Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells

Article abstract of DOI:10.1016/j.bmcl.2004.07.018

Double-stranded short interfering RNAs (siRNAs) mediate post-transcriptional inhibition of gene expression in a variety of biological systems. However, human liver cells show poor uptake of these nucleic acids. In order to improve the delivery of siRNA into these cells without transfection agents, we have synthesized two series of lipophilic siRNAs conjugated with derivatives of cholesterol, lithocholic acid or lauric acid. The lipid moieties were covalently linked to the 5′-ends of the RNAs using phosphoramidite chemistry. The potency of these chemically modified siRNAs to inhibit reporter gene expression was further investigated in vitro with β-galactosidase expressing liver cells.

Full text of DOI:10.1016/j.bmcl.2004.07.018

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4975–4977
Steroid and lipid conjugates of siRNAs to enhance cellular
uptake and gene silencing in liver cells
a
a
Christina Lorenz,a Philipp Hadwiger,a, Matthias John, Hans-Peter Vornlocher and
*
Carlo Unverzagtb
aAlnylam Europe AG, Fritz-Hornschuch-Str. 9, D-95326 Kulmbach, Germany
bLehrstuhl fu¨r Bioorganische Chemie, Universita¨t Bayreuth, D-95440 Bayreuth, Germany
Received 27 May 2004; revised 7 July 2004; accepted 8 July 2004
Available online 29 July 2004
Abstract—Double-stranded short interfering RNAs (siRNAs) mediate post-transcriptional inhibition of gene expression in a variety
of biological systems. However, human liver cells show poor uptake of these nucleic acids. In order to improve the delivery of si-
RNA into these cells without transfection agents, we have synthesized two series of lipophilic siRNAs conjugated with derivatives of
cholesterol, lithocholic acid or lauric acid. The lipid moieties were covalently linked to the 50-ends of the RNAs using phosphoram-
idite chemistry. The potency of these chemically modified siRNAs to inhibit reporter gene expression was further investigated in
vitro with b-galactosidase expressing liver cells.
ꢀ 2004 Elsevier Ltd. All rights reserved.
The phenomenon of RNA interference was first discov-
ered by Fire et al.1 and describes post-transcriptional
gene silencing mediated by long double-stranded RNAs.
During RNA interference these RNAs are processed
into small fragments of approximately 22 nucleotides
by the enzyme complex Dicer.2,3 The so-termed short
interfering RNAs (siRNA) get unwound and are then
incorporated into the RNA-induced silencing complex
(RISC), which uses the single strands to search for com-
plementary mRNA sequences.4 Finally, the homologous
mRNA is cleaved by RISC and further degraded by cel-
lular nucleases, resulting in sequence specific inhibition
of gene expression. As long double-stranded RNAs in-
duce a nonspecific interferon response in mammalian
cells, chemically synthesized siRNAs with 19–24 nucleo-
tides in length provide a possibility to bypass Dicer
processing and thus to induce RNA interference in high-
er biological systems, especially in human cells.5,6
moieties may enhance siRNA uptake via a receptor-
mediated mechanism or by an increased membrane per-
meability of the otherwise negatively charged RNA.
In this study we describe the synthesis of lipophilic si-
RNA conjugates. The cholesterol derivative (Chol 67)
was chosen for modification, as Manoharan and co-
workers have shown an increased uptake of choles-
terol-modified antisense oligonucleotides in liver cells.8
Furthermore, derivatives of lithocholic acid (LithoP 3
and pLitho 5) and 12-hydroxy lauric acid (Laurin 9
and C32 11) were synthesized. The lithocholic acid
derivatives 3 (LithoP) and 5 (pLitho) used in this study
were obtained as outlined in Scheme 1. A C6-linker was
introduced by chemoselective coupling of 6-aminohexa-
nol to acetylated lithocholic acid 19 with ethyl-dimethyl-
aminopropyl-carbodiimide hydrochloride (EDCl) and
p-dimethylaminopyridine (DMAP) in dichloromethane.
Subsequently, the hydroxyl group of compound 2 was
converted to the phosphoramidite 3 (LithoP) using 2-
cyanoethyl N,N-diisopropylchlorophosphoramidite.10
Potential siRNA therapeutics against liver cell-specific
diseases such as hepatitis C, require improved cellular
uptake of siRNA. The liver is involved in lipid and ster-
oid metabolism. Thus, siRNA modified with lipophilic
In order to obtain the derivative 5 (pLitho), the hydrox-
yl group of 2 was first protected by a dimethoxytrityl
group (DMT). After deacetylation of the crude trityl-
ether the resulting alcohol 4 was converted to the desired
phosphoramidite 5. The cholesterol derivative 6 (Chol,
Scheme 2) was obtained according to the literature.7
Keywords: siRNA; RNAi; Cellular uptake; Solid phase synthesis.
*
Corresponding author. Tel.: +49-9221-8276241; fax: +49-9221-
8276299; e-mail: phadwiger@alnylam.de
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.018

4976
C. Lorenz et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4975–4977
CONH-(CH2)6-OR1
CONH-(CH2)6-ODMT
COOH
a
c, d
2
H
H
H
H
H
H
H
H
H
AcO
R2O
AcO
H
H
H
2: R1 = H
3: R1
4: R2 = H
5: R2
1
CN
CN
e
b
O
N
O
N
=
=
P
P
= LithoP
= pLitho
Scheme 1. Synthesis of 3 (LithoP) and 5 (PLitho). Reagents and conditions: (a) 6-aminohexanol, DMAP, EDCl, CH2Cl2 (67%); (b) 2-cyanoethyl-
N,N-diisopropylchlorophosphoramidite, DIPEA, CH2Cl2 (35%); (c) DMT-Cl, pyridine; (d) K2CO3, MeOH/CH2Cl2 (c + d: 83%); (e) 2-cyanoethyl-
N,N-diisopropylchlorophosphoramidite, DIPEA, CH2Cl2 (76%).
a,b
DMTO (CH2)11 CONH (CH2)6 OR1
HO (CH2)11 COOH
8: R1 = H
H
7
CN
c
H
O
9: R1
=
P
H
H
= Laurin
N
O
O
O
O
N
H
CN
P
N
(CH2)9-CH3
6
d
R2O (CH2)11
N
C
O
HO (CH2)11 COOH
= Chol
(CH2)9-CH3
CN
10: R2 = H
11: R2
7
e
O
P
=
= C32
N
Scheme 2. Phosphoramidite 6 (Chol); synthesis of Laurin 9 and C32 11. Reagents and conditions: (a) DMT-Cl, pyridine; (b) 6-aminohexanol,
DMAP, EDCl, CH2Cl2; (a + b: 97%); (c) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, DIPEA, CH2Cl2 (43%); (d) di-n-decylamine,
DMAP, EDCl, CH2Cl2 (90%); (e) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, DIPEA, CH2Cl2 (54%).
The synthesis of the two 12-hydroxy lauric acid deriva-
tives 9 (Laurin) and 11 (C32), mimicking membrane
lipids, was performed as indicated in Scheme 2. Chemo-
selective elongation of the dimethoxytrityl ether of 12-
hydroxy lauric acid 7 with 6-aminohexanol gave the
amide 8. Phosphitylation of the OH-function of 8
yielded compound 9 (Laurin). To obtain the second
derivative, 12-hydroxy lauric acid 7 was reacted with
di-n-decylamine, which was added in 2M excess result-
ing in the branched amide 10. The final conversion of
the hydroxyl function of 10 to a phosphoramidite group
led to compound 11 (C32).
With the five lipid-containing phosphoramidites in
Scheme 3. Shown are the six exemplary siRNA conjugates obtained
hand, a series of 50-modified siRNAs was synthesized
with a Chol 6 modification. The prefixes Gal and HCV indicate the
on solid phase. The siRNA sequences were directed
target mRNA b-galactosidase or the HCV-30-UTR, respectively. The
extensions -s, -as and -2 refer to the modification attached to the sense
(-s) or the antisense (-as) strand or both strands (-2). In a similar
either against the 30-untranslated region of hepatitis C-
RNA (HCV 30-UTR) or against E. coli b-galalactosi-
dase mRNA. The incorporation of the modifications 5
and 9 proceeded in efficiencies similar to commercially
fashion the other four lipophilic compounds LithoP 3, pLitho 5,
Laurin 9 and C32 11 were used to obtain six conjugates each.

C. Lorenz et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4975–4977
4977
2.00
1.50
1.00
0.50
0.00
siRNA 50 nM
Scheme 4. The results of siRNA delivery experiments without transfection agents in b-Gal¯Huh-7 cells with all 30 modified siRNAs are shown in
this figure. The cells were divided into five wells and treated for 4h with siRNAs at a concentration of 50nM in serum free medium. Subsequently, the
incubation medium was replaced by complete medium. After 48h cells were lysed and b-galactosidase activity was measured in a standard
chemoluminescence assay. The four most effective siRNAs for further studies are highlighted. Data are presented as mean values with the
corresponding standard deviation of five assays. Gal3/2 and HCV11/2 are unmodified siRNAs possessing the same sequence as the modified siRNAs.
K22 is an unmodified, nonspecific siRNA. Furthermore the b-gal activity value for untreated cells is shown.
available standard nucleotide phosphoramidites as
judged by colorimetric analysis of the release of the di-
methoxytrityl cation. The oligonucleotides were depro-
tected employing established procedures,14 purified by
IEX HPLC and characterized by electrospray mass
spectrometry (ESI-TOF-MS).
either Chol 6 or C32 11 (HCVChol-s, HCVC32-s, Gal-
Chol-s and GalC32-s) were selected for detailed studies,
which will be reported in due course.
References and notes
Each series of siRNAs contained one of the five possible
modifications either at the 50-end of the sense or the
antisense strand or contained modifications at both
strands. Altogether 15 modified siRNAs directed against
the same sequence of HCV and 15 siRNAs targeting one
region of the b-galactosidase mRNA were assembled
(Scheme 3).
1. Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.;
Driver, S. E.; Mello, C. C. Nature 1998, 391, 806.
2. Zamore, P. D.; Tuschl, T.; Sharp, P. A.; Bartel, D. P. Cell
2000, 101, 25.
3. Elbashir, S. M.; Martinez, J.; Patkaniowska, A.; Lendec-
kel, W.; Tuschl, T. EMBO J. 2001, 20, 6877.
4. Hammond, S. M.; Bernstein, E.; Beach, D.; Hannon, G. J.
Nature 2000, 404, 293.
5. Elbashir, S. M.; Harborth, J.; Lendeckel, W.; Yalcin, A.;
Weber, K.; Tuschl, T. Nature 2001, 411, 494.
6. Caplen, N. J.; Parrish, S.; Imani, F.; Fire, A.; Morgan, R.
A. Proc. Natl. Acad. Sci. U.S.A. 2001, 17, 9742.
7. MacKellar, C.; Graham, D.; Will, D. W.; Burgess, S.;
Brown, T. Nucl. Acid Res. 1992, 20, 3411.
8. (a) Bijsterbosch, M. K.; Rump, E. T.; De Vrueh, R. L.;
Dorland, R.; van Veghel, R.; Tivel, K. L.; Biessen, E. A.;
van Berkel, T. J.; Manoharan, M. Nucl. Acid Res. 2000,
28, 2717–2725; (b) Manoharan, M. Antisense Nucl. Acid
Drug Dev. 2002, 12, 103.
9. Barton, D. H. R.; Boivin, J.; Lelandais, P. J. Chem. Soc.,
Perkin Trans. 1, 1989, 463.
10. Sinha, N. D.; Biernat, J.; Koester, H. Tetrahedron Lett.
1983, 24, 5843.
11. Nakabayashi, H.; Taketa, T.; Miyano, K.; Yamane, T.;
Sato, J. Cancer Res. 1982, 42, 3858.
12. Nakabayashi, H.; Taketa, T.; Yamane, T.; Miyazaki, M.;
Miyano, K.; Sato, J. Gann 1984, 75, 151.
The complete panel of siRNAs was evaluated for the
ability to inhibit gene expression in the human liver cell
line b-Gal¯Huh-7, most importantly, without the use of
any transfection agent. These cells are derived from the
human hepatoma cell line Huh-711–13 and contain a sta-
ble expressed gene fusion consisting of a part of the
HCV 30-UTR and lacZ.15
Incubation of these cells with siRNAs against each of
the two target sequences resulted in a decrease in b-ga-
lactosidase expression. It was found that siRNAs with
a modified sense strand downregulated b-galactosidase
expression to a higher extent than siRNAs with a mod-
ified antisense strand or two modified strands. Unmodi-
fied siRNAs did not reduce gene expression under
identical conditions, although they were equally effective
when transfected with lipofection (data not shown).
Furthermore, two modifications were identified (Chol
6 and C32 11), which were superior in downregulation
of b-galactosidase expression, independent of the si-
RNA sequence they were attached to (Scheme 4). Con-
sequently, the four siRNAs, directed against the HCV
30-UTR or the lacZ sequence and sense modified with
13. Nakabayashi, H.; Taketa, T.; Yamane, T.; Oda, M.; Sato,
J. Cancer Res. 1985, 45, 6379.
14. Wincott, F.; DiRenzo, A.; Shaffer, C.; Grimm, S.; Tracz,
D.; Workman, C.; Sweedler, D.; Gonzalez, C.; Scaringe,
S.; Usman, N. Nucl. Acid Res. 1995, 23, 2677.
15. Kryspin, M. Diploma thesis, University Bayreuth, Ger-
many.