Fatty Acid-Modified Gapmer Antisense Oligonucleotide and Serum Albumin Constructs for Pharmacokinetic Modulation

Abstract

Delivery technologies are required for realizing the clinical potential of molecular medicines. This work presents an alternative technology to preformulated delivery systems by harnessing the natural transport properties of serum albumin using endogenous binding of gapmer antisense oligonucleotides (ASOs)/albumin constructs. We show by an electrophoretic mobility assay that fatty acid-modified gapmer and human serum albumin (HSA) can self-assemble into constructs that offer favorable pharmacokinetics. The interaction was dependent on fatty acid type (either palmitic or myristic acid), number, and position within the gapmer ASO sequence, as well as phosphorothioate (PS) backbone modifications. Binding correlated with increased blood circulation in mice (t increased from 23 to 49 min for phosphodiester [PO] gapmer ASOs and from 28 to 66 min for PS gapmer ASOs with 2× palmitic acid modification). Furthermore, a shift toward a broader biodistribution was detected for PS compared with PO gapmer ASOs. Inclusion of 2× palmitoyl to the ASOs shifted the biodistribution to resemble that of natural albumin. This work, therefore, presents a novel strategy based on the proposed endogenous assembly of gapmer ASOs/albumin constructs for increased circulatory half-life and modulation of the biodistribution of gapmer ASOs that offers tunable pharmacokinetics based on the gapmer modification design.

The how and why of lncRNA function: An innate immune perspective

“…Gapmers offer the benefit of modifications for stability and reduced toxicity, while still allowing engagement of the RNase H pathway (Fig. 3B) [159].…”

Palmitoylated phosphodiester gapmer designs with albumin binding capacity and maintained in vitro gene silencing activity

“…The approach exploits the natural ligand binding and transport properties of albumin as a result of interaction with fatty acid‐modified drug molecules. We showed that palmitoylated gapmers can strongly bind to serum albumin and exhibit a prolonged serum half‐life . An in‐depth investigation into the effect of gapmer designs on gene silencing, however, was not conducted.…”

“…A typical gapmer contains a short central block of antisense DNA nucleotides for activating RNase H‐mediated mRNA degradation, flanked at each end by modified nucleotides such as locked nucleic acid (LNA), 2′‐ O ‐methyl or 2′‐ O ‐methoxyethyl nucleotides for increased stability . Gapmer modifications have been focused toward maximizing silencing activity and a prolonged serum half‐life by inclusion of α‐ l ‐LNA 3 and N2′‐palmitoylated 2′‐amino‐LNA nucleotides (Figure ) . A large number of antisense studies have utilized phosphorothioate (PS) gapmers.…”

“…Previous work from our laboratory has introduced a novel systemic gapmer nanoconstruct delivery platform based on endogenous assembly with human serum albumin (HSA) . The approach exploits the natural ligand binding and transport properties of albumin as a result of interaction with fatty acid‐modified drug molecules.…”

Synthesis of Oligonucleotides Containing 2′-N-alkylaminocarbonyl-2′-amino-LNA (2′-urea-LNA) Moieties Using Post-Synthetic Modification Strategy

“…In particular, 2′amino-LNA, a 2′,4′-bridged nucleic acid, can have various substituents of the 2′-amino group [14]; therefore, 2′-amino-LNA would be a useful scaffold to explore oligonucleotides possessing the desired properties. Previous studies have reported oligonucleotides containing 2′-N-substituted 2′amino-LNA derivatives, such as 2′-N-alkyl, 2′-N-acyl, and 2′-N-alkoxycarbonyl derivatives [15][16][17][18][19][20][21][22]. In general, the synthesis was based on a common method using each modified phosphoramidite; however, post-synthetic approaches using click chemistry [20,21,23] and amidation [24] were also applied to the synthesis of the 2′-N-substituted 2′-amino-LNA derivatives in the oligonucleotides (Figure 1).…”