Synthesis and in vitro inhibition properties of siRNA conjugates carrying glucose and galactose with different presentations

“Synthesis and in vitro inhibition properties of siRNA conjugates carrying glucose and galactose with different presentation” Aviñó, A., Ocampo, S. M., Lucas, R., Reina, J. J., Morales, J. C., Perales, J. C., Eritja, R. Mol. Divers., 15(3), 751-757 (2011). doi: 10.1007/s11030-011-9305-6 Synthesis and in vitro inhibition properties of siRNA conjugates carrying glucose and galactose with different presentation. Anna Aviñó, Sandra M. Ocampo, Ricardo Lucas,$ José J. Reina,$ Juan Carlos Morales,$ José Carlos Perales,# and Ramon Eritja Institute for Research in Biomedicine (IRB Barcelona), Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Networking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Baldiri Reixac 10, E-08028 Barcelona, Spain. #Dpt of Ciencies Fisiológiques. School of Medicine. University of Barcelona (Campus Belvitge). C/ Feixa Llarga, L’Hospitalet de Llobregat. 08907 Barcelona, Spain $Instituto de Investigaciones Químicas, CSIC _ Universidad de Sevilla, 49 Americo Vespucio, 41092 Sevilla, Spain Corresponding author: Ramon Eritja. Phone 34-934039942, Email: re[email protected]sic.es 2 KEYWORDS: siRNA, oligonucleotide-carbohydrate conjugates, RNA interference, tumor necrosis factor, oligonucleotide synthesis. Abstract. Oligoribonucleotides conjugates and the corresponding siRNA duplexes against tumor necrosis factor carrying one, two or four glucose and galactose residues at the 5’-end have been prepared using phosphoramidite chemistry. Carbohydratemodified siRNA duplexes displayed similar or slightly reduced inhibitory properties compared to unmodified RNA duplexes in HeLa cells upon transfection with oligofectamine. When HeLa cells were treated with siRNA carrying one, two or four glucose residues without oligofectamine, no inhibition was observed. The inhibitory properties of siRNA carrying galactose residues without transfecting agent were tested on HuH-7 cells that have abundant asialoglycoprotein receptors. In these cells siRNA carrying galactose residues have slight anti-TNF inhibitory properties (25% in the best case) that are eliminated if the receptors are blocked with a competitor such as a-D- galactopyranosyl-albumine. These results demonstrate receptor-mediated uptake of siRNA carrying galactose residues although the efficacy of the process is not enough for efficient RNA interference experiments. INTRODUCTION Chemically synthesized DNAs and RNAs have shown promising results as potential drugs [1, 2]. In these cases nucleic acids are used for the inhibition of a specific gene by blocking gene translation or gene transcription or by stimulating the degradation of a 3 particular messenger RNA. In the antisense strategy synthetic oligonucleotides complementary to the messenger RNA of a given gene are used to inhibit translation of mRNA to protein [1,3,4]. On the other hand, small interfering RNA duplexes (siRNA) are used to inhibit a specific gene expression by the RNA interference (RNAi) mechanism. This is a cellular post-transcriptional gene silencing mechanism induced by a double stranded siRNA. In this strategy one of these strands binds to a protein called RISC and the complex formed is able to cleave the complementary mRNA sequence so that the corresponding protein is not formed [5-7]. Although large efforts have been devoted in the development of novel siRNA derivatives for gene silencing, the clinical potential of siRNA is still limited mainly due low stability and inefficient delivery of siRNAs. Chemical modification of DNA or RNA strands has been addressed as one promising strategy to solve these inconvenients [8-10]. Conjugation with different molecules such as lipids, peptides, aptamers or polymers has been used to improve pharmacokinetic properties of siRNA without decreasing activity [9]. Synthesis of carbohydrate conjugates is a potential alternative to enhance oligonucleotide uptake by using sugar binding membrane receptors to mediate cell entry [10,11]. Oligonucleotide delivery in the form of covalent conjugates with carbohydrates is not as well explored as in the form of non covalent complexes [10]. Because the interaction between individual carbohydrates and lectins is rather weak, multivalent carbohydrate moieties linked to oligonucleotides may enhace the affinity. In the antisense strategy, galactosylated PEG- conjugates of oligonucleotides phosphorothioates have been shown to enhance uptake by galactose-specific asialoglycoprotein receptor-mediated endocytosis [12]. Multivalent carbohydrate conjugates have been described in order to increase the interaction of carbohydrates with cellular receptors [13-16]. Oishi et al described the 4 synthesis of siRNA conjugates with lactose and polyethyleneglycol at the 3’-end of the passenger strand. This modification improved gene inhibition in hepatoma cells [17]. Glycotargeting is considered a valuable method to enhance delivery of therapeutic oligonucleotides. Moreover, selectivity between different cell types can be achieved if different carbohydrate binding receptors are expressed. This is the case with a membrane-active polymer with N-acetyl galactosamine covalently attached, which preferentially accumulated the oligonucleotides in hepatocyte cells whereas if mannose was attached to the polymer the dsDNA went preferentially to nonparenchymal and Kupffer cells, which possess mannose receptors, and away from hepatocytes [18]. Synthesis of oligonucleotide conjugates with carbohydrates has been reviewed [19,20]. There are three main protocols reported for the solid phase synthesis of glycoconjugates. Carbohydrate phosphoramidite coupling is the oldest and one of the most used so far [13-15, 21-23]. Synthesis of oligonucleotide glycoconjugates can also be performed using a 1,3-dipolar cycloaddition (“click reaction”) of sugar azides and support-anchored alkynylated oligonucleotides [24]. Moreover, sugar oligonucleotide conjugates have also been prepared by solid phase oximation with carbohydrate monosubstitution [25] or displaying sugar multivalency [26]. Few examples for carbohydrate- RNA conjugates have been described. In these cases sugar attachment has been carried out using an amide bond [27], an oxime bond [28], a 3,4-diethoxy-3-cyclobutene-1,2- dione as linker [29], or a long functional spacer built with an amide bond (containing sugar and spacer) and a Michael reaction between the sugar acrylate derivative and a 5’- thiol-modified RNA [17]. In this paper we describe, for the first time, the synthesis of carbohydrate conjugates of oligoribonucleotides (1-6) using phosphoramidite chemistry (scheme 1). These modifications are introduced to the passenger strand of the siRNA for the inhibition of 5 tumor necrosis factor (TNF-α) protein which is involved in apoptosis, inflammation and immunity processes [30]. We envisioned that the introduction of multiple carbohydrates (glucose or galactose) at the 5’-terminus of the passenger strand may favour cellular uptake by receptor-mediated endocitosis as the number of carbohydrate residues is increased. EXPERIMENTAL SECTION Oligonucleotides. The following RNA sequences were obtained from commercial sources (Sigma-Proligo, Dharmacon): sense or passenger scrambled 5’-CAGUCGCGUUUGCGACUGG-dT- dT-3’, antisense or guide scrambled 5’-CCAGUCGCAAACGCGACUG-dT-dT-3’, antisense or guide anti-TNFa: 5’-GAGGCUGAGACAUAGGCAC-dT-dT-3’ and sense or passenger anti-TNFa: 5’-GUGCCUAUGUCUCAGCCUC-dT-dT-3’. RNA monomers in capital letters, dT represents thymidine. The anti-TNFa siRNA has been previously described to efficiently downregulate murine TNFa mRNA [30]. 3-Hydroxypropyl-2,3,4,6-tetra-O-acetyl-b- D -glucopyranoside (7). To a solution of the tetra-O-acetylglucopyranosyl bromide [31] (730 mg, 1.78 mmol) in anhydrous CH2Cl2 (40 mL) was added Na2SO4 (two lips of spatula) and 1,3-propanediol (1.43 mL, 17.8 mmol). Alter stirring at room temperature for 15 min, under argon atmosphere, Ag2CO3 (4.9 g, 17.8 mmol) was added to the reaction mixture and was stirred for 96 hours (TLC hexane-ethyl acetate, 2:3). The mixture was then filtered over celite and the crude was purified by silica gel column chromathography using as eluent (hexane-ethyl acetate, 2:3) to give 7 (390 mg, 54%) as a syrup. [α]D20 -12.3 (c 1 in CHCl3); 1H-NMR 6 (CDCl3, 300 MHz) δ (ppm) 5.15 (t, J = 9.5 Hz, 1H, ), 5.01 (t, J= 9.6 Hz, 1H), 4.91 (m, 1H, ), 4.47 (d, J= 7.9 Hz, 1H, H1), 4.18 (dd, J= 12.3 & 4.7 Hz, 1H, H6), 4.09 (dd, J= 12.3 & 2.3 Hz, 1H, H6), 3.93 (td, J= 9.6 & 5.9 Hz, 1H), 3.63 (m, 4H, 2 x CH2O-), 2.18 (s, 1H, OH), 2.03, 1.98, 1.96, 1.94 (4s, 12H, 4 x OCOCH3), 1.75 (dd, J= 5.7 & 4.7 Hz, 2H, -OCH2CH2CH2O-). 13C-NMR (CDCl3, 75 MHz) δ (ppm) 171.1 (C=O), 170.6 (C=O), 169.9 (C=O), 169.8 (C=O), 101.1 (C1), 73.1, 72.2, 71.6, 68.8, 68.0, 62.3, 60.2, 32.5 (CH2CH2CH2), 21.1 (OCOCH3), 21.0 (OCOCH3), 20.9 (OCOCH3), 20.9 (OCOCH3). HRMS (ES+) Calcd. for C17H26O11 (M+H): 407.1553, found; 407.1555. 3-[(2,3,4,6-Tetra-O-acetyl-b- D -glucopyranosyloxy)]propyl (2-cyanoethyl) (N,N- diisopropyl) phosphoramidite (8). Diisopropylethylamine (DIEA) (0.040 mL, 0.228 mmol) and 2-cyanoethyl-N,N’-diisopropylamino-chlorophosphoramidite (20 mL, 0.092 mmoL) were added to a solution of compound 7 (25.0 mg, 0.061 mmol) in anhydrous CH2Cl2 (2 mL) at room temperature under an argon atmosphere. After 1.0 h no starting material was observed. Solvent was then removed and the crude was purified by silica gel column chromatography by using hexane-ethyl acetate (1:1 with 5% of triethylamine) to give compound 8 (35 mg, 95%) as a syrup. 1H-NMR (CDCl3, 300 MHz) (mix of isomers) δ (ppm) 5.13 (dt, J =4.2 & 9.5 Hz, 1H, H3), 5.01 (t, J= 9.6 Hz, 1H, H4), 4.90 (t, J= 8.6 Hz, 1H, H2), 4.45 (t, J= 8.3 Hz, 1H, H1), 4.20 (dd, J= 12.1 & 4.0 Hz, 1H, H6), 4.05 (d, J= 12.1 Hz, 1H, H6’), 3.93-3.84 (m, 1H, H5), 3.83-3.44 (m, 8H, 2 x CH2O- + -OCH2CH2CN + 2CHisopropyl), 2.63-2.53 (m, 2H, -OCH2CH2CN), 2.01, 1.98, 1.95, 1.93 (4s, 12H, 4 x OCOCH3), 1.80 (m, 2H, -OCH2CH2CH2O-), 1.11 (d, 12H, J=6.5 Hz, 4CH3isopropyl). HRMS (ES+) Calcd. for C26H44N2O12P (M+H): 607.2632, found; 607.2638. 7 Synthesis of oligoribonucleotide-carbohydrate conjugates. Oligoribonucleotide carrying carbohydrates at the 5’-end were synthesized by a DNA synthesizer (Applied Biosystems 3400) using 2-cyanoethyl phosphoramidites and polystyrene solid supports (LV200). The following sequences were prepared: Glucose- C3-5'-GUG CCU AUG UCU CAG CCU C-dT-dT-3’, (Glucose-C2)2-DB-GUG CCU AUG UCU CAG CCU C-dT-dT-3’, (Glucose-C2)4-DB-DB-GUG CCU AUG UCU CAG CCU C-dT-dT-3’, Galactose-C2-5'-GUG CCU AUG UCU CAG CCU C-dT-dT- 3’, (Galactose-C2)2-DB-GUG CCU AUG UCU CAG CCU C-dT-dT-3’, (Galactose- C2)4-DB-DB-GUG CCU AUG UCU CAG CCU C-dT-dT-3’. DB stands for the symmetric doubler phosphoramidite obtained from commercial sources (Glen Research); C3 stands for -CH2CH2CH2-OPO2--; C2 for -CH2CH2-OPO2--; and dT for thymidine. The synthesis of carbohydrate phosphoramidites of Glucose-C2 (9) and Galactose-C2 (10) is described in [14, 15] whereas details for the preparation of Glucose-C3 phosphoramidite (8) are described above. Guanosine was protected with the dimethylaminomethylidene group, cytidine was protected with the acetyl group and adenosine with the benzoyl group. The 2’-OH protecting group for the RNA monomers was the t-butyldimethylsilyl (TBDMS) group. The phosphoramidites were dissolved in dry acetonitrile (0.1M) and a modified cycle was used with increased coupling time (10 min). The solid supports were treated with concentrated aqueous ammonia-ethanol (3:1) for 1hr at 55oC. After filtration of the solid supports, the supports were washed with ethanol and the combined solutions were evaporated to dryness. Sequences were treated with 0.15 ml of triethylamine hydrofluoride / triethylamine / N- methylpyrrolidone (4:3:6) for 2.5 hours at 65 ºC to remove the t-butyldimethylsilyl groups. The reactions were stopped by addition of 0.3 ml of isopropoxytrimethylsilane and 0.75 ml of ether. The resulting mixtures were mixed with the vortex and cooled at 4 8 ºC. A precipitate was formed that was centrifugated at 7000 rpm, 5 min at 4ºC. The precipitates were washed with ether and centrifuged again. The residues were dissolved in water and the conjugates were purified by HPLC. HPLC conditions were: column: Nucleosil 120-10 C18 (250x4 mm); 20 min linear gradient from 0% to 50% B (DMT off conditions); flow rate 3 ml/min; solution A was 5% acetonitrile in 0.1 M aqueous triethylammonium acetate (TEAA) buffer and B 70% acetonitrile in 0.1 M aqueous TEAA. The purified products were analyzed by MALDI-TOF mass spectrometry. MALDI-TOF spectra were performed using a Perseptive Voyager DETMRP mass spectrometer, equipped with nitrogen laser at 337 nm using a 3ns pulse. The matrix used contained 2,4,6-trihydroxyacetophenone (THAP, 10 mg/ml in ACN/ water 1:1) and ammonium citrate (50 mg/ ml in water). Glucose-C3-RNA 1 [M] = 6861 (expected M= 6858). (Glucose-C2)2-DB-RNA 3 [M] = 7476 (expected M= 7478). (Glucose-C2)4- DB-DB-RNA 5 [M] = 8755 (expected M= 8754). Galactose-C2-RNA 2 [M] = 6847 (expected M= 6841). (Galactose-C2)2-DB-RNA 4 [M] = 7461 (expected M= 7478). (Galactose-C2)4-DB-DB-RNA 6 [M] = 8746 (expected M= 8754). Yields (0.2 mmol scale synthesis) were between 15-20 OD units at 260 nm. Melting temperatures of siRNA duplexes (15 mM HEPES-KOH pH 7.4; 1 mM MgCl2, 50 mM potassium acetate) : Unmodified 83.3 ºC; 1 83.7 ºC; 2 84.7 ºC; 5 82.1 ºC; 6 83.0 ºC. Cell culture, transfection and cellular assays. HeLa cells were cultured under standard conditions (37ºC, 5% CO2, Dulbecco’s Modified Eagle Medium, 10% fetal bovine serum, 2mM L-glutamine, supplemented with penicillin (100 U/ml) and streptomycin (100mg/ml). All in vitro experiments were conducted at 40-60% confluence. HeLa cells were transfected with 250 ng of murine expressing TNF-a plasmid using lipofectin (Invitrogen) according to the manufacturer’s 9 instructions. One hour following transfection murine TNF-a expressing HeLa cells were transfected with 50nM of siRNA (5’-GUGCCUAUGUCUCAGCCUC-dT-dT-3’ / 5’ GAGGCUGAGACAUAGGCAC-dT-dT-3’, [30]) against TNF-a, using oligofectamine (Invitrogen). TNF-a concentration was determined from cell culture supernatant by enzyme-linked immunoabsorbent assay kit (Bender MedSystems) according to the manufacturer’s instructions. RESULTS AND DISCUSSION Synthesis of oligoribonucleotide carrying glucose and galactose Among the different approaches described to prepare carbohydrate oligonucleotide conjugates [19,20] we selected the protocol based on the coupling of a sugar phophoramidite to oligonucleotides on a solid support. This approach has been previously used in our group to introduce several sugars in DNA [14,15]. We have extended this approach to prepare RNA-carbohydrate conjugates that possess one, two and four glucose or galactose moieties. Glucose and galactose phosphoramidites carrying C2 and C3 spacers (scheme 2) were synthesized in two steps: classical glycosylation from their corresponding bromo peracetylated sugars and ethylene glycol or propylene glycol and subsequent standard phosphoramidite chemistry as described in [14,15]. Oligoribonucleotide carrying glucose and galactose at the 5’-end were synthesized. using the appropriate carbohydrate phosphoramidites (Scheme 2) and the commercial dendrimer symmetric doubler phosphoramidite. We selected the 21mer RNA sequence 5'-GUGCCUAUGUCUCAGCCUC-dT-dT-3’ for the introduction of one or several 16 oligonucleotides. Tetrahedron Lett. 36: 7333-7336. doi:10.1016/0040-4039(95)01512- G [22] Sheppard TL, Wong C-H, Joyce GF (2000) Nucleoglycoconjugates: design and synthesis of a new class of DNA-carbohydrate conjugates. Angew Chem Int Ed 39: 3660-3663. doi:10.1002/1521-3773(20001016)39:20 [23] Adinolfi M, De Napoli L, Di Fabio G, Iadonisi A, Montesarchio D (2004) Modulating the activity of oligonucleotides by carbohydrate conjugation: solid phase synthesis of sucrose-oligonucleotide hybrids. Org Biomol Chem 2: 1879-1886. doi:10.1039/b401819b [24] Pourceau G, Meyer A, Vasseur JJ, Morvan F (2009) Synthesis of mannose and galactose oligonucleotide conjugates by bi-click chemistry. J Org Chem 74:1218-1222. doi:10.1021/jo802536q [25] Forget D, Renaudet O, Defranq E, Dumy P (2001) Efficient preparation of carbohydrate-oligonucleotide conjugates (COCs) using oxime bond formation. Tetrahedron Lett. 42: 7829-7832. doi:10.1016/S0040-4039(01)01682-3 [26] Katajisto J, Virta H, Lönnberg H (2004) Solid-phase synthesis of multiantennary oligonucleotide glycoconjugates utilizing on-support oximation. Bioconjugate Chem 15: 890-896. doi:10.1021/bc049955n [27] Matulic-Adamic J, Serebryany V, Haeberli P, Mokler VR, Beigelman L (2002) Synthesis of N-acetyl-D-galactosamine and folic acid conjugated ribozymes. Bioconjugate Chem 13: 1071-1078. doi:10.1021/bc025525q [28] Edupuganti OP, Renaudet O, Defrancq E, Dumy P (2004) The oxime bond formation as an efficient chemical tool for the preparation of 3’,5’-bifunctionalised oligodeoxyribonucleotides. Bioorg Med Chem Lett 14: 2839-2842. doi:10.1016/j.bmcl.2004.03.053 17 [29] Zhao Y, Tram K, Yan H (2009) Synthesis and characterization of mannosylated oligoribonucleotides Carbohyd Res 344: 2137–2143. doi:10.1016/j.carres.2009.08.033 [30] Sorensen DR, Leirdal M, Sioud MJ (2003) Gene silencing by systemic delivery of synthetic siRNA in adult mice. J Mol Biol 327: 761-766. doi:10.1016/S0022- 2836(03)00181-5 [31] Karjala S, Link KP (1940) Synthesis of Glycol Glucosides. J Am Chem Soc 62: 917-920. doi:10.1021/ja01861a058 [32] Grimau M, Iacopino D, Aviñó A, de la Torre B G, Ongaro A, Fitzmaurice D, Wessels J, Eritja R (2003) Synthesis of branched oligonucleotides as templates for the assembly of nanomaterials. Helv Chim Acta 86: 2814-2826. doi:10.1002/hlca.200390232 [33] Ren T, Zhang G, Liu D (2001) Synthesis of galactosyl compounds for targeted gene delivery. Bioorg Med Chem 9: 2969-2978. doi:10.1016/S0968-0896(01)00203-6 [34] Biessen EAL, Beuting DM, Roelen HCPF, van der Marel GA, van Boom JH, Van Berkel TJC (1995) Synthesis of cluster galactosides with high affinity for the hepatic asialoglycoprotein receptor. J Med Chem 38: 1538-1546. doi:10.1021/jm00009a014 [35] Moni L, Pourceau G, Zhang J, Meyer A, Vidal S, Souteyrand E, Dondoni A, Morvan F, Chevolot Y, Vasseur JJ, Marra A (2009) Design of triazole-tethered glycoclusters exhibiting three different spacial arrengements and comparative study of their affinities towards PA-IL and RCA120 by using a DNA-based glycoarray. Chembiochem 10: 1369-1378. doi: 10.1002/cbic.200900024 [36] Hangeland JJ, Flesher JE, Deamond SF, Lee YC, Ts’o POP, Frost JJ (1997) Tissue distribution and metabolism of the [32P]-labeled oligodeoxynucleotide methylphosphonate-neoglycopeptide conjugate [YEE(ah-GalNAc)3]-SMSS-AET- pUmpT7 in the mouse. Antisense Nucleic A 7: 141-149. doi:10.1089/oli.1.1997.7.141 18 19 Scheme 1. Structure of the carbohydrate oligodeoxyribonucleotide conjugates prepared. RNA seq is passenger anti-TNF-a sequence: 5’-GUGCCUAUGUCUCAGCCUC-dT- dT-3’. OO O OH HO HO OH PO O- ORNA seq OOO OH HO R1 OH R2 PO -O O OOO OH HO R1 OH R2 PO O- O NH NH O O OPO -O O RNA seq OOO OH HO R1 OH R2 PO -O O OOO OH HO R1 OH R2 PO O- O NH NH O O OPO -O O OOO OH HO R1 OH R2 PO -O O OOO OH HO R1 OH R2 PO O- O NH NH O O OPO -O O NH NH O O OPO -O O RNA seq OOO OH HO OH HO PO O- ORNA seq 1 2 R1=OH, R2=H 3 R1=H, R2=OH 4 R1=OH, R2=H 5 R1=H, R2=OH 6 20 Scheme 2. Sugar phosphoramidites (8-10) used in this work for the preparation of oligoribonucleotides carrying carbohydrates (1-6). PO N(iPr) 2CN O OAc R1 AcO OAc OO R2 O OAc AcO AcO OAc OOPO N(iPr)2 CN R1=OAc, R2=H Glucose-C2 phosphoramidite 9 R1=H, R2=OAc Galactose-C2 phosphoramidite 10 Glucose-C3 phosphoramidite 8 21 Figure 1. HPLC profile of the product obtained after the synthesis of the oligoribonucleotide bearing glucose (5'- Glucose-C3-GUGCCUAUGUCUCAGCCUC- dT-dT-3’) using DMT off conditions (see experimental section). Product eluting at 8 min is the desired oligonucleotide. 0 5 10 15 20 25 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Abs 260 nm Tr(min) 22 Figure 2. In vitro potency of glucose modified siRNAs against murine TNF-a in Hela cells using oligofectamine as transfecting agent for siRNA. The amount of murine TNF- a produced after 24 hrs of transfection of 50nM siRNA unmodified duplex (Unm), 50 nM of the same siRNA duplex carrying a glucose C3 molecule at the 5’-end of the passenger strand (1), 50 nM of the same siRNA duplex carrying two (3) and four (5) glucose C2 molecules at the 5’-end of the passenger strand and 50 nM of a scrambled RNA duplex control sequence (Scr). Error bars represent the s.d of the mean. Statistical analysis was by ANOVA with Bonferroni post-test, one tailed. *** P<0.001 compared with scrambled siRNA. Unm 1 3 5 Scr 0 100 200 300 400 500 600 *** ** *** TNF-a (pg/ml) 23 Figure 3. In vitro potency of galactose modified siRNAs against murine TNF-a in Hela cells using oligofectamine as transfecting agent for siRNA. The amount of murine TNF-a produced after 24 hrs of transfection of 50nM siRNA unmodified duplex (Unm), 50 nM of the same siRNA duplex carrying a galactose C2 molecule at the 5’-end of the passenger strand (2), 50 nM of the same siRNA duplex carrying two (4) and four (6) galactose C2 molecules at the 5’-end of the passenger strand and 50 nM of a scrambled RNA duplex control sequence (Scr). Error bars represent the s.d of the mean. Statistical analysis was by ANOVA with Bonferroni post-test, one tailed. *** P<0.001 compared with scrambled siRNA. Unm 2 4 6 Scr 0 100 200 300 400 500 600 *** ** * TNF-a (pg/ml) 24 Figure 4. In vitro potency of glucose modified siRNAs against murine TNF-a in Hela cells without using oligofectamine as transfecting agent for siRNA. The amount of murine TNF-a produced after 24 hrs of transfection of 100nM siRNA unmodified duplex (Unm), 100 nM of the same siRNA duplex carrying a glucose C3 molecule at the 5’-end of the passenger strand (1), 100nM of the same siRNA duplex carrying two (3) and four (5) glucose C2 molecules at the 5’-end of the passenger strand and 100 nM of a scrambled RNA duplex control sequence (Scr). Error bars represent the s.d of the mean. Unm 1 3 5 Scr 0 200 400 600 800 TNF-a (pg/ml) 25 Figure 5. In vitro potency of galactose modified siRNAs against murine TNF-a in HuH-7 cells without using oligofectamine as transfecting agent for siRNA. The amount of murine TNF-a produced after 24 hrs of transfection of 100nM siRNA unmodified duplex (Unm), 100 nM of the same siRNA duplex carrying a galactose C2 molecule at the 5’-end of the passenger strand (2), 100nM of the same siRNA duplex carrying two (4) and four (6) galactose C2 molecules at the 5’-end of the passenger strand and 100 nM of a scrambled RNA duplex control sequence (Scr). Error bars represent the s.d of the mean. Unm 2 4 6 Scr 0 50 100 150 200 250 TNF-a (pg/ml)