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_1/2 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.

Keywords: albumin; antisense gapmer oligonucleotides; biodistribution; palmitoyl; pharmacokinetics.

Figure 1

Chemical Structure of the N2′-Functionalized Amino-LNA Monomers B, thymin-1-yl or 5-methylcytosin-1-yl; R, palmitoyl or myristoyl; X, O (PO, phosphodiester linkage) or S (PS, phosphorothioate linkage).

Figure 2

Polyacrylamide Gel Electrophoresis of Gapmer ASO/HSA Constructs Upper panels show recombinant human serum albumin (rHSA)-bound gapmer antisense oligonucleotides (ASOs); lower panels show naked gapmer ASOs. Gapmer ASOs without (left) or with (right) phosphorothioate (PS) backbone linkages are shown. (A) No palmitoylated modifications. (B) From top to bottom: 1× 5′-palmitoylated modification, 1× 3′- and 1× 5′- palmitoylated modifications, 2× 3′-palmitoylated modifications, 1 × 5’-myristoylated modification, and 1× 3′- and 1× 5′- myristoylated modifications myristoylated modifications. Gapmer ASOs are visualized by SYBR gold staining. Illustrations depict the different gapmer ASO designs: phosphodiester (PO) backbone is represented by a thin black line, whereas PS backbone is represented by a thick black line. LNA is depicted by black dots, palmitic acid is represented by black ovals, and myristic acid is represented by hollow ovals. For an example of a whole gel, refer to Figure S1.

Figure 3

Plasma Levels of Cy5.5 Fluorescent Gapmer Antisense Oligonucleotides at Selected Time Points following Intravenous Injection in C57BL/6 Mice Fluorescence was quantified using Living Image (Perkin Elmer), and the amounts normalized to the levels after 1 min. Inset in top right corner is a magnification of the first 4 hr. Data are presented as means with SD (n = 5).

Figure 4

Organs from Mice Scanned in the IVIS Scanner 24 hr after Injection of PO, PS, 2× Palmitoyl PO, or 2× Palmitoyl PS-Modified Gapmer ASOs (A–F) Quantification of fluorescence from isolated organs scanned in the In Vivo Imaging Service (IVIS) scanner, 24 hr after injection. The fluorescence intensity was normalized to the signal from the phosphorothioate (PS) antisense oligonucleotide (ASO)-treated mouse kidney and presented as means with SD (n = 5). (A) Kidneys, spleen, liver, lung, and heart. (B) Kidneys. (C) Spleen. (D) Liver. (E) Lung. (F) Heart. PO, phosphodiester.