [10]
A method which allows for characterization of interactions of PS ASOs with individual plasma proteins from the different species used for preclinical development of nucleic acid therapeutics could be beneficial
Summary
The phosphorothioate (PS) backbone modification, where one of the non-bridging oxygen atoms of the phosphodiester (PO) linkage is replaced with sulfur, represents one of the most widely used chemical modifications in nucleic acid therapeutics [1]. While significant progress has been made recently towards understanding cellular uptake of ASOs, including the characterization of several cell surface receptors [6,7,8], it remains unclear what contribution plasma proteins have for ASO tissue distribution beside limiting glomerular filtration and urinary excretion [9]. Interactions of PS ASOs with specific plasma proteins can modulate hematological toxicities in the plasma compartment in a species-dependent manner [11]. Given this background, a method which allows for characterization of interactions of PS ASOs with individual plasma proteins from the different species used for preclinical development of nucleic acid therapeutics could be beneficial. Methods to characterize interactions of PS ASOs with plasma proteins have largely been limited to filter binding assays and size exclusion chromatography which do not inform on interactions with individual proteins [12]
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