Lipid-conjugated oligonucleotides via click chemistry efficiently inhibit hepatitis C virus translation

Article abstract of DOI:10.1021/jm800518u

Conjugation of a lipid moiety via click chemistry potentiates the cellular uptake of oligonucleotides and allows their intracellular delivery. These nontoxic lipid conjugates efficiently inhibit hepatitis C virus internal ribosome entry site (IRES)-mediated translation in human hepatic Huh7 cells. The biological activity of the lipid-conjugated oligonucleotides is not affected by the presence of serum.

Full text of DOI:10.1021/jm800518u

4374
J. Med. Chem. 2008, 51, 4374–4376
Lipid-Conjugated Oligonucleotides via “Click
Chemistry” Efficiently Inhibit Hepatitis C
Virus Translation
Guilhem Godeau,†,¶ Cathy Staedel,†,¶ and
Philippe Barthe´le´my*,†,¶
Inserm, U869, Bordeaux F-33076 France, and UniVersite´ de
Bordeaux, F-33076 Bordeaux, France
ReceiVed May 6, 2008
Abstract: Conjugation of a lipid moiety via “click chemistry” potenti-
ates the cellular uptake of oligonucleotides and allows their intracellular
delivery. These nontoxic lipid conjugates efficiently inhibit hepatitis
C virus internal ribosome entry site (IRES)-mediated translation in
human hepatic Huh7 cells. The biological activity of the lipid-
conjugated oligonucleotides is not affected by the presence of serum.
Figure 1. Synthesis of lipid-oligonucleotide conjugates (LONs): (i)
10 mol % CuSO4, 20 mol % sodium ascorbate, THF/H2O (50/50), 65
°C; (ii) 1.5 equiv of 2-cyanoethyl-N,N-diisopropylchlorophosphora-
midite, 2 equiv of DIEA, DCM, room temp; (iii) solid phase synthesis
with 1H-tetrazole as activator. Oligonucleotides sequences of ONs
(ON17mer and ON17mer3′F*) and LONs (5a-7b) have been selected
to specifically target the subdomain IIId (target) of the hepatitis C virus
RNA.
There is currently substantial interest in the use of nucleic
acids for modifying gene expression for therapeutic purposes.1
The use of oligonucleotide (ONa) analogues as potential
therapeutic agents for modulating the expression of specific
genes has shown promise for different classes of molecules
including aptamers, triplex-forming oligonucleotides, antisense
oligonucleotides, small interfering RNAs (siRNAs), and an-
tagomirs.2 However, one major barrier in the broad utilization
of ON analogues as clinical drugs is their reduced ability to
transverse cell membranes. Cellular uptake and localization of
ONs are crucial problems for their effects on the genetic
expressions. Most approaches to their cellular delivery involve
cationic lipophilic carriers and/or polymers.3 While such
synthetic carriers can be exceptionally effective for delivering
plasmid DNA into the cytoplasm of cells, most are of limited
utility because of their toxicity to cells and poor efficiency in
the case of ONs.4 Alternatively, a lipophilic moiety has been
covalently tethered to the ON structures with the aim of
improving cellular uptake and antisense activity.5 Despite the
different synthetic strategies developed for modification of the
oligonucleotides,6 the synthesis of the lipid-ON conjugates
(LON) is anything but trivial and requires extensive expertise
in organic chemistry and solid phase synthesis.
A real advance would be to use a bio-orthogonal conjugation
reaction that can be utilized for a large variety of lipid and ON
sequences. As a modular approach to reliable chemical trans-
formations, the Huisgen 1,3-dipolar cycloaddition of alkynes
and azides,7 a typical example of a “click” reaction,8 is
especially attractive for irreversibly coupling biomolecules with
ONs under mild conditions. Because of its biocompatibility,
“click” chemistry has recently emerged as an efficient strategy
for synthesizing labeled-carbohydrate ONs,9 peptides and protein
conjugates,10,11 fluorescent ONs,12 self-assembled monolayers
ON probes,13 circular ONs,14 and multimodified ONs.15
Herein, we covalently conjugated lipids moieties to an ON
by using a click chemistry reaction and evaluated the biological
activity of the conjugates on cultured human cells (Figure 1).
The ON sequence we chose is a 17-mer 2′-O-methylribonucle-
otide antisense (ON17mer, Figure 1) of the hepatitis C virus
(HCV) RNA, specifically targeting the subdomain IIId of the
internal ribosome entry site (IRES) at the 5′ end of the viral
RNA. The antisense efficiency of this ON was correlated to its
affinity for the IIId subdomain and to its ability to displace the
40S ribosomal subunit, leading to the inhibition of HCV-IRES-
dependent translation in cultured cells.16
We hypothesized that a lipid modification of a 2′-OMe
oligonucleotide, in particular the ON17mer (Figure 1), could
lead to an improvement of its bioavailability and, consequently,
enhancement of the antisense activity compared to the noncon-
jugated ON. To validate our approach, a family of LON
conjugates was investigated (5a-7b, Figure 1). Interestingly,
the LON conjugates were prepared in three steps starting from
alkyne-modified lipids derived from cholesterol 1a and octa-
decanol 1b (Figure 1). Briefly, 5′-azido-5′-deoxythymidine was
refluxed with propargyl lipids (1a and 1b) in water/THF in the
presence of sodium ascorbate and a catalytic amount of copper
sulfate. The resulting 1,3-dipolar cycloaddition reaction provided
the expected 1,2,3-triazole intermediates 3a, 3b, which were
first converted into the phosphoramidites in one step. The
phosphoramidites 4a, 4b were further coupled to the ON chain
using a classical solid support synthesis, in which the ON is
elongated in the 3′-5′ direction. This lipid modification
noticeably increased the lipophilicity of the oligonucleotides,
as judged by the retention times in reverse-phase (RP C4) HPLC
analysis, which were 9 and 23 min for the parent (ON17mer)
and the conjugated 5b oligonucleotides, respectively, under the
same experimental conditions (Supporting Information, Figure
SI6).
* To whom the correspondence should be addressed. Phone: 33 5 57 57
48 53. Fax: 33 5 57 57 10 15. E-mail: philippe.barthelemy@inserm.fr.
†
Inserm.
Universite´ de Bordeaux.
¶
a Abbreviations: DCM, methylene chloride; DIEA, diisopropylethy-
lamine; IRES, internal ribosome entry site; HCV, herpes virus C; Huh-7,
human hepatoma-7; LON, lipid conjugated oligonucleotide; ODN, oligode-
oxynucleotide; ON, oligonucleotide; THF, tetrahydrofurane; Tm, melting
temperature.
10.1021/jm800518u CCC: $40.75
2008 American Chemical Society
Published on Web 07/08/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4375
Figure 3. (a) Cell viability at different concentrations of LONs 5a
(blue), 5b (red), and 6a (yellow). (b) Inhibition (in %) of the HCV
IRES-dependent translation in the presence of different concentrations
of 5a (blue), 5b (red), and mismatch 6a (yellow).
Figure 2. (a) Percentage of fluorescent cells (Huh7) incubated in the
presence of 0.5 µM LONC183′F* (7a, blue) and LONChol3′F* (7b, red)
at 37 and 4 °C. (b-f) Fluorescence microscopy of Huh7 cells
co-incubated at 37 °C with 0.5 µM LONs or ONs. Cells co-incubated
with (i) 7a at 37 °C for 2 h (b) and 4 h (c), (ii) 7b for 4 h at 4 °C (e)
and 37 °C (f), (iii) ON17mer for 24 h at 37 °C (d).
cholesterol derivative 7b was not internalized to the cytoplasm
and was limited to the cellular membrane (Figure 2e), indicating
a strong affinity of the cholesterol moiety for the bilayers. These
results suggest that LON conjugates enter the cells via an
energy-dependent mechanism rather than by passive diffusion.
Next, we evaluated the cytotoxicity of the LON, assessing
the cell viability after a 4-day incubation of growing Huh7 cells
with increasing concentrations of LONs. Figure 3a indicates
that the LONs were neither toxic nor cytostatic for these cells
at any of the tested LON concentrations. Huh7 cell uptake of
the unconjugated ON17mer required transfecting reagents.
However, these formulations led to toxic effects (data not
shown). This observation highlights the interest of lipid
conjugation for achieving cell permeability.
Finally, the antisense activity of LONC18 (5a) and LONChol
(5b) was evaluated after transient transfection of a bicistronic
luciferase reporter mRNA, in which the second cistron transla-
tion depends on the HCV IRES,16 in Huh7 cells preincubated
with ONs or LONs in the presence of serum for 4 or 24 h.
Whereas the unconjugated antisense ON17mer was ineffective,
both lipid-conjugated 5a and 5b induced a dose-dependent
reduction of HCV IRES-dependent translation: 50% inhibition
(plateau) was reached with about 1 µM 5b and 2 µM 5a
preincubated for 4 h before the RNA target (Figure 3b). A 24 h
preincubation did not improve the antisense activity of the LONs
or the unconjugated ON (data not shown). This inhibition was
selective, as the mismatched 6a induced only a 5% decrease at
2 µM, and related to the lipid modification of the ON, since
the unconjugated ON (ON17mer) and the lipid (3a) added
simultaneously also were inactive (data not shown).
We first studied the thermal denaturation of the complemen-
tary duplexes ON17mer/ODN, 5a/ODN, and 5b/ODN by
measuring the absorbance at 260 nm in a cacodylate aqueous
buffers. Melting temperatures of roughly 70 °C were observed
for any complementary duplex (Supporting Information, Figure
SI9). As expected, with a Tm of 30 °C the mismatch sequence
LONC18mm (6a/ODN) induced a destabilization of the double
helix. The lipid modification appeared to have little or no impact
on the duplex stability and melting temperature values (Tm).
We next used fluorescence microscopy and flow cytometry
to determine whether the LON conjugates penetrated cultured
eukaryotic cells. Human hepatic Huh7 cells were incubated with
0.5 mM of 3′-fluorescein-labeled LONs or unconjugated ONs
for increasing periods of times at 37 and 4 °C (Figure 2). At
37 °C, fluorescein-labeled LONC183′F* (7a) was internalized
within 30 min and accumulated in the cytoplasm as a function
of the incubation time (Figure 2b,c). Cholesterol derivative
LONChol3′F* (7b) was quickly taken up by the cells at 37 °C
(Figure 2f and Figure SI10). By contrast, the nonlipidic labeled
ON17mer3′F* did not penetrate cells (Figure 2d) after 24 h of
incubation in the same conditions, indicating that efficient uptake
requires lipid modification of the ONs. Flow cytometry studies
confirmed the time-dependent uptake of the C18 lipid conjugate
and indicated that 7a (LONC183′F*) penetrated in 80% of cells
after 4 h (Figure 2a and Figure SI11). The mechanism by which
the LON conjugates enter cells is unknown. Recent results
obtained with cholesterol polyanionic derivatives17 suggest that
endocytosis is a possible pathway18 for LON conjugates.
Interestingly, the cellular uptake of the C18 lipid conjugate 7a
was dramatically decreased after incubation at 4 °C as shown
by flow cytometry (Figure 2a). Surprisingly, at 4 °C the
In conclusion, we have developed a straightforward procedure
for creating lipid-oligonucleotide conjugates. The incorporation
of a lipid moiety via a nonscissile triazole linker potentiates

4376 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Letters
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microscopy images. This material is available free of charge via
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JM800518U