NOVEL MRNA COMPOSITION AND PRODUCTION METHOD FOR USE IN ANTI-VIRAL AND ANTI-CANCER VACCINES

Abstract

This invention relates to a novel mRNA composition and its production method useful for developing and manufacturing RNA-based anti-viral and/or anti-cancer vaccines and medicines. This invention includes two types of mRNA constructs, namely “5′-hairpin messenger RNA (5hmRNA)” and “messenger-hairpin-messenger RNA (mhmRNA)”, respectively. Both of 5hmRNA and mhmRNA contain at least a hairpin-like stem-loop RNA structure. The 5hmRNA contains at least a stem-loop RNA structure in the 5′-UTR of a protein/peptide-coding mRNA, while the mhmRNA contains a middle stem-loop structure flanked with two protein/peptide-coding mRNA sequences on both sides. In mhmRNA, the first 5′-mRNA preferably encodes an RNA replicase, for amplifying the second 3′-mRNA in transfected cells. After transfection into target cells, 5hmRNA and mhmRNA can be further translated into at least a desired protein/peptide. To produce highly structured 5hmRNA and mhmRNA, a novel PCR-IVT methodology has been developed and used with a specially designed RNA polymerase-helicase mixture reaction

Claims

1. A novel mRNA composition for use in anti-viral and anti-cancer vaccine manufacture and development, comprising: at least a 5′-hairpin messenger RNA (5hmRNA) and/or messenger-hairpin-messenger RNA (mhmRNA), or both, wherein the 5hmRNA contains at least a stem-loop RNA structure in the 5′-UTR region of a desired mRNA and the mhmRNA contains at least a stem-loop RNA structure flanked with two same or different mRNA sequences of interest on its both sides, and wherein said 5hmRNA and mhmRNA are produced using a novel polymerase chain reaction (PCR)-in vitro transcription (IVT) reaction with at least an additional helicase activity and improved buffer system.

2. The composition as defined in claim 1, wherein said 5hmRNA mainly consists of two parts: 5′-stem-loop RNA and 3′-mRNA.

3. The composition as defined in claim 2, wherein said 5′-stem-loop RNA contains at least a perfectly or imperfectly matched either single or multiple hairpin structure, ranging about 10˜800 nucleotides in length, and a short sequence located between the stem-loop structure and the start codon of the following 3′-mRNA, ranging about 1˜500 nucleotides apart in length.

4. The composition as defined in claim 3, wherein said multiple hairpin structures in the 5′-stem-loop RNA further contains a spacer sequence in between every two hairpin RNA structures, ranging about 2-500 nucleotides apart in length.

5. The composition as defined in claim 2, wherein said 3′-mRNA not only encodes at least one desired protein or peptide but also contains either SEQ.ID.NO.1 or SEQ.ID.NO.2 in its 3′-end.

6. The composition as defined in claim 2, wherein said 5′-stem-loop RNA structure further functions as an artificial internal ribosome entry site (IRES) mimic for initiating and enhancing the translation of the 3′-mRNA.

7. The composition as defined in claim 1, wherein said mhmRNA is formed by adding an additional mRNA (5′-mRNA) sequence in the 5′-end of 5hmRNA, wherein the stem-loop RNA structure of said mhmRNA is served as a middle separator to set apart the individual translation of the first 5′-mRNA and the next 3′-mRNA into either same or different proteins/peptides, respectively.

8. The composition as defined in claim 7, wherein said stem-loop RNA structure further functions as an artificial internal ribosome entry site (IRES) mimic for initiating and enhancing the translation of the 3′-mRNA.

9. The composition as defined in claim 7, wherein the first 5′-mRNA of said mhmRNA encodes an RNA replicase, for amplifying the second 3′-mRNA sequence in transfected cells, leading to a mRNA self-amplification mechanism in the transfected cells.

10. The composition as defined in claim 1, wherein the stem-loop RNA structure of 5hmRNA and mhmRNA contain at least a sequence selected from SEQ.ID.NO.3, SEQ.ID.NO.4, SEQ.ID.NO.5, SEQ.ID.NO.6, SEQ.ID.NO.7, SEQ.ID.NO.8, SEQ.ID.NO.9, SEQ.ID.NO.10, SEQ.ID.NO.11, SEQ.ID.NO.12, SEQ.ID.NO.13, SEQ.ID.NO.14, SEQ.ID.NO.15, and a combination thereof.

11. The composition as defined in claim 1, wherein after transfection into target cells, both of 5hmRNA and mhmRNA are capable of being further translated into at least a desired protein/peptide for eliciting a pre-designed, desired biological effect or cellular function.

12. The composition as defined in claim 1, wherein said 5hmRNA and mhmRNA is further processed in transfected cells to generate at least a shRNA and/or piRNA useful for silencing at least a specific target gene.

13. The composition as defined in claim 12, wherein said specific target genes include a variety of disease-associated cellular and viral genes.

14. The composition as defined in claim 1, wherein said helicase activity is capable of unwinding the secondary structures of both DNA and RNA.

15. The composition as defined in claim 1, wherein the design of said 5hmRNA and mhmRNA is useful for developing new pharmaceutical and therapeutic applications.

16. The composition as defined in claim 1, wherein said 5hmRNA or mhmRNA, or both, is used as an ingredient of an anti-viral vaccine.

17. The composition as defined in claim 1, wherein said 5hmRNA or mhmRNA, or both, is used as an ingredient of an anti-cancer medicine.

18. The composition as defined in claim 1, wherein said 5hmRNA or mhmRNA, or both, is further mixed with at least a delivery agent for cellular transfection in vitro, ex vivo or in vivo.

19. The composition as defined in claim 18, wherein said delivery agent includes glycylglycerins, liposomes, nanoparticles, liposomal nanoparticles, conjugating molecules, infusion chemicals, gene gun materials, electroporation particles, transposon, and a combination thereof.

20. The composition as defined in claim 1, wherein said improved buffer system contains 1× transcription buffer with additional 0.001-10 mM of betaine (trimethylglycine, TMG), dimethylsulfoxide (DMSO), or 3-(N-morpholino)propane sulfonic acid (MOPS), or a combination thereof.

21. The composition as defined in claim 1, wherein said 5hmRNA or mhmRNA, or both, further contains at least a modified nucleotide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Referring particularly to the drawings for the purpose of illustration only and not limitation, there is illustrated:

[0075] FIG. 1 depicts the step-by-step procedure of the PCR-IVT methodology. For mRNA production, a part or whole procedure of this PCR-IVT method can be adopted for either single or multiple cycle amplification of desired mRNA products.

[0076] FIG. 2 depicts the designed structures of 5hmRNA and mhmRNA.

[0077] FIG. 3 depicts an example structure of designed red fluorescent RGFP-coding 5hmRNA. As shown, the stem-loop RNA structure of this RGFP-coding 5hmRNA contains a combination of SEQ.ID.NO.14/15, SEQ.ID.NO.3, SEQ.ID.NO.4, and SEQ.ID.NO.5 sequences located in the 5′-UTR of the encoded RGFP mRNA. Herein, just to serve as an example but not to limit the present invention, it is understandable that the stem-loop RNA structure can be replaced by any hairpin-like RNA, shRNA, pre-miRNA and/or IRES-like RNA structures for eliciting a desired biological effect or cellular function in vitro, ex vivo as well as in vivo. By the same token, the enconded RGFP mRNA can also be replaced by any other desired protein-/peptide-coding mRNA to eliciting at least a desired biological and/or cellular effect in vitro, ex vivo as well as in vivo, depending on the resulting protein/peptide function.

[0078] FIG. 4 shows the resulting promoter-incorporated PCR product and its derived 5hmRNA made by the invented PCR-IVT methodology with a novel helicase activity. Following an increase of starting amounts of the PCR product, the amplified 5hmRNA molecules are proportionally increased in a dose-dependent matter. Accordingly, an optimal generation rate of 30˜2000 fold increase can be reached, resulting in a maximal up to 0.6˜0.9 mg mRNA production per 1 mL IVT reaction (0.6˜0.9 mg/mL mRNA).

[0079] FIG. 5 shows the hairpin-like/IRES-like stem-loop RNA structures of PCR-IVT-made 5hmRNA (i.e. RGFP-coding 5hmRNA) sequences after processed by a Drosha-like RNaseIII enzyme activity (Ambion RNase III, ThermoFisher Scientific, MA, USA) in vitro. As shown in this example, the Drosha-processed hairpin-like RNA structures contain a designed IRES-like stem-loop RNA and precursors of miR-302b, miR-302c, and miR-302a. It is known that miR-302a, b and c are tumor-suppressor microRNAs, which are useful for anti-cancer therapy.

[0080] FIG. 6 shows the resulting RGFP protein (red fluorescent color) production in the A549 cells transfected by the invented PCR-IVT-made 5hmRNA (from FIG. 3), indicating that desired proteins/peptides can be successfully produced from the 5hmRNA in the transfected cells, so as to deliver a designed biological and/or cellular effect. It is conceivable that the encoded RGFP mRNA can be replaced by any other protein/peptide-coding mRNA sequence in the 5hmRNA and mhmRNA, so as to deliver a different biological effect or cellular function of interest.

[0081] FIG. 7 shows Northern blot analysis result of anti-viral (COVID-19) 5hmRNA.

[0082] FIG. 8 shows Northern blot analysis result of anti-viral mhmRNA.

[0083] FIG. 9 shows the immunostaining results of coronaviral (e.g. COVID-19) S 2 proteins produced in the BEAS-2B cells after transfected with the anti-viral 5hmRNA (from FIG. 7), indicating that the delivery of anti-viral 5hmRNA into BEAS-2B lung epithelial cell line can induce intracellular production of coronaviral S 2 protein, which is useful for trigger antiviral immune responses in vivo.

EXAMPLES

1. Human Cell Culture and In-Vitro RNA Transfection

[0084] For culturing keratinocytes, cells are isolated from skin tissues and cultivated in EpiLife serum-free cell culture medium supplemented with human keratinocyte growth supplements (HKGS, Invitrogen, Carlsbad, Calif.) in the presence of proper antibiotics at 37° C. under 5% CO.sub.2. Culture cells are passaged at 50%-60% confluency by exposing cells to trypsin/EDTA solution for 1 min and rinsing once with phenol red-free DMEM medium (Invitrogen), and the detached cells are replated at 1:10 dilution in fresh EpiLife medium with HKGS supplements. Human cancer and normal cell lines MCF7, HepG2, A549 and BEAS-2B were obtained either from the American Type Culture Collection (ATCC, Rockville, Md.) or our collaborators and maintained according to manufacturer's suggestions. For RNA transfection, 0.5˜500 μg of isolated mRNA (i.e. either 5hmRNA or mhmRNA) is dissolved in 0.5 ml of fresh EpiLife medium and mixed with 1˜50 μl of In-VivoJetPEI transfection reagent. After 10˜30 min incubation, the mixture is added into a cell culture containing 50%-60% confluency of the cultivated cells. The medium is reflashed every 12 to 48 hours, depending on cell types. This transfection procedure may be performed repeatedly to increase transfection efficiency. The transfection results are shown in FIG. 6 and FIG. 9, respectively.

2. Novel PCR-IVT Protocol

[0085] Reverse transcription (RT) of desired mRNA is performed by adding about 0.01 ng-10 microgram (μs) of isolated mRNA into a 20˜50 μL RT reaction (SuperScript III RT kit, ThermoFisher Scientific, MA, USA), following the manufacturer's suggestions. Depending on the mRNA amount, the RT reaction mixture contains the mRNA, about 0.01˜20 nmole RT primer, a proper amount of dNTPs and reverse transcriptase in 1×RT buffer. Then, the RT reaction is incubated at 46˜65° C. for 1˜3 hours (hr), depending on the structure and length of the desired mRNA, so as to make at least a complementary DNA (cDNA) template for the next step of PCR. Regarding RT primer designs, for serving as an example but not limited to this example, we use 5′-CAGTTCCAAT TGTGAAGATT CTC-3′ (SEQ.ID.NO.16) for RT of a desired COVID-19 viral mRNA sequence and used another 5′-CTTGATGACG TTCTCAGTGC-3′ (SEQ.ID.NO.17) for RT of another anti-cancer microRNA (miRNA) stem-loop-containing red fluorescent protein (RGFP)-coding mRNA (i.e. RGFP-coding 5hmRNA) of interest, respectively (FIG. 3).

[0086] Next, polymerase chain reaction (PCR) is performed by adding about 0.01 pg-10 μg of the RT-derived cDNA into a 50 μL PCR mixture (High-Fidelity PCR Enzyme kit, ThermoFisher Scientific, MA, USA), following the manufacturer's suggestions. Then, the PCR mixture is first incubated in five to twenty (5˜20) cycles of denaturation at 94° C. for 1 mim, annealing at 30˜55° C. for 30 sec˜1 min, and then extension at 72° C. for 1-3 min, depending on the structure and length of the desired DNA and primers. After that, another ten to twenty (10˜20) cycles of PCR are performed with a series of sequential steps of denaturation at 94° C. for 1 mim, annealing at 50˜55° C. for 30 sec, and then extension at 72° C. for 1˜3 min, depending on the structure and length of the resulting PCR products. Finally, the resulting PCR products are used as templates for IVT. For serving as an example but not limited to this example, we have designed two sets of PCR pair primers for amplifying viral promoter-containing DNA templates, including a pair of 5′-GATATCTAAT ACGACTCACT ATAGGGAGAG GTATGGTACT TGGTAGTT-3′ (SEQ.ID.NO.18) and SEQ.ID.NO.16 (for amplifying an about 12.5 k-nucleotide (nt) COVID-19 RdRp/helicase/S protein-coding mhmRNA (result shown in FIG. 8)) and another pair primers of 5′-GATATCTAAT ACGACTCACT ATAGGGAGAC TAGTGATGTT CTTGTTAACA ACT-3′ (SEQ.ID.NO.19) and SEQ.ID.NO.16 (for amplifying an about 2.8 k-nt COVID-19 S 2 protein-coding 5hmRNA (result shown in FIG. 7)). Moreover, we use another pair of PCR primers for amplifying anti-cancer miRNA-stem-loop-containing RGFP-coding DNA templates (i.e. the RGFP-coding 5hmRNA), including a promoter-containing forward primer 5′-GATATCTAAT ACGACTCACT ATAGGGAGAG GTATGGTACT TGGTAGTT-3′ (SEQ.ID.NO.20) and SEQ.ID.NO.17 (results shown in FIG. 4 and FIG. 5). In principal design, all designed forward PCR primers encode at least a conserved promoter sequence for IVT, such as, but not limited to T7, T3, and/or SP6 promoter sequences, particularly 5′-TCTAATACGA CTCACTATAG GGAGA-3′ (SEQ.ID.NO.21). Furthermore, there may be at least a restriction site in the 3′-end of the forward promoter-primers for insertion of either at least a hairpin-like stem-loop structure or at least an IRES-like stem-loop structure in the PCR products.

[0087] For mRNA production, since a promoter-primer has been incorporated into the resulting PCR products, an improved IVT reaction can be performed to amplify desired mRNA sequences, using the PCR products as templates. The IVT reaction mixture contains 0.01 ng˜10 μg of the PCR product, 0.1˜10 U of helicase (Creative Enzymes, NY), a proper amount of NTPs and RNA polymerase (i.e. T7, T3, or SP6) in 1× transcription buffer. The contents of 1× transcription buffer may be adjusted according to the used RNA polymerase, following the manufacturer's suggestions. Additionally, the 1× transcription buffer may further contain 0.001˜10 mM of betaine (trimethylglycine, TMG), dimethylsulfoxide (DMSO), and/or 3-(N-morpholino)propane sulfonic acid (MOPS), and/or a combination thereof, which facilitates the denaturation of highly structured RNA/DNA sequences, such as hairpins and IRES. Then, the IVT reaction is incubated at 37° C. for 1˜6 hr, depending on the stability and activity of the used RNA polymerase(s). In this improved novel IVT reaction, at least an additional helicase enzyme is added in order to facilitate the unwinding of RNA/DNA secondary structures, such as hairpin-like and IRES-like stem-loop structures, so as to overcome the low efficiency problem of hairpin-like RNA production in vitro. Notably, the helicase enzyme can unwind the secondary structures in both DNA and RNA strands.

3. RNA Purification, Northern Blot Analysis and RNA Sequencing

[0088] Desired mRNAs (10 μg) are isolated with a mirVana™ RNA isolation kit (Ambion, Austin, Tex.), following the manufacturer's protocol, and then further purified by using either 15% TBE-urea polyacrylamide gel or 3.5% low melting point agarose gel electrophoresis. For Northern blot analysis, the gel-fractionated mRNAs are electroblotted onto a nylon membrane. Detection of the mRNA and its IVT template (the PCR product) is performed with a labeled [LNA]-DNA probe complementary to a desired target sequence of the mRNA. The probe is further purified by high-performance liquid chromatography (HPLC) and tail-labeled with terminal transferase (20 units) for 20 min in the presence of either a dye-labeled nucleotide analog or [.sup.32P]-dATP (>3000 Ci/mM, Amersham International, Arlington Heights, Ill.). For determining mRNA/miRNA sequences, the designed SEQ.ID.NO.21 and either SEQ.ID.NO.16 or SEQ.ID.NO.17 are used as primers, separately, for performing RNA sequencing from the 5′-end and 3′-end of the mRNA/miRNA sequences, respectively.

4. Protein Extraction and Western Blot Analysis

[0089] Cells (10.sup.6) are lysed with a CelLytic-M lysis/extraction reagent (Sigma) supplemented with protease inhibitors, Leupeptin, TLCK, TAME and PMSF, following the manufacturer's suggestion. Lysates are centrifuged at 12,000 rpm for 20 min at 4° C. and the supernatant is recovered. Protein concentrations are measured using an improved SOFTmax protein assay package on an E-max microplate reader (Molecular Devices, CA). Each 30 μg of cell lysate are added to SDS-PAGE sample buffer under reducing (+50 mM DTT) and non-reducing (no DTT) conditions, and boiled for 3 min before loading onto a 6-8% polyacylamide gel. Proteins are resolved by SDS-polyacrylamide gel electrophoresis (PAGE), electroblotted onto a nitrocellulose membrane and incubated in Odyssey blocking reagent (Li-Cor Biosciences, Lincoln, NB) for 2 hr at room temperature. Then, a primary antibody is applied to the reagent and incubated the mixture at 4° C. After overnight incubation, the membrane is rinsed three times with TBS-T and then exposed to goat anti-mouse IgG conjugated secondary antibody to Alexa Fluor 680 reactive dye (1:2,000; Invitrogen—Molecular Probes), for 1 hr at the room temperature. After three additional TBS-T rinses, fluorescent scanning of the immunoblot and image analysis are conducted using Li-Cor Odyssey Infrared Imager and Odyssey Software v.10 (Li-Cor).

5. Immunostaining Assay

[0090] Cell/Tissue samples are fixed in 100% methanol for 30 min at 4° C. and then 4% paraformaldehyde (in 1×PBS, pH 7.4) for 10 min at 20° C. After that, the samples are incubated in 1×PBS containing 0.1-0.25% Triton X-100 for 10 min and then washed in 1×PBS three times for 5 min. For immunostaining, primary antibodies include, but not limited to, anti-DsRed/RGFP (Clontech, Palo Alto, Calif.) and anti-COVID-19 S (Invitrogen) monoclonal antibodies. Dye-labeled goat anti-rabbit or horse anti-mouse antibody are used as the secondary antibody (Invitrogen—Molecular Probes, Carlsbad, Calif.). Results are examined and analyzed at 100× or 200× magnification under a fluorescent 80i microscopic quantitation system with a Metamorph imaging program (Nikon). Positive results are shown in FIG. 6 and FIG. 9.

6. In Vivo Transfection Assay

[0091] Isolated mRNA or miRNA from Examples 2 and 3 is mixed well with a proper amount of delivery agent, such as an In-VivoJetPEI transfection reagent, following the manufacturer's protocol, and then injected into blood veins or muscles of an animal, depending the purpose of applications. The delivery agent is used for mixing, conjugating, encapsulating or formulating the desired 5hmRNA or mhmRNA, so as to not only protect the 5hmRNA or mhmRNA from degradation but also facilitate the delivery of the 5hmRNA or mhmRNA into specific target cells of interest in vitro, ex vivo and/or in vivo.

7. Statistic Analysis

[0092] Any change over 75% of signal intensity in the analyses of immunostaining, western blotting and northern blotting is considered as a positive result, which in turn is analyzed and presented as mean±SE. Statistical analysis of data is performed by one-way ANOVA. When main effects are significant, the Dunnett's post-hoc test is used to identify the groups that differ significantly from the controls. For pairwise comparison between two treatment groups, the two-tailed student t test is used. For experiments involving more than two treatment groups, ANOVA is performed followed by a post-hoc multiple range test. Probability values of p<0.05 is considered significant. All p values are determined from two-tailed tests.

REFERENCES

[0093] 1. U.S. Pat. No. 7,662,791 to Shi-Lung Lin et al. [0094] 2. U.S. Pat. No. 8,080,652 to Shi-Lung Lin et al. [0095] 3. U.S. Pat. No. 8,372,969 to Ying S Y and Shi-Lung Lin. [0096] 4. U.S. Pat. No. 8,609,831 to Shi-Lung Lin and Ying S Y. [0097] 5. U.S. Pat. No. 9,637,747 to Shi-Lung Lin et al. [0098] 6. U.S. Pat. No. 9,783,811 to Shi-Lung Lin et al. [0099] 7. U.S. Provisional Patent Application No. 60/222,479 to Shi-Lung Lin. [0100] 8. U.S. Provisional Patent Application No. 60/290,902 to Shi-Lung Lin. [0101] 9. Shi-Lung Lin and Ji H; Replicase cycling reaction amplification. WO2002/092774. [0102] 10. Shi-Lung Lin; Peptide library construction from RNA-PCR-derived RNAs. Methods Mol Biol. 221:289-293, 2003. [0103] 11. Shi-Lung Lin and Ji H; cDNA library construction using in-vitro transcriptional amplification. Methods Mol Biol. 221:93-101, 2003. [0104] 12. McDowell et al.; Determination of intrinsic transcription termination efficiency by RNA polymerase elongation rate. Science 266:822-825, 1994. [0105] 13. Schlake et al.; Developing mRNA-Vaccine Technologies. RNA biology 9:1319-1330, 2012. [0106] 14. Ko et al.; Development of an RNA Expression Platform Controlled by Viral Internal Ribosome Entry Sites. J. Micobiol. Biotechnol. 29:127-140, 2019.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

[0107] (iii) NUMBER OF SEQUENCES: 21

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[0117] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AAUAAA

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[0118] (i) SEQUENCE CHARACTERISTICS: [0119] (A) LENGTH: 7 base pairs [0120] (B) TYPE: nucleic acid [0121] (C) STRANDEDNESS: single [0122] (D) TOPOLOGY: linear [0123] (ii) MOLECULE TYPE: RNA [0124] (A) DESCRIPTION:/desc=“synthetic” [0125] (iii) HYPOTHETICAL: NO [0126] (iv) ANTI-SENSE: NO

[0127] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: AAUUAAA

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[0128] (i) SEQUENCE CHARACTERISTICS: [0129] (A) LENGTH: 73 base pairs [0130] (B) TYPE: nucleic acid [0131] (C) STRANDEDNESS: single [0132] (D) TOPOLOGY: hairpin [0133] (ii) MOLECULE TYPE: RNA [0134] (A) DESCRIPTION:/desc=“synthetic” [0135] (iii) HYPOTHETICAL: NO [0136] (iv) ANTI-SENSE: NO

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[0138] (i) SEQUENCE CHARACTERISTICS: [0139] (A) LENGTH: 68 base pairs [0140] (B) TYPE: nucleic acid [0141] (C) STRANDEDNESS: single [0142] (D) TOPOLOGY: hairpin [0143] (ii) MOLECULE TYPE: RNA [0144] (A) DESCRIPTION:/desc=“synthetic” [0145] (iii) HYPOTHETICAL: NO [0146] (iv) ANTI-SENSE: NO [0147] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CCUUUGCUUU AACAUGGGGG UACCUGCUGU GUGAAACAAA AGUAAGUGCU UCCAUGUUUC AGUGGAGG 68

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[0148] (i) SEQUENCE CHARACTERISTICS: [0149] (A) LENGTH: 69 base pairs [0150] (B) TYPE: nucleic acid [0151] (C) STRANDEDNESS: single [0152] (D) TOPOLOGY: hairpin [0153] (ii) MOLECULE TYPE: RNA [0154] (A) DESCRIPTION:/desc=“synthetic” [0155] (iii) HYPOTHETICAL: NO [0156] (iv) ANTI-SENSE: NO [0157] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CCACCACUUA AACGUGGAUG UACUUGCUUU GAAACUAAAG AAGUAAGUGC UUCCAUGUU UUGGUGAUGG 69

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[0178] (i) SEQUENCE CHARACTERISTICS: [0179] (A) LENGTH: 65 base pairs [0180] (B) TYPE: nucleic acid [0181] (C) STRANDEDNESS: single [0182] (D) TOPOLOGY: stem-loop [0183] (ii) MOLECULE TYPE: RNA [0184] (A) DESCRIPTION:/desc=“synthetic” [0185] (iii) HYPOTHETICAL: NO [0186] (iv) ANTI-SENSE: NO [0187] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CUGUGUGGCU GUCACUCGGC UGCAUGCUUA GUGCACUCAC GCAGUAUAAU UAAUAACUAA UUACU

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[0198] (i) SEQUENCE CHARACTERISTICS: [0199] (A) LENGTH: 88 base pairs [0200] (B) TYPE: nucleic acid [0201] (C) STRANDEDNESS: single [0202] (D) TOPOLOGY: stem-loop [0203] (ii) MOLECULE TYPE: RNA [0204] (A) DESCRIPTION:/desc=“synthetic” [0205] (iii) HYPOTHETICAL: NO [0206] (iv) ANTI-SENSE: NO [0207] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: AAUUAUAAAU UACCAGAUGA UUUUACAGGC UGCGUUAUAG CUUGGAAUUC UAACAAUCUU GAUUCUAAGG UUGGUGGUAA UUAUAAUU

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[0208] (i) SEQUENCE CHARACTERISTICS: [0209] (A) LENGTH: 129 base pairs [0210] (B) TYPE: nucleic acid [0211] (C) STRANDEDNESS: single [0212] (D) TOPOLOGY: multiple stem-loop [0213] (ii) MOLECULE TYPE: RNA [0214] (A) DESCRIPTION:/desc=“synthetic” [0215] (iii) HYPOTHETICAL: NO [0216] (iv) ANTI-SENSE: NO [0217] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CACAAAUAUU ACCAGAUCCA UCAAAACCAA GCAAGAGGUC AUUUAUUGAA GAUCUACUUU UCAACAAAGU GACACUUGCA GAUGCUGGCU UCAUCAAACA AUAUGGUGAU UGCCUUGGUG AUAUUGCUG

(2) INFORMATION FOR SEQ ID NO:12:

[0218] (i) SEQUENCE CHARACTERISTICS: [0219] (A) LENGTH: 189 base pairs [0220] (B) TYPE: nucleic acid [0221] (C) STRANDEDNESS: single [0222] (D) TOPOLOGY: multiple stem-loop [0223] (ii) MOLECULE TYPE: RNA [0224] (A) DESCRIPTION:/desc=“synthetic” [0225] (iii) HYPOTHETICAL: NO [0226] (iv) ANTI-SENSE: NO [0227] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GCAAAAAUGU GAUCUUGCUU GUAAAUACAA UUUUGAGAGG UUAAUAAAUU ACAAGUAGUG CUAUUUUUGU AUUUAGGUUA GCUAUUUAGC UUUACGUUCC AGGAUGCCUA GUGGCAGCCC CACAAUAUCC AGGAAGCCCU CUCUGCGGUU UUUCAGAUUC GUUAGUCGAA AAACCUAAGA AAUUUAAUG

(2) INFORMATION FOR SEQ ID NO:13:

[0228] (i) SEQUENCE CHARACTERISTICS: [0229] (A) LENGTH: 294 base pairs [0230] (B) TYPE: nucleic acid [0231] (C) STRANDEDNESS: single [0232] (D) TOPOLOGY: multiple stem-loop [0233] (ii) MOLECULE TYPE: RNA [0234] (A) DESCRIPTION:/desc=“synthetic” [0235] (iii) HYPOTHETICAL: NO [0236] (iv) ANTI-SENSE: NO [0237] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CACUCCCCUG UGAGGACUAC UGUCUUCACG CAGAAAGCGU CUAGCCAUGG CGUUAGUAUG AGUGUCGUGC AGCCUCCAGG ACCCCCCCUC CCGGGAGAGC CAUAGUGGUC UGCGGAACCG GUGAGUACAC CGGAAUUGCC AGGACGACCG GGUCCUUUCU UGGAUCAACC CGCUCAAUGC CUGGAGAUUU GGGCGUGCCC CCGCGAGACU GCUAGCCGAG UAGUGUUGGG UCGCGAAAGG CCUUGUGGUA CUGCCUGAUG GGUGCUUGCG AGUGCCCCGG GAGGUCUCGU AGAC 294

(2) INFORMATION FOR SEQ ID NO:14:

[0238] (i) SEQUENCE CHARACTERISTICS: [0239] (A) LENGTH: 55 base pairs [0240] (B) TYPE: nucleic acid [0241] (C) STRANDEDNESS: single [0242] (D) TOPOLOGY: stem-loop [0243] (ii) MOLECULE TYPE: RNA [0244] (A) DESCRIPTION:/desc=“synthetic” [0245] (iii) HYPOTHETICAL: NO [0246] (iv) ANTI-SENSE: NO [0247] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GGACACGAGU AACUCGUCUA UCUUCUGCAG GCUGCUUACG GUUUCGUCCG UGUUG 55

(2) INFORMATION FOR SEQ ID NO:15:

[0248] (i) SEQUENCE CHARACTERISTICS: [0249] (A) LENGTH: 52 base pairs [0250] (B) TYPE: nucleic acid [0251] (C) STRANDEDNESS: single [0252] (D) TOPOLOGY: stem-loop [0253] (ii) MOLECULE TYPE: RNA [0254] (A) DESCRIPTION:/desc=“synthetic” [0255] (iii) HYPOTHETICAL: NO [0256] (iv) ANTI-SENSE: NO [0257] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: CAGCCGAUCA UCAGCACAUC UAGGUUUUGU CCGGGUGUGA CCGAAAGGUA AG 52

(2) INFORMATION FOR SEQ ID NO:16:

[0258] (i) SEQUENCE CHARACTERISTICS: [0259] (A) LENGTH: 23 base pairs [0260] (B) TYPE: nucleic acid [0261] (C) STRANDEDNESS: single [0262] (D) TOPOLOGY: linear [0263] (ii) MOLECULE TYPE: DNA [0264] (A) DESCRIPTION:/desc=“synthetic” [0265] (iii) HYPOTHETICAL: NO [0266] (iv) ANTI-SENSE: YES [0267] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CAGTTCCAAT TGTGAAGATT CTC

(2) INFORMATION FOR SEQ ID NO:17:

[0268] (i) SEQUENCE CHARACTERISTICS: [0269] (A) LENGTH: 20 base pairs [0270] (B) TYPE: nucleic acid [0271] (C) STRANDEDNESS: single [0272] (D) TOPOLOGY: linear [0273] (ii) MOLECULE TYPE: DNA [0274] (A) DESCRIPTION:/desc=“synthetic” [0275] (iii) HYPOTHETICAL: NO [0276] (iv) ANTI-SENSE: YES [0277] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CTTGATGACG TTCTCAGTGC

(2) INFORMATION FOR SEQ ID NO:18:

[0278] (i) SEQUENCE CHARACTERISTICS: [0279] (A) LENGTH: 48 base pairs [0280] (B) TYPE: nucleic acid [0281] (C) STRANDEDNESS: single [0282] (D) TOPOLOGY: linear [0283] (ii) MOLECULE TYPE: DNA [0284] (A) DESCRIPTION:/desc=“synthetic” [0285] (iii) HYPOTHETICAL: NO [0286] (iv) ANTI-SENSE: NO [0287] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GATATCTAAT ACGACTCACT ATAGGGAGAG GTATGGTACT TGGTAGTT 48

(2) INFORMATION FOR SEQ ID NO:19:

[0288] (i) SEQUENCE CHARACTERISTICS: [0289] (A) LENGTH: 53 base pairs [0290] (B) TYPE: nucleic acid [0291] (C) STRANDEDNESS: single [0292] (D) TOPOLOGY: linear [0293] (ii) MOLECULE TYPE: DNA [0294] (A) DESCRIPTION:/desc=“synthetic” [0295] (iii) HYPOTHETICAL: NO [0296] (iv) ANTI-SENSE: NO [0297] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GATATCTAAT ACGACTCACT ATAGGGAGAC TAGTGATGTT CTTGTTAACA ACT 53

(2) INFORMATION FOR SEQ ID NO:20:

[0298] (i) SEQUENCE CHARACTERISTICS: [0299] (A) LENGTH: 48 base pairs [0300] (B) TYPE: nucleic acid [0301] (C) STRANDEDNESS: single [0302] (D) TOPOLOGY: linear [0303] (ii) MOLECULE TYPE: DNA [0304] (A) DESCRIPTION:/desc=“synthetic” [0305] (iii) HYPOTHETICAL: NO [0306] (iv) ANTI-SENSE: NO [0307] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: GATATCTAAT ACGACTCACT ATAGGGAGAG GTATGGTACT TGGTAGTT 48

(2) INFORMATION FOR SEQ ID NO:21:

[0308] (i) SEQUENCE CHARACTERISTICS: [0309] (A) LENGTH: 25 base pairs [0310] (B) TYPE: nucleic acid [0311] (C) STRANDEDNESS: single [0312] (D) TOPOLOGY: linear [0313] (ii) MOLECULE TYPE: DNA [0314] (A) DESCRIPTION:/desc=“synthetic” [0315] (iii) HYPOTHETICAL: NO [0316] (iv) ANTI-SENSE: NO [0317] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: TCTAATACGA CTCACTATAG GGAGA