Patent Application
20090298913
The invention relates generally to small interfering RNA duplexes (siRNA) containing at least one arabinose modified nucleotide, as well as small interfering 2′-deoxy-2′-fluoroarabinonucleic acid:RNA hybrids for the downregulation of gene expression.
Numerous strategies for silencing gene expression with nucleic acid-based molecules are under development [Stephenson, M. L. & Zamecnik, P. C. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxynucleotide. Proc. Natl. Acad. Sci. USA 74, 4370-4373 (1977); Opalinska, J. B. & Gewirtz, A. M. Nucleic-acid therapeutics: basic principles and recent applications. Nature Rev. 1 (July), 1-10 (2002)]. Of these, the hybridization-driven “antisense” strategies, using ribozymes, DNAzymes, and antisense oligonucleotides such as chimeric RNA-DNA (gapmers) or phosphorothioate DNA have received the greatest attention and are the subject of numerous reviews [Stull, R. A. & Szoka, F. C. Antigene, ribozyme and aptamer nucleic acid drugs: progress and prospects. Pharmaceutical Res. 12, 465-483 (1995); Uhlmann E. and Peyman, A. Antisense oligonucleotides: a new therapeutic principle. Chem. Rev. 90, 544-584 (1990)]. More recently, post-transcription gene silencing or RNA interference (RNAi) has emerged as an exciting potential alternative to these more classical approaches [Elbashir, S. M, Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188-200 (2001); Caplen, N. J. et al. Specific inhibition of gene expression by small dsRNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA 98, 9742-9747 (2001); Nishikura, K. A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell 107, 415-418 (2001); Tuschl, T. Expanding small RNA interference. Nature Biotechnol. 20, 446-448 (2002); Mittal, V. Improving the efficiency of RNA interference in mammals. Nature Rev. 5, 355-365 (2004); Nykanen A., Haley, B. & Zamore, P. D. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321 (2001)]. There are numerous reports describing the utility of this method for silencing genes in living organisms ranging from yeast to mammals [Yu, J. Y., S. L. DeRuiter, and D. L. Turner, RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 6047 (2002); Donze, O. and D. Picard, RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Res. 30, e46 (2002); Sui, G., C. Soohoo, B. Affar el, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 5515 (2002); Paddison, P. J., A. A. Caudy, E. Bernstein, et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948 (2002)].
The utility of siRNA in vivo and its possible application in pharmacotherapy, as with other oligonucleotide-based therapies, faces some key hurdles (e.g., delivery, cellular uptake and biostability of oligonucleotides). There is a need to develop chemical modifications that result in clinically useful molecules. Initial work with antisense and siRNA oligonucleotides was undertaken with unmodified, natural molecules. It soon became clear however, that native oligonucleotides were subject to relatively rapid degradation, primarily through the action of 3′ exonucleases, but as a result of endonuclease attack as well. Oligoribonucleotides (RNA) are, in fact, generally more susceptible to nuclease degradation relative to DNA.
Antisense and siRNA molecules are now routinely modified to enhance their stability, as well as the strength of their hybridization with RNA since these physical attributes are often necessary for their therapeutic application [Mangos, M. M. & Damha, M. J. Flexible and frozen sugar-modified nucleic acids—modulation of biological activity through furanose ring dynamics in the antisense strand, Curr. Top. Med. Chem. 2, 1145-1169 (2002); Agrawal, S. and Q. Zhao. Mixed backbone oligonucleotides: improvement in oligonucleotide-induced toxicity in vivo. Antisense Nucleic Acid Drug Dev. 8, 135 (1998); Crooke, S. T. Molecular mechanisms of action of antisense drugs. Biochim. Biophys. Acta 1489, 31 (1999); Micklefield, J. Backbone modification of nucleic acids: synthesis, structure and therapeutic applications. Curr. Med. Chem. 8, 1157 (2001); Nielsen, P. E., Antisense peptide nucleic acids. Curr. Opin. Mol. Ther. 2, 282 (2000); Braasch, D. A., S. Jensen, Y. Liu, et al., RNA interference in mammalian cells by chemically-modified RNA. Biochemistry 42, 7967 (2003)]. In the presence of a delivery vehicle, both types of molecules are able to cross cell membranes and then to hybridize with their intended RNA target. RNA tertiary structure is an important factor governing the ability of antisense oligonucleotides [Opalinska, J. B., A. Kalota, L. K. Gifford, et al. Oxetane modified, conformationally constrained, antisense oligodeoxyribonucleotides function efficiently as gene silencing molecules [Nucleic Acids Res. 32, 5791 (2004). Scherr, M., J. J. Rossi, G. Sczakiel, et al., RNA accessibility prediction: a theoretical approach is consistent with experimental studies in cell extracts. Nucleic Acids Res. 28, 2455 (2000). Sokol, D. L., X. Zhang, P. Lu, et al., Real time detection of DNA.RNA hybridization in living cells. Proc. Natl. Acad. Sci. USA 95, 11538 (1998)] and siRNA [Opalinska, J. B., A. Kalota, L. K. Gifford, et al. Oxetane modified, conformationally constrained, antisense oligodeoxyribonucleotides function efficiently as gene silencing molecules. Nucleic Acids Res. 32, 5791 (2004); Scherr, M., J. J. Rossi, G. Sczakiel, et al., RNA accessibility prediction: a theoretical approach is consistent with experimental studies in cell extracts. Nucleic Acids Res. 28, 2455 (2000); Sokol, D. L., X. Zhang, P. Lu, et al., Real time detection of DNA.RNA hybridization in living cells. Proc. Natl. Acad. Sci. USA 95, 11538 (1998)] to hybridize with their target. It goes without saying that it is undesirable for either type of molecule to exert non-sequence specific binding. Meeting all these requirements has turned out to be a demanding task.
Unmodified siRNA duplexes have been used with success for gene silencing, however, chemical modification of one or both of the strands will likely be necessary for therapeutic applications in order to improve biostability and pharmacokinetic properties. Numerous chemical modifications have been tested for effects on siRNA activity, although it is not clear yet which of these modifications will be the most advantageous. In designing new analogues, it is important to recognize that two key features of siRNA differ from traditional antisense approaches: (i) duplex RNAs are recognized and (ii) gene inhibition involves RISC(RNA-Induced Silencing Complex)—rather than RNase H—to promote recognition and cleavage of the mRNA target [Elbashir, S. M, Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188-200 (2001); Caplen, N. J. et al. Specific inhibition of gene expression by small dsRNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA 98, 9742-9747 (2001); Nishikura, K. A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell 107, 415-418 (2001); Tuschl, T. Expanding small RNA interference. Nature Biotechnol. 20, 446-448 (2002); Mittal, V. Improving the efficiency of RNA interference in mammals. Nature Rev. 5, 355-365 (2004)]. As such, RNA-like oligonucleotides are prime candidates for introducing sugar or backbone modifications without perturbing the overall A-form helical structure they require for activity. A promising modification is Locked Nucleic Acids (LNA), in which key benefits were achieved with relatively few modifications that do not significantly compromise siRNA activity (e.g., improved thermal stability and biostability, and reduced off target-effects) [Elmen, J. et al. Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality. Nucl. Acids Res. 33, 439-447 (2005)]. However, the activity and specificity of such compounds was found to be highly dependent on the site and degree of the LNA modifications. A single LNA substitution at the 5′-end of the antisense strand abolished activity. Moreover, activity was significantly impaired when the antisense strand was modified, whereas sense strand LNA modifications were only tolerated with slightly modified oligonucleotides, displaying equal or lower activity than unmodified siRNA. There appears to be limitations with other chemistries, including toxicity (phosphorothioate-RNA) and impaired activity (2′F-RNA, boranophosphate-RNA), with increasing degrees of modification [Amarzguioui, M. et al. Tolerance for mutations and chemical modifications in a siRNA, Nucl. Acids. Res. 31, 589-595 (2003)]. While this may in principle be compensated by the nuclease stability and/or specificity imparted by certain oligonucleotide chemistries, the prediction of effective siRNA chemistries remains an active focus of continued studies.
There is a need for chemically modified siRNAs that have nuclease stability and/or the ability to inhibit gene expression.
According to one broad aspect of the invention, a small interfering RNA (siRNA) for modulating expression of a target gene in a sequence-specific manner comprising a double stranded duplex wherein at least one ribonucleic acid nucleotide of the siRNA is substituted with an arabinose modified nucleotide is provided. The arabinose modified nucleotide is 2′-deoxy-2′-fluoroarabinonucleotide (FANA).
Preferably, the siRNA is 15-30 nucleotides in length and has 1-3 nucleotide overhangs at the 3′ and 5′ termini.
In specific embodiments, the duplex may have any number of arabinonucleotides at any location at either the sense or the antisense strand, for example:
etc.
wherein A is an arabinonucleotide and R is a ribonucleotide.
In other embodiments of the invention, the sense strand is fully substituted with arabinonucleotides. For example:
5′-AAAAAAAAAAAAAAAAAAAAA-3′
and the antisense strand is an all-RNA strand or partially substituted RNA strand, for example:
5′-RRRRRRRRRRRRRRRRRRRRRRR-3′
5′-RRRRRRRRRRRRRRRRRRRRRAA-3′
5′-AARRRRRRRRRRRRRRRRRRRRR-3′
etc.
In other embodiments of the invention, the arabinonucleotide comprises a 2′ substituent selected from the group consisting of fluorine, hydroxyl, amino, azido, alkyl, alkoxy, and alkoxyalkyl groups. In a further embodiment of the invention, the alkyl group is selected from the group consisting of methyl, ethyl, propyl, butyl, and functionalized alkyl groups such as ethylamino, propylamino and butylamino groups. In embodiments, the alkoxy group is selected from the group consisting of methoxy, ethoxy, proproxy and functionalized alkoxy groups such as —O(CH2)q—R, where q=2-4 and —R is a —NH2, —OCH3, or —OCH2CH3 group. In embodiments, the alkoxyalkyl group is selected from the group consisting of methoxyethyl, and ethoxyethyl. In embodiments, the 2′ substituent is fluorine and the arabinonucleotide is a 2′-fluoroarabinonucleotide (FANA). Preferably, the FANA nucleotide is araF-G, araF-T, araF-U, araF-A, araF-5-methyl-C.
According to some embodiments of the invention, the siRNA is for decreasing any one of luciferase expression, CCR3 expression, or PDE4D expression.
According to another embodiment of the invention, the siRNA is for decreasing Respiratory Syncytial Virus replication.
In other embodiments of the invention, the duplex comprises one or more internucleotide linkages selected from the group consisting of:
a) phosphodiester;
b) phosphotriester;
c) phosphorothioate;
d) methylphosphonate;
e) boranophosphate and
f) any combination of (a) to (e).
According to another broad aspect of the invention, a method is provided for increasing at least one of nuclease stability and modulation of target gene activity of an siRNA comprising replacing at least one nucleotide of the siRNA with an arabinose modified nucleotide, preferably 2′-deoxy-2′-fluoroarabinonucleotide (FANA).
According to another broad aspect of the invention a pharmaceutical composition is provided, comprising the siRNA of the present invention along with a pharmaceutically acceptable carrier.
According to another broad aspect of the invention, use of the siRNA of the present invention is provided for the preparation of a medicament for modulating expression of a target gene, preferably one of CCR3 and PDE4D.
According to another embodiment of the invention, use of the siRNA of the present invention is provided for the preparation of a medicament for decreasing Respiratory Syncytial Virus replication.
According to another broad aspect of the invention, a method of modulating gene expression in a patient in need thereof is provided. The method comprises administering to the patient a therapeutically effective amount of the pharmaceutical composition of the invention. Preferably, the pharmaceutical composition comprises a siRNA for any one of decreasing expression of CCR3, decreasing expression of PDE4D, and decreasing Respiratory Syncytial Virus replication.
According to another broad aspect of the invention a commercial package is provided. The commercial package comprises the pharmaceutical composition of the present invention together with instructions for its use for modulating gene expression. Preferably, the pharmaceutical composition comprises an siRNA for any one of decreasing CCR3 expression, decreasing expression of PDE4D and decreasing Respiratory Syncytial Virus replication.
The invention will now be described in greater detail having regard to the appended drawings in which:
This invention relates to modified oligonucleotide duplexes designed to target mRNA and promote mRNA degradation via the RNAi mechanism. In particular, selective inhibition of luciferase activity, rat CCR3 expression and RSV viral replication using short interfering RNA duplexes containing modified arabinonucleotides (FANA) is shown. The methods of RNAi described herein are in contrast to the common methods described above, which have concentrated on the use of modified nucleotides derived from the naturally occurring units (i.e., DNA, RNA, 2′-OMe-RNA, 2′F-RNA nucleotides) [Allerson, C. R. et al. Fully 2′-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA. J. Med. Chem. 48, 901-904 (2005)].
This invention encompasses the characterization of a series of sugar modified duplexes that inhibit gene expression in a human cell line. These small interfering duplexes contain arabinose modified nucleotides conferring improved characteristics on the duplex, such as improved stability against nucleases present in body fluid. Preferably, the sugar modified nucleotides are 2′-deoxy-2′-fluoroarabinonucleotides (FANA). The method for generating the FANA modified duplexes necessitates the substitution of RNA nucleotides for FANA residues.
Activity of the modified siRNAs was evaluated using a modified HeLa cell line engineered to over-express luciferase. Luciferase mRNA expression levels and luciferase activity levels were determined using real-time PCR and luciferase assay techniques, respectively. Design and selection of the actual siRNA base sequence was performed according to Mittal et al. [Mittal, V. Improving the efficiency of RNA interference in mammals. Nature Rev. 5, 355-365 (2004)] utilizing the Ambion and Qiagen algorithms and NCBI Blast searches. At least three candidate siRNA duplexes were selected and tested as described above. Once the most active siRNA duplex was identified (EC50 ˜0.5 nM), preliminary experiments were carried out to assess the impact of arabinose modification on siRNA activity. Selective, specific and efficient inhibition of luciferase activity of such FANA modified duplexes is demonstrated (
The compounds disclosed here represent the first examples of FANA modified duplexes (FANA modified siRNAs, and FANA:RNA hybrids) capable of inhibiting gene expression selectively via the RNAi mechanism.
Specifically, this invention provides FANA nucleotides that are compatible with the activity of siRNA duplexes. In addition, it is shown that an entire FANA sense strand can bind to a complementary unmodified RNA antisense strand generating a duplex that enters the RNAi pathway to selectively and efficiently target a mRNA and promote its degradation (
This invention also provides RNA duplexes in which an unmodified sense strand is annealed to an antisense strand in which the dangling dN terminal residues (3′ or 5′-termini) are replaced with FANAs without affecting activity (
This invention also provides RNA duplexes in which both sense and antisense strand contain modified residues while maintaining RNAi activity (
Similar to unmodified siRNAs, sustained inhibition of luciferase activity was observed when arabinose modified duplexes were transfected into cells for up to 4 days post transfection (
Herein is presented evidence that the nuclease stability of FANA containing siRNA duplexes is improved over unmodified siRNA duplexes (
A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a modified nucleic acid of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the modified nucleic acid to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.
As used herein “pharmaceutically acceptable carrier” or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, an oligonucleotide duplex of the invention can be administered in a time release formulation, for example in a composition which includes a slow release polymer. The modified oligonucleotide can be prepared with carriers that will protect the modified oligonucleotide duplex against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.
Sterile injectable solutions can be prepared by incorporating an active compound, such as an oligonucleotide duplex of the invention, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. In accordance with an alternative aspect of the invention, an oligonucleotide duplex of the invention may be formulated with one or more additional compounds that enhance its solubility.
Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”.
The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.
The sequence and composition of the oligomers prepared in this study are shown in Table 1. Syntheses of oligoribonucleotides, FANA modified oligoribonucleotides, as well as all-FANA oligonucleotides were carried out on a 1 μmol scale on an Applied Biosystems (ABI) synthesizer using the standard β-cyanoethylphosphoramidite chemistry according to published protocols [E. Viazovkina, M. M. Mangos, M. I. Elzagheid, and M. J. Damha (2002) Current Protocols in Nucleic Acid Chemistry, Unit 4.15); M. J. Damha and K. K. Ogilvie (1993) Oligoribonucleotide synthesis—the silyl-phosphoramidite method in “Protocols for Oligonucleotide and Analogs: Synthesis and Properties” S. Agrawal (ed.), Methods in Molecular Biology pp. 81-114, The Humana Press Inc., Totowa, N.J.]. FANA modified oligoribonucleotides and oligoribonucleotides were deprotected, purified and handled identically. All oligonucleotides were purified by anion exchange HPLC or gel electrophoresis, and desalted via size-exclusion chromatography using Sephadex G-25 beads. Stock solutions of duplexes were prepared by mixing the sense and corresponding antisense strands (1:1 stoichiometric ratio), lyophilizing the samples, and adding sufficient resuspension/annealing buffer to make a 20 μM solution. The composition of the siRNA resuspension/annealing buffer is 100 mm potassium acetate, 30 mM HEPES-KOH, 2 mM magnesium acetate, pH 7.4.
This example relates to the efficacy of FANA-containing siRNAs with respect to the specific knockdown of the target mRNA and reduction of luciferase activity in HeLa X1/5 cells. The HeLa X1/5 cell line was obtained from ECACC (ECACC No. 95051229) and maintained in EMEM media supplemented (Invitrogen, Burlington ON) with 10% fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, 1% vitamins, 500 μg/ml G418 and 300 μg/ml Hygromycin. For transfection, 1.0×105 cells/well were plated onto 24-well plates 24 hours prior to transfection. The day of transfection, cells were transfected with 0.21 μg of siRNA using Lipofectamine 2000 (Invitrogen, Burlington ON) at a siRNA:Lipofectamine 2000 ratio of 1:2 according to the manufacturers' recommendations. Cells were harvested 24 h post-transfection. Cell metabolic activity, as an indicator of cellular toxicity resulting from siRNA transfection, was assessed using the alamar Blue™ fluorimetric assay (Medicorp, Montreal QC) as per the manufacturers' recommendations.
Luciferase activity assays were performed using the luciferase assay system (BD Bioscience, Mississauga, ON) according to the manufacturer's protocol. Briefly, following exposure to the siRNA, cells were washed with phosphate-buffered saline (Invitrogen, Burlington ON) and lysed. Cell lysates were centrifuged to remove cellular debris and 20 μl aliquots were transferred to 96-well lumitrac plates (Ultident; Greiner Bio-one). Luminescence was measured using a microplate luminometer (Luminoskan Ascent, Thermo LabSystem) immediately following addition of the luciferin substrate solution. Luminescence values were then normalized to the cell metabolic activity values (alamar Blue™) to compensate for toxicity resulting from transfection.
For real-time PCR analysis, total RNA was extracted using the RNeasy mini kit (Qiagen, Mississauga ON) according to the manufacturers' protocol. cDNA was prepared from 1 μg total RNA using the SuperScrip™ II Reverse Transcriptase and random primers (Invitrogen, Burlington ON). Quantitative real-time PCR was performed using gene-specific primers and probes for the luciferase gene (LUC5013 F1: 5′-acgctgggcgttaatcagag-3′; LUC5013 R1: 5′-gtcgaagatgttggggtgttg-3′; TIB MOLBIOL) and the housekeeping gene GAPDH (huGAPD for: 5′-ggtggtctcctctgacttc-3′; huGAPD rev: 5′-ctcttcctcttgtgctcttg-3′; TIB MOLBIOL) using previously optimized conditions and the LightCycler (Roche, Laval QC).
Results presented in
This example relates to the potency of FANA-containing siRNAs with respect to the specific knockdown of luciferase activity in HeLa X1/5 cells. Dose-response studies were performed using a total amount of siRNA of 0.21 μg whereby the effective siRNA was serially diluted with a control siRNA, reducing the effective amount of active oligonucleotide while keeping the final amount of siRNA constant. Cells were harvested 24 h post-transfection and luciferase activity determined.
Results indicate that an siRNA having two deoxynucleotides of the 3′-overhang of the antisense strand replaced with FANAs and having an unmodified (O/F4) or fully modified (F3/F4) sense strand inhibits luciferase activity in a concentration-dependent manner with increased potency over the counterpart unmodified siRNA (
This example shows that FANA-containing siRNAs have sustained inhibitory activity up to 96 h. Luciferase activity was measured at different time points following exposure to the different modified and unmodified siRNAs (
This example relates to siRNA duplex stability in the presence of fetal bovine serum. Results of experiments are presented in
Results show that incorporation of FANAs in the sense strand confers significant resistance to serum-mediated siRNA degradation. Introduction of FANAs significantly enhances serum resistance of siRNAs. A representative gel is shown in
This example relates to the efficacy of FANA-containing siRNAs in specific knockdown of the expression levels of CCR3 mRNA in NIH-3T3 cells. The NIH-3T3 cell line was obtained from ATCC (ATCC CRL-1658) and maintained in DMEM medium (Invitrogen, Burlington ON) supplemented with 10% calf bovine serum, 4 mM L-glutamine, 3.7 g/L sodium bicarbonate, 4.5 g/l glucose and 1% Penicillin/Streptomycin. 1.0×105 cells/well were seeded onto 24-well plates one day prior to transfection. Cells were transfected with 0.2 μg of plasmid expressing the rat CCR3 gene, 0.2 μg of plasmid expressing luciferase (reference gene) and 0.01, 0.1 or 0.2 μg of siRNA using Lipofectamine 2000 (Invitrogen, Burlington ON) at a DNA/siRNA:Lipofectamine 2000 ratio of 1:2 according to the manufacturers' recommendations. Cells were harvested 24 h following transfection. Expression levels of CCR3 and luciferase were quantified using the Quantigene method (Panomics, Fremont Calif.). CCR3 expression levels were then normalized to the levels measured for luciferase.
Results presented in
This example relates to the efficacy of siRNA duplexes containing FANA residues to inhibit replication of respiratory syncytial virus (RSV) in A549 cells. The A549 cell line (ATCC, # CCL-185) was maintained in Ham F12 medium (HyClone, Logan Utah) supplemented with 10% non-inactivated FBS (HyClone). 1.0×105 cells were seeded into individual wells of 24-well plates one day prior to transfection. On the day of transfection, cells were transfected with 0.05 μg, 0.2 μg or 0.4 μg of siRNA at a 1:3 ratio of siRNA to transfection reagent (Lipofectamine 2000 (Invitrogen, Burlington ON)) according to the manufacturers' recommendations. 24 hours post-transfection cells were infected with RSV at a multiplicity of infection (M.O.I.) of 1 and the viral infection was allowed to proceed for one day. 24 hours after exposure to virus, cell supernatants were harvested and RSV levels were assessed using an ELISA method to detect RSV proteins.
Results indicate that an siRNA duplex, wherein the two deoxynucleotides of the 3′ overhang of the antisense strand are substituted with FANAs and the sense strand remains unmodified (O/F4), inhibits RSV replication in a concentration-dependent manner having increased inhibitory activity compared to unmodified siRNA at lower doses (
All references cited are incorporated by reference herein. Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.
auppercase letters = RNA; lowercase letters = DNA; bold uppercase letters = FANA
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA06/01760 | 10/26/2006 | WO | 00 | 10/15/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60730876 | Oct 2005 | US | |
| 60741544 | Dec 2005 | US |
