Targeted intracellular delivery of antisense oligonucleotides via conjugation with small-molecule ligands

Article abstract of DOI:10.1021/ja102635c

Selective delivery of antisense or siRNA oligonucleotides to cells and tissues via receptor-mediated endocytosis is becoming an important approach for oligonucleotide-based pharmacology. In most cases receptor targeting has been attained using antibodies or peptide-type ligands. Thus, there are few examples of delivering oligonucleotides using the plethora of small-molecule receptor-specific ligands that currently exist. In this report we describe a facile approach to the generation of mono- and multivalent conjugates of oligonucleotides with small-molecule ligands. Using the sigma-receptor ligand anisamide as an example, we describe conversion of the ligand to a phosphoramidite and direct incorporation of this moiety into the oligonucleotide by solid-phase DNA synthesis. We generated mono- and trivalent conjugates of anisamide with a splice switching antisense oligonucleotide (SSO) and tested their ability to modify splicing of a reporter gene (luciferase) in tumor cells in culture. The trivalent anisamide-SSO conjugate displayed enhanced cellular uptake and was markedly more effective than an unconjugated SSO or the monovalent conjugate in modifying splicing of the reporter. Significant biological effects were attained in the sub-100 nM concentration range.

Full text of DOI:10.1021/ja102635c

Published on Web 06/15/2010
Targeted Intracellular Delivery of Antisense Oligonucleotides via Conjugation
with Small-Molecule Ligands
Osamu Nakagawa, Xin Ming, Leaf Huang, and Rudolph L. Juliano*
DiVision of Molecular Pharmaceutics, Eshelman School of Pharmacy, UniVersity of North Carolina at Chapel Hill,
1072 Genetic Medicine Building, Chapel Hill, North Carolina 27599
Received April 12, 2010; E-mail: arjay@med.unc.edu
Scheme 1. Synthesis of 5′ Mono- and Trivalent
Targeted delivery is a key issue for the pharmacology of antisense
and siRNA oligonucleotides.1 Receptor-specific ligands can be
linked to various nanocarriers that contain oligonucleotides, or they
can be directly conjugated to the oligonucleotide itself. For example,
we recently showed that conjugation of a dimeric RGD (arginine-
glycine-aspartic acid) peptide, a high-affinity ligand for the integrin
Rvꢀ3, to an oligonucleotide (ON) increased cellular uptake via
receptor-mediated endocytosis and enhanced the biological effect
of the ON.2 Thus, direct mono- or multivalent ligand conjugation
to ONs seems an attractive strategy for enhancing receptor-specific
delivery of ONs to cells and tissues while using well-defined
chemical moieties. In this report, we describe the synthesis of mono-
and multivalent oligonucleotide conjugates using anisamide, a high-
affinity ligand for sigma receptors, and evaluate the function of
these conjugates in tumor cells in culture.
Anisamide-Conjugated Oligonucleotidesa
Sigma receptors (σ1, σ2) are transmembrane proteins, found on
the endoplasmic reticulum and on plasma membranes, that seem
to play a role in regulating ion channels.3 High-level expression of
sigma receptors has been observed for a diverse set of human and
rodent tumor cell lines.4 Small molecules such as haloperidol,
SA4503, and opipramol have been reported as sigma-receptor
ligands.3 Several σ1 ligands have been developed as radioimaging
agents for tumors and successfully tested in ViVo.5 These observa-
tions suggested that sigma-receptor ligands could also be used for
targeted drug delivery. Huang and colleagues have reported that
the high-affinity sigma-receptor ligand anisamide, when conjugated
to lipid nanocarriers, could be used to deliver doxorubicin6 or
siRNA7 to tumors in animals. Mukherjee et al. reported that
haloperidol conjugated lipoplexes showed 10-fold greater delivery
of DNA to breast carcinoma cells than did control lipoplexes.8
For liposome-conjugated anisamide [N-(2-aminoethyl)-4-meth-
oxybenzamide], the 4-methoxybenzamide unit itself is very im-
portant for binding activity to sigma receptor. The aminoethyl
moiety, especially the terminal nitrogen, is also important.6,9
Interestingly, DeSimone et al. developed a new anisamide ligand
that used a modification with an oxygen atom, instead of a nitrogen
atom, in the aminoethyl moiety.10
With the intent of establishing an efficient strategy for
ligand-oligonucleotide conjugation, we designed the direct incor-
poration of anisamide ligand into antisense oligonucleotides using
a DNA synthesizer. Thus, we used N-[2-(2-hydroxyethoxy)ethyl]-
4-methoxybenzamide (1) for direct conjugation to ONs. This allows
solid-phase synthesis of anisamide-ON conjugates, since a protec-
tive group is not needed during DNA synthesis (Scheme 1 and
Supporting Information). The phosphitylation of the anisamide 1
successfully afforded the phosphoramidite 2. The anisamide phos-
phoramidite 2 was incorporated into oligonucleotides using con-
ventional phosphoramidite chemistry on an automated DNA
synthesizer. For the monoanisamide conjugate, 2 was directly
incorporated at the 5′-terminal of the ON. The trivalent conjugate
a ON (623): 5′-GTT ATT CTT TAG AAT GGT GC-TAMRA-3′ (2′-
O-Me-RNA with phosphorothioate backbone). Reagents and conditions:
(a) iPr2NP(Cl)OCH2CH2CN, iPr2NEt, CH2Cl2; (b) DNA synthesizer, HPLC
purification.
was synthesized using a three-branched linker, readily introduced
into the 5′-terminal of the ON. The facile synthesis described here
should be readily applicable to various other types of oligonucle-
otides such as siRNA, miRNA, triplex-forming oligonucleotides,
and molecular decoys.
To evaluate the biological activity of the conjugates, we utilized
sigma-receptor-expressing cells (PC3, human prostate carcinoma
cells) that contain a luciferase reporter gene interrupted by an
abnormal intron that prevents expression of functional luciferase
protein.11 Upon adequate delivery of an appropriate splice-switching
antisense oligonucleotide (an SSO, here designated ON 623) to the
nucleus, the intron is spliced out and luciferase is expressed.11 The
623 ONs were 2′-O-methyl-RNA with a phosphorothioate backbone
and a 3′-TAMRA fluorophore. Uptake of the fluorescent ONs was
monitored in cells by flow cytometry.2 As shown in Figure 1A,
there was slightly higher uptake of the monoanisamide conjugate
as compared to the unconjugated ON control, while the trivalent
anisamide conjugate showed significantly greater uptake than
control, possibly reflecting greater avidity of the multivalent version
for the sigma receptor. The increased uptake could be partially
blocked by co-incubation with excess free haloperidol (Figure 1B),
a strong sigma-receptor antagonist. Since the overall uptake process
likely involves both receptor-mediated endocytosis and nonspecific
fluid-phase pinocytosis, it is expected that the blocking effect of
an antagonist would be partial.
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10.1021/ja102635c  2010 American Chemical Society

C O M M U N I C A T I O N S
The biological effect (luciferase induction) paralleled the uptake
data but was more pronounced. The trivalent conjugate displayed
a significantly greater effect than the monovalent compound or the
unconjugated control (Figure 1C). Consistent with our previous
observations,2 the biological effects of oligonucleotides entering
cells by a receptor-mediated process seem to be greater than those
of oligonucleotides entering by pinocytosis. Thus, as compared to
the unmodified oligonucleotide, the trivalent conjugate displayed
an approximately 2-fold increase in uptake but a 4-fold increase in
luciferase induction. No toxicities were apparent with use of these
conjugates, as judged by the retention of normal cell morphology
and lack of effect on total cell protein recovered.
the ligand to a phosphoramidite followed by conventional solid-
phase ON synthesis. The multivalent anisamide conjugate signifi-
cantly enhanced receptor-specific cell uptake and biological effect.
While there has been considerable work on peptide-oligonucleotide
conjugates,12 relatively little has been done with oligonucleotides
linked to small organic molecules. The novel conjugation approach
described here should create opportunities to utilize a variety of
highly selective small-molecule ligands to target various receptor
types, including members of the vast G-protein-coupled receptor
family,13 thus enhancing possibilities for receptor-selective ON
delivery to a wide variety of cells and tissues.
Acknowledgment. This work was supported by a grant from
the NIH (PO1GM 26599) to R.L.J.
Supporting Information Available: Synthesis of mono- and
trivalent conjugated oligonucleotides and cell culture experiments. This
material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 1. (A) Initial cellular uptake. Cells were treated with 50 nM
monoanisamide-623-TAMRA, trianisamide-623-TAMRA, or 623-TAMRA
for 4 h in OptiMEM at 37 °C. The cells were rinsed in buffered saline
solution and then trypsinized. Total cellular uptake of the TAMRA-labeled
conjugate was measured by flow cytometry. (B) Effect of sigma-receptor
inhibitor on initial uptake. Cells were treated with 50 nM mono- or
trianisamide-623-TAMRA in the absence or in the presence of 50 µM
haloperidol. After 4 h, the cellular uptake was measured by flow cytometry.
Results are normalized on the basis of cells receiving no inhibitor as 100%.
(C) Luciferase induction. Cells were treated with monoanisamide-623-
TAMRA, trianisamide-623-TAMRA, or 623-TAMRA for 24 h, and
luciferase activity was determined 48 h after treatment. Results are the means
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Thus, we have demonstrated the facile production of mono- and
trivalent anisamide-conjugated oligonucleotides via conversion of
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