Targeted delivery of antisense oligonucleotides by chemically self-assembled nanostructures

Abstract

Synthetic nucleic acids have shown great potential in the treatment of various diseases. Nevertheless, the selective delivery to a target tissue has proved challenging. The coupling of nucleic acids to targeting peptides, proteins, and antibodies has been explored as an approach for their selective tissue delivery. Nevertheless, the preparation of covalently coupled peptides and proteins that can also undergo intracellular release as well as deliver more than one copy of the nucleic acid has proved challenging. Recently, we have developed a novel method for the rapid noncovalent conjugation of nucleic acids to targeting single chain antibodies (scFv) using chemically self-assembled nanostructures (CSANs). CSANs have been prepared by the self-assembly of two dihydrofolate reductase molecules (DHFR(2)) and a targeting scFv in the presence of bis-methotrexate (bis-MTX). The valency of the nanorings can be tuned from one to eight subunits, depending on the length and composition of the linker between the dihydrofolate reductase molecules. To explore their potential for the therapeutic delivery of nucleic acids as well as the ability to expand the capabilities of CSANs by incorporating smaller cyclic targeting peptides, we prepared DHFR(2) proteins fused through a flexible peptide linker to cyclic-RGD, which targets αvβ3 integrins, and a bis-MTX chemical dimerizer linked to an antisense oligonucleotide (bis-MTX-ASO) that has been shown to silence expression of eukaryotic translation initiation factor 4E (eIF4E). Monomeric and multimeric cRGD-CSANs were then prepared with bis-MTX-ASO and shown to undergo endocytosis in the breast cancer cell line, MDA-MB-231, which overexpresses αvβ3. The bis-MTX-ASO was shown to undergo endosomal escape resulting in the knock down of eIF4E with at least the same efficiency as ASO delivered by oligofectamine. The modularity, flexibility, and common method of conjugation may prove to be a useful general approach for the targeted delivery of ASOs, as well as other nucleic acids to cells.

Figure 1

(a) Scheme for the formation of Oligo functionalized monomeric (upper) and multimeric (lower) nanostructures. DHFR²RGD4C contains two DHFR proteins (gray) and cRGD peptide (blue ring with 3 red dots). Bis-MTX-ASO has a bis-MTX, shown in green, attached to the ASO (blue). Size exclusion chromatography (SEC) trace of 13DHFR²RGD4C (b) or 1DHFR²RGD4C (c) alone (black) and after incubation with bis-MTX-ASO (red)

Figure 2

Subcellular localization by Confocal microscopy: (a) MDA-MB-231 cells: differential interference contrast (first column), FITC fluorescence confocal channel (second column) and overlay of both channels (third column) images for bis-MTX-ASO-FITC incubated with either 1DHFR²RGD4C (upper panel) or 13DHFR²RGD4C (lower panel) at 37 °C. (b) FITC fluorescence channel for 1DHFR²RGD4C nanostructures (first column), cholera toxin Alexa Fluor 594 (second column), overlay of green and red channel (third column).

Figure 3

Translational reporter assay data with cell viability (grey bars) and % expression (white bars) of luciferase in MDA-MB 231 cells at 48 hr, no treatment a; 1DHFR²RGD4C, b; 13DHFR²RGD4C, c; Bis-MTX-ASO, d; ASO with Oligofectamine, e; 0.5 μM multimer, f; 1.0 μM multimer, g; 0.5 μM monomer, h; 1.0 μM monomer, i.

Figure 4

Western blotting data with cell viability (grey bars) and % expression of eIF4E/ actin (white bars) of MDA-MB-231 cells at 48 hr, a; 1DHFR²RGD4C, b; 13DHFR²RGD4C, c; Bis-MTX-ASO, d; 1.0 μM multimer scrambled, e; 1.0 μM monomer scrambled, f; 1.0 μM ASO with Oligofectamine, g; 1.0 μM multimer, h; 1.0 μM monomer, i. (* p > 0.01)