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Evaluation of Locked Nucleic Acid–Modified Small Interfering RNA in Vitro and in Vivo

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Evaluation of Locked Nucleic Acid–Modified Small Interfering RNA in Vitro and in Vivo 833 Evaluation of locked nucleic acid–modified small interfering RNA in vitro and in vivo Olaf R. Mook, Frank Baas, Marit B. de Wissel, model. Therefore, LNA-modified siRNA should be pre- and Kees Fluiter ferred over unmodified siRNA. [Mol Cancer Ther 2007; 6(3):833–43] Department of Neurogenetics, Academic Medical Center, Amsterdam, the Netherlands Introduction RNA interference (RNAi) is a natural process that affects Abstract gene silencing in eukaryotic systems at transcriptional, RNA interference has become widely used as an experi- posttranscriptional, and/or translational levels (1). Small mental tool to study gene function. In addition, small interfering RNA (siRNA) molecules are the key intermedi- interfering RNA (siRNA) may have great potential for the ates in this process, which can potentially inhibit the treatment of diseases. Recently, it was shown that siRNA expression of any given target gene. siRNA molecules hold can be used to mediate gene silencing in mouse models. great promise as biological tools and as potential thera- Locally administered siRNAs entered the first clinical trials, peutic agents for targeted inhibition of disease-causing but strategies for successful systemic delivery of siRNA genes. are still under development. Challenges still exist about the To optimize the use of siRNA as a therapeutic therapy, stability, delivery, and therapeutic efficacy of siRNA. In several modes of delivery have been tested in vivo. the present study, we compare the efficacy of two Improved delivery in animals has been achieved by methods of systemic siRNA delivery and the effects of complexation with cationic liposomes (2), polyethyleni- siRNA modifications using locked nucleic acids (LNA) in a mine (3), and Arg-Gly-Asp–polyethylene glycol–polyethy- xenograft cancer model. Low volume tail vein bolus lenimine (4) or by conjugation of cholesterol to siRNA (5). injections and continuous s.c. delivery using osmotic However, Ma et al. (6) has shown that complexation to minipumps yielded similar uptake levels of unmodified cationic liposomes resulted in a potent induction of both siRNA by tumor xenografts. Both routes of administration type I and type II IFN responses in mice. In line, sequence- mediated sequence-specific inhibition of two unrelated specific and Toll-like receptor-7–dependent induction of targets inside tumor xenografts. Previous studies have IFNs has been shown on liposome-mediated siRNA shown that LNA can be incorporated into the sense strand transfection in mice (7). Furthermore, conjugation of of siRNA while the efficacy is retained. Modification of cholesterol to a siRNA against apolipoprotein B could siRNA targeting green fluorescent protein with LNA results specifically degrade its mRNA in vivo (5). in a significant increase in serum stability and thus may be Another approach to improve potency and efficacy of beneficial for clinical applications. We show that minimal siRNA in vivo is by the introduction of other chemical 3¶ end LNA modifications of siRNA are effective in modifications in siRNA. In vitro studies showed that sev- stabilization of siRNA. Multiple LNA modifications in the eral modifications are allowed in functional siRNAs. Modi- ¶ accompanying strand further increase the stability but fications of phosphorothioate (8, 9) 2 -O-methyl (10, 11), ¶ ¶ negate the efficacy in vitro and in vivo. In vivo, LNA- 2 -O-allyl (10), and 2 -deoxy-fluorouridine (8, 9) have been modified siRNA reduced off-target gene regulation com- examined for potential in vivo use. Some of the modified pared with nonmodified siRNA. End-modified siRNA siRNAs were found to exhibit enhanced serum stability (11) ¶ targeting green fluorescent protein provides a good and longer duration of action (10). Modification of the 5 end ¶ trade-off between stability and efficacy in vivo using the of the antisense strand with 2 -O-allyl (10) or chemical ¶ two methods of systemic delivery in the nude mouse blocking of the 5 -hydroxyl group (11) resulted in a dramatic loss in activity consistent with the proposed in vivo requirement for 5¶ end phosphorylation (12). In addition, more substantial modifications, such as total modification by 2¶-O-methyl (8) or phosphorothioate modifications of Received 4/10/06; revised 11/23/06; accepted 1/31/07. every second or all internucleoside linkages (8, 9), increased Grant support: Dutch Cancer Society project no. 2003-2968. cytotoxic effects and resulted in a significant decrease or The costs of publication of this article were defrayed in part by the complete loss of activity. In contrast, totally modified payment of page charges. This article must therefore be hereby marked duplexes containing a combination of 2¶-O-methyl and advertisement in accordance with 18 U.S.C. Section 1734 solely to 2¶-fluoro modifications were shown to be effective in vitro indicate this fact. and in vivo (13). Requests for reprints: Olaf R. Mook, Department of Neurogenetics, Academic Medical Center, Meibergdreef 9, Amsterdam, the Netherlands Locked nucleic acid (LNA) is a novel nucleotide analogue 1105 AZ. Phone: 312-056-64540. E-mail: [email protected] that contains a methylene bridge that connects the 2¶- Copyright C 2007 American Association for Cancer Research. oxygen of the ribose with the 4¶-carbon. The bicyclic doi:10.1158/1535-7163.MCT-06-0195 structure locks the furanose ring of the LNA molecule in Mol Cancer Ther 2007;6(3). March 2007 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. 834 Evaluation of LNA-Modified siRNA In vitro and In vivo a3¶-endo conformation, thereby structurally mimicking the AS 5¶-gaugaacuucagggucagcTT-3¶ and heavily modified standard RNA monomers. LNA induce an increase in siGFP SS 5¶-GCTgacCcuGaagTTcaucTT-3¶ and AS 5¶-gau- ¶ thermal stability (melting temperature, Tm) when bound to gaacuucagggucagcTT-3 (LNA nucleotides are depicted in a matching RNA sequence. Introduction of LNA into capitals) were synthesized by Santaris A/S (Hørsholm, classic antisense oligonucleotides has been shown to Denmark) as described previously (15). increase its serum stability (14). In analogy, Braasch et al. Cell Line (8) showed that LNA can be used to thermally stabilize The pancreatic cancer cell line MiaPaca-II and MiaPaca-II siRNAs without losing their function. Recently, a system- stably expressing eGFP (17) were maintained at 37jC and atic study on LNA containing siRNAs has identified the 5% CO2 by serial passage in DMEM supplemented with number and positions of LNA molecules within the siRNA, 10% FCS, 2 mmol/L L-glutamine, 100 units/mL penicillin, which still allow a functional siRNA (15). Incorporation of and 100 Ag/mL streptomycin. LNA molecules into siRNA significantly increased its Application of Unmodified and LNA-Modified siRNA serum stability, which potentially favors successful in vivo In vivo applications (15). Furthermore, LNA modifications at the S.c. tumors were induced in 8- to 10-week-old NMRI nu/ 5¶ end of the sense strand of siRNA has been reported to nu mice (Charles River, Maastricht, the Netherlands) as favor incorporation of the antisense strand into the RNA- described previously (14). One week after tumor cell induced silencing complex (RISC), thereby reducing se- injection, when tumor take was positive, administration quence related off-target effects (15). A second potential of the siRNA or LNA-modified siRNA started. benefit of LNA-modified siRNA is the protection of the two For POLR2A inhibition, siPOLR2A and the mismatch nt 3¶ overhang of siRNA. Recently, it has been shown that control containing two central mismatches were adminis- in certain cell types blunt-ended double-stranded RNA tered via tail vein injections at a dosage of 0.15 mg/kg (200 (dsRNA; lacking 3¶ overhangs) <30 bp induced dsRNA- ALof1.4Amol/L siPOLR2A solution) twice weekly for 3 mediated signaling (16). Therefore, protection of those weeks as described previously (18). During treatment, overhangs with LNA potentially increases the specificity of tumor growth was monitored as described previously (17). siRNA. In all experiments targeting GFP, administration was In this study, we show that systemic administration of either via tail vein injection as described above or via unmodified siRNA induced specific RNAi effects of two osmotic minipumps (model 1007D; Alzet Corp., Palo Alto, independent targets in tumor xenografts. Different routes CA) dosed at 0.25 mg/kg/d for 7 days. For each treatment, of administration resulted in comparable target knock- five mice per group were used. All animal experiments down. To test whether LNA-modified siRNA contributes to were conducted under the institutional guidelines and the efficacy in vivo, we have designed an end-modified according to the law; they were sanctioned by the animal siRNA targeting green fluorescent protein (siGFP) and a ethics committee. heavily modified siGFP and compared their characteristics Whole-Body Imaging and Tissue Processing with unmodified siGFP both in vitro and in vivo. In the eGFP fluorescence was visualized with whole-body present article, we show that minimal modification of imaging using the GFP fluorescence mode of a LAS3000 siGFP with LNA greatly enhanced its serum stability and (Fuji, Tokyo, Japan). Parts of the tumors were fixed in was compatible with the silencing machinery. End-modi- formaldehyde (4%)/sucrose (20%). Other parts of the fied siGFP effectively lowered its target in vivo after different routes of administration. Target knockdown was not associated with dsRNA-dependent protein kinase activation or induction of the IFN response. Introduction of LNA into siRNA resulted in significantly less off-target regulated genes. These results showthat end-modified siRNA holds promise for in vivo applications. Materials and Methods siRNA and LNA-Modified siRNA Synthesis siRNA against large subunit of RNA polymerase II (siPOLR2A) SS 5¶-gcugcgcuauggcgaagacgg-3¶ and AS 5¶- gucuucgccauagcgcagctg-3 and the mismatch control siPOLR2A mismatch SS 5¶-gcugcgcuacugcgaagacgg-3¶ and AS 5¶-gucuucgcaguagcgcagctg-3¶ (DNA nucleotides are depicted in italics) were purchased from Proligo (Boulder, Figure 1.
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