AU2018101287A4 - Method and pharmaceutical composition for treating cancer - Google Patents

Method and pharmaceutical composition for treating cancer Download PDF

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AU2018101287A4
AU2018101287A4 AU2018101287A AU2018101287A AU2018101287A4 AU 2018101287 A4 AU2018101287 A4 AU 2018101287A4 AU 2018101287 A AU2018101287 A AU 2018101287A AU 2018101287 A AU2018101287 A AU 2018101287A AU 2018101287 A4 AU2018101287 A4 AU 2018101287A4
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methyladenosine
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Kai-yue CAO
Zhi-hong JIANG
Tong-Meng YAN
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Increasepharm Hk Ltd
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Abstract

A method of treating a subject suffering from cancer comprising a step of administering an effective amount of a RNA molecule to the subject, wherein the RNA 5 molecule is isolated or derived from a plant of the genus Taxus. A method of inhibiting growth or proliferation of cancer cells comprising a step of contacting said cells with said RNA molecule; and a pharmaceutical composition for treating cancer comprising said RNA molecule and a pharmaceutically tolerable excipient. Also a double stranded RNA molecule and a recombinant vector comprising the double-stranded 10 RNA molecule. rCl r-r spe8Cojqn

Description

METHOD AND PHARMACEUTICAL COMPOSITION FOR TREATING CANCER
TECHNICAL FIELD
The present invention relates to a method of treating a subject suffering from cancer by administering a nucleic acid to the subject. Said nucleic acid is in particular but not exclusively a RNA molecule. The invention further relates to a pharmaceutical composition comprising a nucleic acid for the treatment and use thereof.
BACKGROUND OF THE INVENTION
Cancer has become the most common disease causing death worldwide. Traditional Chinese medicines (TCMs) have been applied for treating and preventing cancer whereas lots of research efforts have been contributed to investigate the effectiveness of isolated small molecules such as alkaloids, terpenoids, flavonoids or the like in treating cancer. Some alkaloids are found to have effect in inhibiting cancer such as by enhancing the efficacy of an anti-cancer drug. However, most of them are often toxic to human. Also, macromolecules such as DNAs, RNAs, and proteins are generally considered unstable and have poor effect in living human body and therefore have not been widely considered as suitable in said treatment.
Currently, some studies show that non-coding RNAs (ncRNAs) such as microRNAs have diverse regulatory roles through targeting different aspects of RNA transcription or post-transcription process in nearly all eukaryotic organisms. Mlotshwa, S. et al. (Cell research 2015, 25 (4), 521-4) suggested that exogenous plant microRNAs in foods could be taken up by the mammalian digestive tract and trafficked via the bloodstream to a variety of tissue cells, where they are capable of regulating the expression of mammalian genes. Goodarzi, H. et al. (Cell 2015, 161 (4), 790-802) revealed that endogenous tRNA derived fragments could suppress the stability of multiple oncogenic transcripts in breast cancer cells through binding and antagonizing activities of pathogenesis-related RNA-binding proteins. Nevertheless, there still remains a need to derive effective molecules from various sources such as plants for treatments.
Taxus chinensis (Pilger) Rehd. var. mairei, a species from the family of Taxaceae, is an ornamental evergreen shrub or tree widely distributed in high elevations of China.
2018101287 04 Sep 2018
As an important medicinal plant, it has been exploited for production of small molecular anti-cancer drugs such as paclitaxel which is also called Taxol. However, patients have been found to develop resistance against commonly used drugs including Taxol and therefore there remains a continuing need for new and improved 5 treatments for patients with cancer and for those having resistance against commonly used drugs and/or associated with different complications.
SUMMARY OF THE INVENTION
In a first aspect, the invention provides a method of treating a subject suffering from cancer, said method comprising the step of administering an effective amount of a RNA molecule said subject. The RNA molecule administered according to the invention is isolated or derived from a plant of the genus Taxus.
In an embodiment, the RNA molecule comprises a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue thereof.
Preferably, the RNA molecule of the invention has a sequence length of from about 50 to 200 nucleotides or 10 to 30 base pairs.
In an embodiment, the RNA molecule is a non-coding molecule in particular a transfer RNA molecule.
In an alternative embodiment, the RNA molecule is a double-stranded RNA molecule 25 comprising a sense sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue therefore, and a complementary antisense sequence.
In another aspect, the invention provides a method of inhibiting growth or proliferation of cancer cells comprising a step of contacting said cancer cells with an effective 30 amount of a RNA molecule isolated or derived from a plant of the genus Taxus.
In an example embodiment, the cancer cells of the present invention are ovarian cancer cells, liver cancer cells, breast cancer cells, colorectal cancer cells, or lung cancer cells.
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In a further aspect, the invention pertains to a pharmaceutical composition for treating cancer. The pharmaceutical composition comprises an RNA molecule and a pharmaceutically tolerable excipient, wherein said RNA molecule is isolated or derived from a plant of the genus Taxus.
Still further, the invention relates to a double-stranded RNA molecule comprising a sense sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue therefore, a complementary antisense sequence, and optionally a 3’ overhang.
In another aspect, the invention pertains to a recombinant vector comprising the double-stranded RNA molecule.
The invention provides a novel and effective approach for treating cancers from 15 various origins by administration of a RNA molecule that is isolated or derived from a plant of the genus Taxus, or in particular a RNA molecule comprising a sequence selected from SEQ ID NO: 1 to 100. Administration of said RNA molecule is also suitable for inhibiting growth or proliferation of cancer cells. The inventors have found that non-coding RNA molecules isolated from a plant of the genus Taxus, particularly 20 transfer RNA molecules, and RNA molecules derived from Taxus are particularly useful in treatment of cancer. The RNA molecules with a sequence length of about 10 to 200 nucleotides are highly effective at inhibiting growth and proliferation of cancer cells in vitro and exhibit an antitumor effect in vivo. Said RNA molecules are also effective against Taxol-resistant cell lines. Further, the pharmaceutical composition 25 comprising the RNA molecule that is isolated or derived from a plant of the genus
Taxus and a pharmaceutically tolerant excipient can act directly on the cancer cells or tumor, and therefore can have a faster-acting therapeutic effect.
Those skilled in the art will appreciate that the invention described herein is 30 susceptible to variations and modifications other than those specifically described.
The invention includes all such variations and modifications. The invention also includes all steps and features referred to or indicated in the specification, individually or collectively, and any and all combinations of the steps or features.
Other features and aspects of the invention will become apparent by consideration of the following detailed description and accompanying drawings.
2018101287 04 Sep 2018
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows gel electrophoresis profiles of RNA molecules from Taxus Chinensis (Pilger) Rehd. var. mairei, including low range RNA markers (denoted as “Ladder”), small RNA molecules, and transfer RNATrp(CCA) in accordance with an example embodiment.
Fig. 2 is a bar chart showing read length distribution of transfer RNAs from Taxus 10 chinensis (Pilger) Rehd. var. mairei in accordance with an example embodiment.
Fig. 3 is a bar chart showing the cytotoxicity of 25 nM RNA molecules tRNAHis(GUG), tRNAGIU(UUC), tRNATrp(CCA), tRNALeu(CAA), or tRNAArg(ACG) from Taxus chinensis (Pilger) Rehd. var. mairei on A2780 cell line, HepG2 cell line, and MCF-7 cell line compared to 15 a control group and a RNAiMAX group where a transfection reagent was added to the cells, in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
Fig. 4A is a bar chart showing the cell viability of A2780 cells after treatment with a 20 RNA molecule tRNATrp(CCA) at different concentrations, i.e. 0.78 nM, 1.56 nM, 3.13 nM,
6.25 nM, 12.5 nM and 25 nM, compared to a control group and a RNAiMAX group in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
Fig. 4B is a bar chart showing the cell viability of A2780 cells after treatment with Taxol at different concentrations, i.e. 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM, compared to a control group and a RNAiMAX group in accordance with an example embodiment (meant SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
Fig. 5A is a bar chart showing the cell viability of A2780 cells after treatment with different RNA molecules derived from Taxus Chinensis (Pilger) Rehd. var. mairei with a sequence length of 22 bp at a dose of 50 nM, compared to a control group and a RNAiMAX group in accordance with an example embodiment (meant SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
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Fig. 5B is a bar chart showing the cell viability of A2780 cells after treatment with different RNA molecules derived from Taxus Chinensis (Pilger) Rehd. var. mairei with a sequence length of 19 bp at a dose of 50 nM, compared to a control group and a
RNAiMAX group in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
Fig. 5C is a bar chart showing the cell viability of Taxol-resistant A2780 cells (denoted as A2780T cells) after treatment with different RNA molecules derived from Taxus Chinensis (Pilger) Rehd. var. mairei with a sequence length of 22 bp at a dose of 50 10 nM, compared to a control group and a RNAiMAX group in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
Fig. 5D is a bar chart showing the cell viability of Taxol-resistant A2780T cells after treatment with different RNA molecules derived from Taxus Chinensis (Pilger) Rehd.
var. mairei with a sequence length of 19 bp at a dose of 50 nM, compared to a control group and a RNAiMAX group in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
Fig. 5E is a bar chart showing cell viability of HCT-8 cells after treatment with different 20 RNA molecules derived from Taxus Chinensis (Pilger) Rehd. var. mairei with a sequence length of 22 bp at a dose of 50 nM, compared to a control group and a RNAiMAX group in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
Fig. 5F is a bar chart showing cell viability of Taxol-resistant HCT-8 cells (denoted as HCT-8T cells) after treatment with different RNA molecules derived from Taxus Chinensis (Pilger) Rehd. var. mairei with a sequence length of 22 bp at a dose of 50 nM, compared to a control group and a RNAiMAX group in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
Fig. 6A is a bar chart showing the cell viability of A2780 cells after treatment with RNA molecule HC11 at different concentrations, i.e. 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM, compared to a control group and a RNAiMAX group in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
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Fig. 6B is a bar chart showing the cell viability A2780 cells after treatment with Taxol at different concentrations, i.e. 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM and 200 nM, compared to a control group in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
Fig. 6C is a bar chart showing the cell viability of Taxol-resistant A2780T cells after treatment with RNA molecule HC11 at different concentrations, i.e. 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM and 200 nM, compared to a control group and a RNAiMAX group in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, 10 p<0.001 vs. vehicle control).
Fig. 6D is a bar chart showing the cell viability of Taxol-resistant A2780T cells after treatment with Taxol at different concentrations, i.e. 0.16 μΜ, 0.8 μΜ, 4μΜ, 20 μΜ and 100 μΜ, compared to a control group in accordance with an example embodiment 15 (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
Fig. 6E is a bar chart showing the cell viability of Taxol-resistant A549T cells after treatment with RNA molecule HC11 at different concentrations, i.e. 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM and 200 nM, compared to a control group and a RNAiMAX 20 group in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
Fig. 6F is a bar chart showing the cell viability of Taxol-resistant A549T cells after treatment with Taxol at different concentrations, i.e. 0.032 μΜ, 0.16 μΜ, 0.8 μΜ, 4 μΜ, 25 20 μΜ and 100 μΜ, compared to a control group in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
Fig. 6G is a bar chart showing the cell viability of HCT-8 cells after treatment with RNA molecule HC36 at different concentrations, i.e. 6.25 nM, 12.5 nM, 25 nM, 50 nM and 30 100 nM, compared to a control group and a RNAiMAX group in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
Fig. 6H is a bar chart showing the cell viability of HCT-8 cells after treatment with RNA molecule HC37 at different concentrations, i.e. 6.25 nM, 12.5 nM, 25 nM, 50 nM and 35 100 nM, compared to a control group and a RNAiMAX group in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
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Fig. 61 is a bar chart showing the cell viability of HCT-8 cells after treatment with Taxol at different concentrations, i.e. 50 nM, 100 nM, 200 nM, 300 nM, 400 nM and 500 nM, compared to a control group in accordance with an example embodiment (mean+ SD n=3; *, p<0.05, **, p<0.001 vs. vehicle control).
Fig. 7A is a line graph showing the ratio of tumor volume of xenograft implanted A2780 cells in mice over time, in which the mice were treated with RNA molecule HC11 or HC30 with atelocollagen at a dose of 2.4mg/kg once a week, compared to 10 1mg/kg Taxol and a control group.
Fig. 7B is a line graph showing the ratio of weight changes of mice having xenograft implanted A2780 cells, in which the mice were treated with RNA molecule HC11 or HC30 with atelocollagen at a dose of 2.4mg/kg once a week, compared to 1mg/kg 15 Taxol and a control group.
DETAILED DESCRIPTION OF THE EMBODIMENTS
Unless otherwise defined, all technical terms used herein have the same meaning as 20 commonly understood by one skilled in the art to which the invention belongs.
As used herein, “comprising” means including the following elements but not excluding others. “Essentially consisting of” means that the material consists of the respective element along with usually and unavoidable impurities such as side 25 products and components usually resulting from the respective preparation or method for obtaining the material such as traces of further components or solvents. “Consisting of” means that the material solely consists of, i.e. is formed by the respective element. As used herein, the forms “a,” “an,” and “the,” are intended to include the singular and plural forms unless the context clearly indicates otherwise.
The present invention in the first aspect provides a method of treating a subject suffering from cancer. The method comprises a step of administering an effective amount of a RNA molecule to said subject. The RNA molecule administered according to the present invention may be naturally present, modified or artificially 35 synthesized according to the sequences disclosed in the present invention, and
2018101287 04 Sep 2018 preferably the RNA molecule is isolated or derived from a plant of the genus Taxus. The RNA molecule of the present invention is not provided in the form of boiled extract obtained from the plant such as decoction, as it would be appreciated that RNA molecule is susceptible to spontaneous degradation at elevated temperature, alkaline pH, and the presence of nucleases or divalent metal ions. In an embodiment, the RNA molecule of the present invention is provided together with a gene delivery carrier which will be described in detail later.
The RNA molecule of the present invention has a sequence length of from about 10 to 200 nucleotides which can be regarded as a small RNA molecule. Preferably, the RNA molecule has a sequence length of from about 50 to about 200 nucleotides, from about 60 to about 150 nucleotides, in particular from about 70 to about 100 nucleotides.
The RNA molecule of the present invention comprises a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue thereof. The term “functional variant” of the RNA molecule refers to a molecule substantially similar to said RNA molecule with one or more sequence alterations that do not affect the biological activity or function of the RNA molecule. The alterations in sequence that do not affect the functional properties of the resultant RNA molecules are well known in the art. For example, nucleotide changes which result in alteration of the -5’-terminal and - 3’-terminal portions of the molecules would not be expected to alter the activity of the polynucleotides. In an embodiment, the RNA molecule of the present invention comprises at least one modified nucleoside selected from inosine, 1-methyladenosine, 2-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, 2'-Omethyladenosine, N6-acetyladenosine, 1-methylinosine, pseudouridine, dihydrouridine, or 2-methylthio-N6-methyladenosine.
In particular, the functional variant of the RNA molecule has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the non-variant RNA molecule according to the present invention.
The term “homologue” used herein refers to nucleotides having a sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, at least 90 % or at least 95% to the RNA molecules according to the present invention. In an embodiment, the 8
2018101287 04 Sep 2018 homologue of the RNA molecule has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the RNA molecule.
In an embodiment, the RNA molecule is a non-coding molecule preferably selected from a transfer RNA molecule, a ribosomal RNA molecule, a micro RNA molecule, a siRNA molecule, or a piwi-interacting RNA molecule; and more preferably is a transfer RNA molecule. tRNA molecules are highly conserved RNAs with function in various cellular processes such as reverse transcription, porphyrin biosynthesis or the like. In a particular embodiment, the RNA molecule of the invention comprises a sequence 10 selected from SEQ ID NO: 201 to SEQ ID NO: 225 or a functional variant or homologue thereof; or the RNA molecule comprises SEQ ID NO: 201 to SEQ ID NO: 205 or a functional variant or homologue thereof; or the RNA molecule consists of a sequence selected from SEQ ID NO: 201 to SEQ ID NO: 225 or SEQ ID NO: 201 to SEQ ID NO: 205 or a functional variant or homologue thereof.
In an alternative embodiment where the RNA molecule is a small RNA molecule having a sequence length of from about 10 to about 30 base pairs, from about 15 to about 25 base pairs, from about 19 to about 22 base pairs, 19 base pairs or 22 base pairs. The RNA molecule comprises a sequence selected from SEQ ID NO: 1 to SEQ 20 ID NO: 100 or a functional variant or homologue thereof, in particular SEQ ID NO: 1 to
SEQ ID NO: 36 or a functional variant or homologue thereof; or consists of a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100, in particular SEQ ID NO: 1 to SEQ ID NO: 36 or a functional variant or homologue thereof. Preferably, the RNA molecule is a double-stranded RNA molecule having a sense sequence selected from 25 SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue thereof, and a complementary antisense sequence. The antisense sequence is complementary to the sense sequence and therefore the antisense sequence is preferably selected from SEQ ID NO: 101 to 200 or functional variant or homologue thereof. In a particular embodiment, the double-stranded RNA molecule of the present invention has a sense 30 sequence selected from SEQ ID NO: 1 to SEQ ID NO: 36 or a functional variant or homologue thereof, and a complementary antisense sequence selected from SEQ ID NO: 101 to SEQ ID NO: 136 or a functional variant or homologue thereof. The inventors unexpectedly found that the double-stranded RNA molecules of the present invention are particularly useful in treatment of cancer such as Taxol-resistant cancer 35 as described in detail below.
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The RNA molecule of the present invention is preferably isolated or derived from the plant of the genus Taxus. The plant of the genus Taxus includes but is not limited to Taxus baccata, Taxus brevifolia, Taxus chinensis, Taxus chinensis (Pilger) Rehd. var. mairei, Taxus yunanensis, Taxus wallischiana, Taxus cuspidate, Taxus sumatrana, 5 Taxus globasa, Taxus canadensis, and Taxus floridana. The plant of the genus Taxus may be the source of Taxol. In an embodiment, the RNA molecule is isolated or derived from Taxus chinensis.
In more detail, the preferred sequences of the RNA molecules of the present invention 10 are listed in Tables 1 and 2 below. In an embodiment, RNA molecules of SEQ ID NO:
201 to 225 as shown in Table 1 are isolated from a plant of genus Taxus in particular from Taxus chinensis. These sequences are obtained by extraction, RNA isolation and purification of the plant. The inventors determined these RNA molecules are associated with chloroplasts. One possible approach to obtain the RNA molecules 15 from a particular plant Taxus chinensis (Pilger) Rehd. var. mairei is illustrated in
Example 1. It would be appreciated that other suitable methods for obtaining the isolated and purified RNA molecules of the present invention according to the disclosure herein can be applied, and the methods can be subject to appropriate modification to obtain an improved yield of the RNA molecules, without departing from 20 the scope of the present invention.
Table 1. RNA molecules in particular tRNAs isolated from Taxus chinensis (Pilger) Rehd. var. mairei according to the present invention.
SEQ ID Length (mer)
NO. tRNA(s) Sequence (5' to 3')
201 tRNAHis<GUG) GCGGACGUAGCCAAGUGGUCCAAAGGCAGUGGAUUGUGA AUCCACCACGCGCGGGUUCAAUCCCCGUCGUUCGCCCCA 78
202 tRNAGlu(UUC) GCCCCUAUCGUCUAGUGGCCCAGGACAUCUCUCUUUCAA GGAGGCAACGGGGAUUCGAUUUCCCCUAGGGGUACCA 76
203 tRNATrp(CCA) GCGCUCUUAGUUCAGUGCGGUAGAACGCAGGUCUCCAAA ACCUGAUGCCGUAGGUUCAAAUCCUACAGAGCGCCA 75
GCCUUGAUGGUGAAAUGGUAGACACGCGAGACUCAAAAUC
204 tRNALeu(CAA) UCGUGCUAAACAGCGUGGAGGUUCGAAUCCUCUUCAAGG 84
CACCA
205 tRNAArg(ACG) GGGCCUGUAGCUCAGAGGAUUAGAGCACGUGGUUGCGAA CCACGGUGUCGGGGGUUCGAAUCCCUCCUCGCCCACCA 77
206 tRNAAsp(GUC) GGGAUUGUAGUUCAAUUGGUUAGAGUACCGCCCUGUCAA GACGGAAGUUGCGGGUUCGAGCCCCGUCAGUCCCGCCA 77
2018101287 04 Sep 2018
207 tRNAAsn(GUU) UCCUCAGUAGCUCAGUGGUAGAGCGGUCGGCUGUUAACC GAUUGGUCGUAGGUUCAAAUCCUAUUUGAGGAGCCA 75
208 tRNACys(GCA) GGCGACAUAGCCAAGUGGUAAGGCAGGGGACUGCAAAUC CCCCAUCCCCAGUUCAAAUCCGGGUGUCGCCUCCA 74
209 tRNAGln(UUG) GGGGCGUGGCCAAGCGGUAAGGCAACAGGUUUUGGUCCU GUUAUUGCGAAGGUUCGAAUCCUUUCGUCCCAGCCA 75
210 tRNAGly(GCC) GGGUAUUGUUUAAUGGAUAAAAUUUAUUCUUGCCAAGGAU AAGAUGCGGGUUCGAUUCCCGCUACCCGCCCA 72
211 tRNAlle(UAU) AGGGAUAUAACU CAG UAGUAGAGU GU CACCU U UAU GU GG UGAAAGUCAUCAGUUCAAACCUGAUUAUCCCUACCA 75
GCCGCCAUGGUGAAAUUGGUAGACACGCUGCUCUUAGGA
212 tRNALeu(UAG) AGCAGUGCUAGAGCAUCUCGGUUCGAAUCCGAGUGGUGG 83
CACCA
GGGGAUAUGGCGGAAUUGGUAGACGCUACGGACUUAAAA
213 tRNALeu(UAA) AAUCCGUUGGUUUUAUAAACCGUGAGGGUUCAAGUCCCU 90
CUAUCCCCACCA
214 tRNALys(uuu) GGGUUGUUAACUCAAUGGUAGAGUACUCGGCUUUUAACC GAcGAGUUCCGGGUUCAAGUCCCGGGCAACCCACCA 75
215 tRNAMet(CAU) GCAUCCAUGGCUGAAUGGUCAAAGCACCCAACUCAUAAUU GGGAAGUCGCGGGUUCAAUUCCUGCUGGAUGCACCA 76
216 tRNAMet(CAU) CGCGGAGUAGAGCAGUUUGGUAGCUCGCAAGGCUCAUAA CCUUGAAGUCACGGGUUCAAAUCCCGUCUCCGCAACCA 77
217 tRNAphe(GAA) GUCGGGAUAGCUCAGUUGGUAGAGCAGAGGACUGAAAAU CCUCGUGUCACCAGUUCAAAUCUGGUUCCUGGCACCA 76
218 tRNAPro(UGG) AGGGAUGUAGCGCAGCUUGGUAGCGCGUUUGUUUUGGGU ACAAAAUGUCGCAGGUUCAAAUCCUGUCAUCCCUACCA 77
219 tRNAPro(GGG) CGGAGCAUAACGCAGUUUGGUAGCGUGCCAUCUUGGGGU GAUGGAGGUCGCGGGUUCAAAUCCUGUUGCUCCGACCA 77
GGAGAGAUGGCCGAGUGGUUGAUGGCUCCGGUCUUGAAA
220 tRNASer(UGA) ACCGGUAUAGUUUUAAAAACUAUCGAGGGUUCGAAUCCCU 91
CUCUCUCCUCCA
GGAGAGAUGGCUGAGCGGACUAAAGCGGUGGAUUGCUAA
221 tRNASer(GCU) UCCGUUGUACAGACUAUCUGUACCGAGGGUUCGAAUCCC 91
UCUUUCUCCGCCA
222 tRNAThr(UGU) GCCUGCUUAGCUCAGAGGUUAGAGCAUCGCACUUGUAAU GCGACGGUCAUCGGUUCGAUCCCGAUAGAAGGCUCCA 76
223 tRNAThr(GGU) GCACUUUUAACUCAGUGGUAGAGUAACGCCAUGGUAAGG CGUAAGUCAUCGGUUCAAGCCCGAUAAAGGGCUCCA 75
GGGUCGAUGCCCGAGUGGCUAAUGGGGACGGACUGUAAA
224 tRNATyr(GUA) UCCGUUGGCAAUAUGCUUACGCUGGUUCAAAUCCAGCUC 87
GGCCCACCA
225 tRNAArg(cuc) GCGUCCAUCGUCUAAUGGAUAGGACAGAGGUCUUCUAAA CCUUAGGUAUAGGUUCAAAUCCUAUUGGACGUACCA 75
2018101287 04 Sep 2018
The sense sequences of SEQ ID NO: 1 to SEQ ID NO: 100 and the antisense sequences of SEQ ID NO: 101 to SEQ ID NO: 200 as shown in Table 2 are artificially synthesized in accordance with the present invention. In particular, these sequences are derived sequence fragments prepared according to the sequences in Table 1 isolated from Taxus chinensis (Pilger) Rehd. var. mairei. Each of the sense sequences together with the corresponding antisense sequence form a doublestranded RNA molecule. As shown in Table 2, the sense sequence of SEQ ID NO: 1 and the antisense sequence of SEQ ID NO: 101 form a double-stranded RNA molecule with a length of 22 base pairs, and the resultant RNA molecule is denoted as HC11 for easy reference. Similarly, the sense sequence of SEQ ID NO: 2 and the antisense sequence of SEQ ID NO: 102 form a double-stranded RNA molecule with a length of 19 base pairs, and the resultant RNA molecule is denoted as HC20. Other RNA molecules of the present invention are presented in the Table.
The double-stranded RNA molecules are classified into 2 groups, namely a 5’-terminal group (5’-t), and a 3’-terminal group (3’-t). The 5’-t group RNA molecules contain a 5’ terminal portion of the corresponding full-length RNA molecules isolated from the plant; and the 3’-t group RNA molecules contain a 3’ terminal portion of the corresponding full-length RNA molecules isolated from the plant. In another embodiment, RNA molecules may contain the anticodon loop portion of the corresponding full-length RNA molecules isolated from the plant and referred as anticodon group RNA molecules. The sense sequences of SEQ ID NO: 1 to SEQ ID NO: 100 can be generated by cleavage at different sites on the full-length RNA molecules SEQ ID NO: 201 to 225.
Further, the RNA molecule of the present invention may comprise a 3' overhang, preferably comprise 2 mer 3' overhangs. The provision of the 3’ overhang improves the stability of the RNA molecules.
Table 2. RNA molecules derived from the sequences in Table 1 through artificial synthesis according to the present invention.
SEQ Sense sequence SEQ Antisense Length
Source Code ID ID sequence Group
(5' to 3') (bp)
NO. NO. (5' to 3')
2018101287 04 Sep 2018
tRNAHis<GUG> 1 2 GCGGACGUAGCC UGGACCACUUGGC
HC11 HC20 AAGUGGUCCA GCGGACGUAGCC AAGUGGU 101 102 UACGUCCGC ACCACUUGGCUAC GUCCGC 22
19 5'-t
UCAAUCCCCGUC UGGGGCGAACGAC
HC12 3 GUUCGCCCCA 103 GGGGAUUGA 22 3'-t
AUCCCCGUCGUU UGGGGCGAACGAC
HC42 4 CGCCCCA 104 GGGGAU 19
tRNAGlu(UUC) HC16 5 GCCCCUAUCGUC 105 UGGGCCACUAGAC 22
UAGUGGCCCA GAUAGGGGC 5'-t
GCCCCUAUCGUC GCCACUAGACGAU
HC25 6 UAGUGGC 106 AGGGGC 19
UCGAUUUCCCCU UGGUACCCCUAGG
HC17 7 AGGGGUACCA 107 GGAAAUCGA 22 3'-t
AUUUCCCCUAGG UGGUACCCCUAGG
HC43 8 GGUACCA 108 GGAAAU 19
tRNATrp(CCA) HC30 9 GCGCUCUUAGUU 109 UACCGCACUGAAC 22
CAGUGCGGUA UAAGAGCGC 5'-t
GCGCUCUUAGUU CGCACUGAACUAA
HC23 10 CAGUGCG 110 GAGCGC 19
GUUCAAAUCCUA UGGCGCUCUGUA
HC31 11 CAGAGCGCCA 111 GGAUUUGAAC 22 3'-t
CAAAUCCUACAGA UGGCGCUCUGUA
HC46 12 GCGCCA 112 GGAUUUG 19
tRNALeu(CAA) HC18 13 GCCUUGAUGGUG 113 UCUACCAUUUCAC 22 5'-t
AAAUGGUAGA CAUCAAGGC
HC22 14 GCCUUGAUGGUG 114 ACCAUUUCACCAU 19
AAAUGGU CAAGGC
HC19 15 UCGAAUCCUCUU 115 UGGUGCCUUGAA 22 3'-t
CAAGGCACCA GAGGAUUCGA
HC44 16 AAUCCUCUUCAA 116 UGGUGCCUUGAA 19
GGCACCA GAGGAUU
tRNAAr9(ACG) HC32 17 GGGCCUGUAGCU 117 UAAUCCUCUGAGC 22 5'-t
CAGAGGAUUA UACAGGCCC
HC24 18 GGGCCUGUAGCU 118 UCCUCUGAGCUAC 19
CAGAGGA AGGCCC
HC33 19 UCGAAUCCCUCC 119 UGGUGGGCGAGG 22 3'-t
UCGCCCACCA AGGGAUUCGA
2018101287 04 Sep 2018
HC47 20 AAUCCCUCCUCG CCCACCA 120 UGGUGGGCGAGG AGGGAUU 19
tRNAAsp(GUC) HC28 21 GGGAUUGUAGUU 121 UAACCAAUUGAAC 22 5'-t
CAAUUGGUUA UACAAUCCC
HC21 22 GGGAUUGUAGUU 122 CCAAUUGAACUAC 19
CAAUUGG AAUCCC
HC29 23 UCGAGCCCCGUC 123 UGGCGGGACUGA 22 3'-t
AGUCCCGCCA CGGGGCUCGA
HC45 24 AGCCCCGUCAGU 124 UGGCGGGACUGA 19
CCCGCCA CGGGGCU
tRNACys(GCA) HC34 25 GGCGACAUAGCC 125 CUUACCACUUGGC 22 5'-t
AAGUGGUAAG UAUGUCGCC
HC26 26 GGCGACAUAGCC 126 ACCACUUGGCUAU 19
AAGUGGU GUCGCC
HC35 27 UCAAAUCCGGGU 127 UGGAGGCGACAC 22 3'-t
GUCGCCUCCA CCGGAUUUGA
HC48 28 AAUCCGGGUGUC 128 UGGAGGCGACAC 19
GCCUCCA CCGGAUU
tRNAAsn(GUU) HC36 29 CCUCAGUAGCUC 129 CUCUACCACUGAG 22 5'-t
AGUGGUAGAG CUACUGAGG
HC27 30 CCUCAGUAGCUC 130 UACCACUGAGCUA 19
AGUGGUA CUGAGG
HC37 31 GGUUCAAAUCCU 131 CUCCUCAAAUAGG 22 3'-t
AUUUGAGGAG AUUUGAACC
HC49 32 UCAAAUCCUAUU 132 CUCCUCAAAUAGG 19
UGAGGAG AUUUGA
tRNAMet(CAU) HC38 33 CGCGGAGUAGAG 133 UACCAAACUGCUC 22 5'-t
CAGUUUGGUA UACUCCGCG
HC40 34 CGCGGAGUAGAG 134 CAAACUGCUCUAC 19
CAGUUUG UCCGCG
HC39 35 GGUUCAAAUCCC 135 UUGCGGAGACGG 22 3'-t
GUCUCCGCAA GAUUUGAACC
HC41 36 UCAAAUCCCGUC 136 UUGCGGAGACGG 19
UCCGCAA GAUUUGA
tRNAThr(UGU) HC50 37 GCCUGCUUAGCU 137 CUAACCUCUGAGC 22 5'-t
CAGAGGUUAG UAAGCAGGC
HC52 38 GCCUGCUUAGCU 138 ACCUCUGAGCUAA 19
CAGAGGU GCAGGC
HC51 39 UCGAUCCCGAUA 139 UGGAGCCUUCUAU 22 3'-t
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HC53 GAAGGCUCCA CGGGAUCGA UGGAGCCUUCUAU CGGGAU 19
40 AUCCCGAUAGAA GGCUCCA 140
tRNAPro(UGG) HC54 41 AGGGAUGUAGCG 141 UACCAAGCUGCGC 22 5'-t
CAGCUUGGUA UACAUCCCU
HC56 42 AGGGAUGUAGCG 142 CAAGCUGCGCUAC 19
CAGCUUG AUCCCU
HC55 43 UCAAAUCCUGUC 143 UGGUAGGGAUGA 22 3'-t
AUCCCUACCA CAGGAUUUGA
HC57 44 AAUCCUGUCAUC 144 UGGUAGGGAUGA 19
CCUACCA CAGGAUU
tRNAG'y(GCC) HC58 45 GGGUAUUGUUUA 145 UUUUAUCCAUUAA 22 5'-t
AUGGAUAAAA ACAAUACCC
HC60 46 GGGUAUUGUUUA 146 UAUCCAUUAAACA 19
AUGGAUA AUACCC
HC59 47 UUCGAUUCCCGC 147 UGGGCGGGUAGC 22 3'-t
UACCCGCCCA GGGAAUCGAA
HC61 48 GAUUCCCGCUAC 148 UGGGCGGGUAGC 19
CCGCCCA GGGAAUC
tRNATyr(GUA) HC62 49 GGGUCGAUGCCC 149 UUAGCCACUCGG 22 5'-t
GAGUGGCUAA GCAUCGACCC
HC64 50 GGGUCGAUGCCC 150 GCCACUCGGGCA 19
GAGUGGC UCGACCC
HC63 51 UCAAAUCCAGCU 151 UGGUGGGCCGAG 22 3'-t
CGGCCCACCA CUGGAUUUGA
HC65 52 AAUCCAGCUCGG 152 UGGUGGGCCGAG 19
CCCACCA CUGGAUU
tRNALeu(UAA) HC66 53 GGGGAUAUGGCG 153 CUACCAAUUCCGC 22 5'-t
GAAUUGGUAG CAUAUCCCC
HC68 54 GGGGAUAUGGCG 154 CCAAUUCCGCCAU 19
GAAUUGG AUCCCC
HC67 55 UCAAGUCCCUCU 155 UGGUGGGGAUAG 22 3'-t
AUCCCCACCA AGGGACUUGA
HC69 56 AGUCCCUCUAUC 156 UGGUGGGGAUAG 19
CCCACCA AGGGACU
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tRNASer(UGA) HC70 HC72 57 58 GGAGAGAUGGCC GAGUGGUUGA GGAGAGAUGGCC GAGUGGU 157 158 UCAACCACUCGGC CAUCUCUCC ACCACUCGGCCAU CUCUCC 22 19 5'-t
HC71 59 UCGAAUCCCUCU 159 UGGAGGAGAGAG 22 3'-t
CUCUCCUCCA AGGGAUUCGA
HC73 60 AAUCCCUCUCUC 160 UGGAGGAGAGAG 19
UCCUCCA AGGGAUU
tRNAGln(UUG) HC74 61 GGGGCGUGGCCA 161 CCUUACCGCUUG 22 5'-t
AGCGGUAAGG GCCACGCCCC
HC76 62 GGGGCGUGGCCA 162 UACCGCUUGGCCA 19
AGCGGUA CGCCCC
HC75 63 UCGAAUCCUUUC 163 UGGCUGGGACGA 22 3'-t
GUCCCAGCCA AAGGAUUCGA
HC77 64 AAUCCUUUCGUC 164 UGGCUGGGACGA 19
CCAGCCA AAGGAUU
tRNAAr9(CUC) HC78 65 GCGUCCAUCGUC 165 CUAUCCAUUAGAC 22 5'-t
UAAUGGAUAG GAUGGACGC
HC80 66 GCGUCCAUCGUC 166 UCCAUUAGACGAU 19
UAAUGGA GGACGC
HC79 67 UCAAAUCCUAUU 167 UGGUACGUCCAAU 22 3'-t
GGACGUACCA AGGAUUUGA
HC81 68 AAUCCUAUUGGA 168 UGGUACGUCCAAU 19
CGUACCA AGGAUU
tRNAMet(CAU) HC82 69 GCAUCCAUGGCU 169 UUGACCAUUCAGC 22 5'-t
GAAUGGUCAA CAUGGAUGC
HC84 70 GCAUCCAUGGCU 170 ACCAUUCAGCCAU 19
GAAUGGU GGAUGC
HC83 71 UCAAUUCCUGCU 171 UGGUGCAUCCAG 22 3'-t
GGAUGCACCA CAGGAAUUGA
HC85 72 AUUCCUGCUGGA 172 UGGUGCAUCCAG 19
UGCACCA CAGGAAU
tRNALeu(UAG) HC86 73 GCCGCCAUGGUG 173 CUACCAAUUUCAC 22 5'-t
AAAUUGGUAG CAUGGCGGC
HC88 74 GCCGCCAUGGUG 174 CCAAUUUCACCAU 19
AAAUUGG GGCGGC
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HC87 HC89 75 76 UCGAAUCCGAGU GGUGGCACCA AAUCCGAGUGGU GGCACCA 175 176 UGGUGCCACCACU CGGAUUCGA UGGUGCCACCACU CGGAUU 22 19 3'-t
tRNALys(UUU) HC90 77 GGGUUGUUAACU 177 UCUACCAUUGAGU 22 5'-t
CAAUGGUAGA UAACAACCC
HC92 78 GGGUUGUUAACU 178 ACCAUUGAGUUAA 19
CAAUGGU CAACCC
HC91 79 UCAAGUCCCGGG 179 UGGUGGGUUGCC 22 3'-t
CAACCCACCA CGGGACUUGA
HC93 80 AGUCCCGGGCAA 180 UGGUGGGUUGCC 19
CCCACCA CGGGACU
tRNAPhe(GAA) HC94 81 GUCGGGAUAGCU 181 CUACCAACUGAGC 22 5'-t
CAGUUGGUAG UAUCCCGAC
HC96 82 GUCGGGAUAGCU 182 CCAACUGAGCUAU 19
CAGUUGG CCCGAC
HC95 83 UCAAAUCUGGUU 183 UGGUGCCAGGAA 22 3'-t
CCUGGCACCA CCAGAUUUGA
HC97 84 AAUCUGGUUCCU 184 UGGUGCCAGGAA 19
GGCACCA CCAGAUU
tRNAPro(GGG) HC98 85 CGGAGCAUAACG 185 UACCAAACUGCGU 22 5'-t
CAGUUUGGUA UAUGCUCCG
HC100 86 CGGAGCAUAACG 186 CAAACUGCGUUAU 19
CAGUUUG GCUCCG
HC99 87 UCAAAUCCUGUU 187 UGGUCGGAGCAA 22 3'-t
GCUCCGACCA CAGGAUUUGA
HC101 88 AAUCCUGUUGCU 188 UGGUCGGAGCAA 19
CCGACCA CAGGAUU
tRNASer(GCU) HC102 89 GGAGAGAUGGCU 189 UAGUCCGCUCAGC 22 5'-t
GAGCGGACUA CAUCUCUCC
HC104 90 GGAGAGAUGGCU 190 UCCGCUCAGCCAU 19
GAGCGGA CUCUCC
HC103 91 UCGAAUCCCUCU 191 UGGCGGAGAAAGA 22 3'-t
UUCUCCGCCA GGGAUUCGA
HC105 92 AAUCCCUCUUUC 192 UGGCGGAGAAAGA 19
UCCGCCA GGGAUU
tRNAThr(GGU) HC106 93 GCACUUUUAACU 193 UCUACCACUGAGU 22 5'-t
2018101287 04 Sep 2018
HC108 CAGUGGUAGA UAAAAGUGC ACCACUGAGUUAA AAGUGC 19
94 GCACUUUUAACU CAGUGGU 194
HC107 95 UCAAGCCCGAUA 195 UGGAGCCCUUUAU 22 3'-t
AAGGGCUCCA CGGGCUUGA
HC109 96 AGCCCGAUAAAG 196 UGGAGCCCUUUAU 19
GGCUCCA CGGGCU
tRNAe(UAU) HC110 97 AGGGAUAUAACU 197 UCUACUACUGAGU 22 5'-t
CAGUAGUAGA UAUAUCCCU
HC112 98 AGGGAUAUAACU 198 ACUACUGAGUUAU 19
CAGUAGU AUCCCU
HC111 99 UCAAACCUGAUU 199 UGGUAGGGAUAAU 22 3'-t
AUCCCUACCA CAGGUUUGA
HC113 100 AACCUGAUUAUC 200 UGGUAGGGAUAAU 19
CCUACCA CAGGUU
The inventors unexpectedly found that the RNA molecules isolated or derived from a plant of genus Taxus in particular Taxus chinensis (Pilger) Rehd. var. mairei are effective against cancer cells, in particular they are capable of inhibiting the growth, 5 proliferation and/ or metastasis of cancer cells.
Turning back to the method of treatment, the method comprises the step of administering an effective amount of a RNA molecule as described above to the subject suffering from a cancer. In an embodiment, the step of administering the RNA 10 molecule to the subject comprises contacting cancer cells of the subject with the RNA molecule.
The term “cancer” describes a physiological condition in subjects in which a population of cells are characterized by unregulated malignant (cancerous) cell growth.
In an embodiment, the cancer to be treated is ovarian cancer, liver cancer, breast cancer, colorectal cancer, or lung cancer. In a particular embodiment, the cancer is ovarian cancer, colorectal cancer or lung cancer. In an alternative embodiment, the RNA molecules of the present invention are effective in treating cancer which is resistant against currently existing drugs such as Taxol, i.e. can be used to treat cancer which is resistant against Taxol. Specifically, the RNA molecules of the present invention can be used to treat Taxol-resistant lung cancer, Taxol-resistant
2018101287 04 Sep 2018 colorectal cancer or Taxol-resistant ovarian cancer. Accordingly, the method of the present invention can be applied to treat a subject suffering from a multi-drug resistant cancer and related disorders.
The term “subject” used herein refers to a living organism and can include but is not limited to a human and an animal. The subject is preferably a mammal, preferably a human. The RNA molecules may be administered through injection to the subject, preferably a human. The term injection encompasses intravenous, intramuscular, subcutaneous and intradermal administration. In an embodiment, the RNA molecule 10 of the present invention is administered together with suitable excipient(s) to the subject through intravenous injection. For instance, the RNA molecule may be delivered to the subject or cells via transfection, electroporation or viral-mediated delivery.
The expression effective amount generally denotes an amount sufficient to produce therapeutically desirable results, wherein the exact nature of the result varies depending on the specific condition which is treated. In this invention, cancer is the condition to be treated and therefore the result is usually an inhibition or suppression of the growth or proliferation of cancer cells, a reduction of cancerous cells or the 20 amelioration of symptoms related to the cancer cells, in particular inhibition of the proliferation of the cancer cells or induction of cell death, i.e. apoptosis of the cancer cells. In an embodiment where the cancer is metastatic cancer, the result is usually an inhibition of migration of cancer cells, suppression of the invasion of cancer cells to other tissues, inhibition of formation metastasis cancer cells at a secondary site 25 distant from the primary site, or amelioration of symptoms related to metastatic cancer.
The effective amount of the RNA molecules of the present invention may depend on the species, body weight, age and individual conditions of the subject and can be determined by standard procedures such as with cell cultures or experimental animals.
A dosage of the RNA molecule such as RNA molecule HC11 (formed by SEQ ID NO: 1 and SEQ ID NO: 101) or HC30 (formed by SEQ ID NO: 9 and SEQ ID NO: 109) may, for example, be at least about 0.1 mg/kg to 5 mg/kg, or about 2 mg/kg to 5 mg/kg, in particular 2.4 mg/kg.
The RNA molecule of the present invention may be administered in form of a pharmaceutical composition comprising the RNA molecule and at least one
2018101287 04 Sep 2018 pharmaceutically tolerable excipient. The pharmaceutically tolerable excipient may be one or more of a diluent, a filler, a binder, a disintegrant, a lubricant, a coloring agent, a surfactant, a gene delivery carrier and a preservative. The pharmaceutical composition can be present in solid, semisolid or liquid form, preferably in liquid form.
The pharmaceutical composition may comprise further pharmaceutical effective ingredients such as therapeutic compounds which are used for treating cancer such as Taxol. The skilled person is able to select suitable pharmaceutically tolerable excipients depending on the form of the pharmaceutical composition and is aware of methods for manufacturing pharmaceutical compositions as well as able to select a 10 suitable method for preparing the pharmaceutical composition depending on the kind of pharmaceutically tolerable excipients and the form of the pharmaceutical composition.
In an embodiment, the RNA molecule is provided in a pharmaceutical composition 15 comprising a gene delivery carrier. The gene delivery carrier refers to any molecules that can act as a carrier for delivering a gene into a cell. In an embodiment where the RNA molecule is transfected into a cell, the gene delivery carrier is considered as a transfecting agent. In an embodiment where the RNA molecule is delivered through a recombinant viral vector, the gene delivery carrier is a viral vector carrying the double20 stranded RNA molecule of the present invention. The gene delivery carriers include, but is not limited to, a vector such as a viral vector, a collagen such as atelocollagen, a polymer such as polyethylenimine (PEI), a polypeptide such as poly (L-lysine) and protamine, and a lipid for forming a liposome such as Lipofectamine. The gene delivery carriers may be commercially available such as LipofectamineRNAiMAX 25 Transfection Reagent, Lipofectamine 3000 Reagent, and Lipofectamine® 2000
Transfection Reagent from Thermo Fisher, U.S.A.; RNAi-Mate from GenePharma, China; atelocollagen from Koken Co., Ltd., Japan); and Histidine-Lysine peptide copolymer from siRNAomics, China. The gene delivery carriers may be viral vectors based on retrovirus, adeno-associated virus, adenovirus, and lentivirus. The gene 30 delivery carriers should have a low toxicity and cannot induce significant immune response in the subject. In an embodiment, the RNA molecule is provided in a pharmaceutical composition comprising atelocollagen, wherein atelocollagen forms a complex with the RNA molecule for delivery. In another embodiment, the RNA molecule is provided in a pharmaceutical composition comprising Lipofectamine such 35 as Lipofectamine® RNAiMAX transfection reagent for delivering the RNA molecule to
2018101287 04 Sep 2018 the cells. In a further embodiment, the RNA molecule is inserted into a plasmid and form recombinant vector.
In an embodiment, the pharmaceutical composition may further comprise a nucleic acid stabilizer. The nucleic acid stabilizer refers to any chemicals that are capable of maintaining the stability of the RNA molecule in the composition to minimize or avoid degradation, in particular those having ability to deactivate activity of nucleases or the like degrading the RNA molecules.
Accordingly, the present invention also pertains to a pharmaceutical composition as described above, in particular comprising the RNA molecule and a pharmaceutically tolerable excipient as defined above. In an embodiment, the RNA molecule comprises at least one sequence selected from SEQ ID NO: 1 to 100 or a functional variant or homologue thereof. Preferably, the RNA molecule is isolated or derived from a plant of the genus Taxus as described above, in particular from Taxus chinensis.
The administration step of the RNA molecule according to the method of the present invention may be performed by injecting a pharmaceutical composition containing the RNA molecule to the target site of the subject, i.e. where cancer cells exist or body tissue adjacent to cancer cells. This is advantageous in that the RNA molecule can be directly delivered to the cancer cells before any cellular degradation such as first pass metabolism.
The RNA molecules of the present invention are also suitable for inhibiting growth or proliferation of cancer cells. In another aspect of the invention, there is provided a method of inhibiting growth or proliferation of cancer cells comprising a step of contacting said cells with an effective amount of a RNA molecule as defined above. Preferably the RNA molecule is isolated or derived from a plant of the genus Taxus or comprises a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue thereof. The cancer cells are as defined above. Preferably, the cancer cells are ovarian cancer cells, liver cancer cells, breast cancer cells, colorectal cancer cells, or lung cancer cells. The cancer cells may be resistant against currently existing cancer drugs such as but are not limited to Taxol.
In an embodiment, the RNA molecule has a sequence length of from about 50 to 200 nucleotides, more preferably has a length of from about 60 to about 150 nucleotides, 21
2018101287 04 Sep 2018 in particular from about 70 to about 100 nucleotides. The RNA molecule is a noncoding molecule preferably a transfer RNA molecule. Preferably, the RNA molecule comprises a sequence selected from SEQ ID NO: 201 to SEQ ID NO: 225 or a functional variant or homologue thereof; or the RNA molecule comprises SEQ ID NO: 201 to SEQ ID NO: 205 or a functional variant or homologue thereof; or the RNA molecule consists of a sequence selected from SEQ ID NO: 201 to SEQ ID NO: 225 or SEQ ID NO: 201 to SEQ ID NO: 205 or a functional variant or homologue thereof.
In an alternative embodiment, the RNA molecule has a sequence length of from about 10 to about 30 base pairs, from about 15 to about 25 base pairs, from about 19 to about 22 base pairs, 19 base pairs or 22 base pairs. Preferably, the RNA molecule is a double-stranded RNA molecule comprising a sense sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue thereof, in particular SEQ ID NO: 1 to SEQ ID NO: 36 or a functional variant or homologue thereof; or consists of a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100, in particular SEQ ID NO: 1 to SEQ ID NO: 36 or a functional variant or homologue thereof. The double-stranded RNA molecule comprises a complementary antisense sequence. The RNA molecule may further comprise 2 mer 3’ overhangs.
The step of contacting the cancer cells with the RNA molecule of the present invention may be carried out by applying a composition in particular an incubation solution comprising the RNA molecule to said cancer cells which incubation solution may further comprise suitable excipients as defined above, a buffer or a suitable growth medium. In such embodiment of the present invention, the cancer cells are taken from a subject such as an animal or human, in particular a human. The RNA molecule is provided in the composition at a concentration of at least 3 nM, at least 5 nM, from about 5 nM to about 200 nM, from about 10 nM to about 100 nM, or from about 25 nM to about 50 nM. Further, the excipients may include a gene delivery carrier such as but is not limited to a collagen based carrier or a liposome forming agent. In an embodiment, the collagen based carrier is atelocollagen and the liposome forming agent is Lipofectamine.
In addition to the above, the present invention pertains to a double-stranded RNA molecule as described above, i.e. comprising a sense sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue thereof, and a complementary antisense sequence. In particular, the double-stranded RNA molecule 22
2018101287 04 Sep 2018 consists of a sense sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue thereof, a complementary antisense sequence selected from SEQ ID NO: 101 to SEQ ID NO: 200, and optionally a 3’ overhang. Example embodiments of the double-stranded RNA molecule are presented in Table 5 2. The double-stranded RNA may be subject to modification and therefore may carry at least one modified nucleoside selected form inosine, 1-methyladenosine, 2methyladenosine, A^-methyladenosine, A^-isopentenyladenosine, 2'-Omethyladenosine, A^-acetyladenosine, 1-methylinosine, pseudouridine, dihydrouridine, or 2-methylthio-/\/6-methyladenosine.
In further aspect of the invention, there is provided a vector comprising a nucleic acid molecule, wherein the nucleic acid molecule is a RNA molecule as described above.
In particular, the RNA molecule having a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue thereof. In an embodiment, the 15 vector is a recombinant vector comprising the double-stranded RNA molecule as described above. The vector may be viral-based vector derived from retrovirus, adeno-associated virus, adenovirus, or lentivirus. An ordinary skilled in the art would appreciate suitable approach to incorporate the RNA molecule of the present invention into a vector.
Still further, the present invention pertains to use of a nucleic acid molecule in the preparation of a medicament for treating cancer. The nucleic acid is a RNA molecule as described above including a functional variant or homologue thereof. It would also be appreciated that the RNA molecule of the present invention can be used as a small 25 interfering RNA molecule to interfere the expression of certain genes in the target cancer cells, thereby to cause gene silencing, apoptosis, inhibition of cell growth and proliferation, or the like to achieve the desired therapeutic effect.
Accordingly, the present invention provides a novel and effective approach for treating cancers from various origins by administration of a RNA molecule that is isolated or derived from a plant of the genus Taxus, or in particular a RNA molecule comprising a sequence selected from SEQ ID NO: 1 to 100. Administration of said RNA molecule is also suitable for inhibiting growth or proliferation of cancer cells. The RNA molecules are found to be highly effective at inhibiting growth and proliferation of cancer cells in vitro and exhibit an antitumor effect in vivo. Said RNA molecules are also effective against Taxol-resistant cell lines.
2018101287 04 Sep 2018
The invention is now described in the following non-limiting examples.
EXAMPLES
Chemicals and materials
Fresh branches of Taxus chinensis (Pilger) Rehd. var. mairei were collected from Sanming City in the year of 2017 from Fujian Province, China. Cetrimonium bromide (CTAB) and sodium chloride were purchased from-Kingdin Industrial Co., Ltd. (Hong Kong, China). Water-saturated phenol was purchased from Leagene Co., Ltd. (Beijing, China). Chloroform and ethanol were purchased from Anaqua Chemicals Supply Inc. Ltd. (U.S.A.). Isopentanol and guanidinium thiocyanate were purchased from Tokyo Chemical Industry CO., Ltd. (Japan). Tris-HCI and ethylenediaminetetraacetic acid (EDTA) were purchased from Acros Organics (U.S.A), low range ssRNA ladder was purchased from New England Biolabs (Beverly, MA, U.S.A.). mirVanaTM miRNA isolation kit, SYBR gold nucleic acid gel stain and gel loading buffer II were purchased from Thermo Fisher Scientific (U.S.A.). 40% acrylamide/bis solution (19:1), tris/borate/EDTA (TBE), ammonium persulphate (APS) and tetramethylethylenediamine (TEMED) were purchased from Biorad Laboratories Inc. (U.S.A). Taxol-resistance adenocarcinomic human alveolar basal epithelial cell line (A549T) and human ovarian carcinoma cell line (A2780) were purchased from KeyGen Biotech Co. Ltd. (Nanjing, China), human hepatocellular carcinoma cell line (HepG2) and human breast cancer cell line (MCF-7) were purchased from ATCC (Manassas, Virginia, U.S.A.). Opti-MEM I Reduced Serum Media, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), RPMI Medium 1640, Fetal Bovine Serum (FBS), Penicillin-Streptomycin were purchased from Gibco, (Life Technologies, Auckland, New Zealand). 3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide (MTT) was purchased from Sigma (St. Louis, MO, U.S.A.).
EXAMPLE 1
Isolation of RNA molecules from a plant of genus Taxus
Branches of Taxus chinensis (Pilger) Rehd. var. mairei were freshly collected and immediately stored in liquid nitrogen until use. RNAs having a length of 200 nucleotides or below, i.e. small RNAs species, were extracted from Taxus chinensis (Pilger) Rehd. var. mairei by using an optimized CTAB method combined with a commercial small RNA isolation kit, which method is described by Patel, R. S. et al. in 24
2018101287 04 Sep 2018
Arch Oral Biol 2011, 56 (12), 1506-1513. Briefly, plant tissues were ground into a fine powder in liquid nitrogen and then homogenized in preheated (65°C) CTAB extraction buffer using a digital dispersing device (IKA, Germany). After incubation for 2 min at 65°C, the tissue lysate was cooled down immediately in an ice bath for 10 min, followed by centrifugation at 12,000 xg for 15 min at 4°C. The supernatant was collected and extracted with an equal volume of phenol:chloroform:isopentanol (50:48:1) by vortexing vigorously. Phases were separated at 4°C by centrifugation at 12,000 xg for 15 min and the supernatant was extracted again as described above with chloroform:isopentanol (24:1). The supernatant was collected and mixed with an equal volume of 6 M guanidinium thiocyanate, followed by adding 100% ethanol to a final concentration of 55%. The mixture was passed through a filter cartridge containing a silica membrane, which immobilizes the RNAs. The filter was then washed for several times with 80% (v/v) ethanol solution, and finally all RNAs were eluted with a low ionic-strength solution or RNase-free water. The small RNA species were isolated and enriched by using a mirVanaTM miRNA isolation kit following the manufacturer’s instruction.
Further, the total tRNAs in the isolated small RNA species were separated by electrophoresis in 6% polyacrylamide TBE gels containing 8 M urea prepared according to the manufacturer’s protocol (Biorad, U.S.A.). After staining with SYBR Gold nucleic acid gel stain, polyacrylamide gels were examined using a UV lamp and the region of gels containing total tRNAs were cut off by using a clean and sharp scalpel. Fig. 1 shows gel electrophoresis profiles of small RNA species from Taxus Chinensis (Pilger) Rehd. var. mairei, including low range RNA markers (denoted as “Ladder”), small RNA species, and transfer RNATrp(CCA). The band was sliced into small pieces and the total tRNAs were recovered from the gel by electroelution in a 3 kD molecular weight cut-off dialysis tubing (Spectrum, C.A.) at 100 V for 50 min in 1X TAE buffer. The eluents in the dialysis tubing were recovered and the total tRNAs were desalted and concentrated by using the mirVanaTM miRNA isolation kit. The quality and purity of the RNA products were then confirmed using a Nanodrop Spectrophotometer (Thermo Scientific, U.S.A.) and Agilent 2100 Bioanalyzer (Agilent, U.S.A.).
The inventors then constructed the total tRNAs library and performed sequencing. Sequencing libraries were generated by using TruSeq small RNA Library Preparation Kit (Illumina, U.S.A.), followed by a round of adaptor ligation, reverse transcription and 25
2018101287 04 Sep 2018
PCR enrichment. PCR products were then purified and libraries were quantified on the Agilent Bioanalyzer 2100 system (Agilent Technologies, U.S.A.). The library preparations were sequenced at the Novogene Bioinformatics Institute (Beijing, China) on an Illumina HiSeq platform using the 150 bp paired-end (PE150) strategy to generate over 15 million raw paired reads. 1,729,438 clean reads were obtained by removing low quality regions and adaptor sequences. Fig. 2 is a bar chart showing read length distribution of tRNAs. The tRNA genes were identified by using the tRNAscan-SE 2.0 program (http://lowelab.ucsc.edu/tRNAscan-SE/) and annotated by searching the Nucleotide Collection (nr/nt) database using Basic Local Alignment Search Tool (BLAST) program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). 25 tRNA sequences from Taxus chinensis (Pilger) Rehd. var. mairei were identified and listed in Table 1.
Each of the tRNAs was then isolated from a mixture of small RNAs (<200 mer) from Taxus chinensis (Pilger) Rehd. var. mairei by immobilization of the target tRNAs onto the streptavidin-coated magnetic beads with specific biotinylated capture DNA probes. To bind specific tRNA molecules, a corresponded single stranded DNA oligonucleotide (20 to 45-mer) were synthesized, which was designed based on the sequence information of Illumina sequencing and should be complementary to a unique segment of the target tRNA. Cognate DNA probes were incubated with small RNA mixture for about 1.5 h in annealing buffer and allowed to hybridize to the targeted tRNA molecules in solution at the proper annealing temperatures that were generally 5°C lower than the melting temperature (Tm). Streptavidin-coated magnetic beads were then added to the mixture and incubated for 30 min at the annealing temperatures. After the hybridized sequences are immobilized onto the magnetic beads via the streptavidin-biotin bond, the biotinylated DNA/tRNA coated beads were separated with a magnet for 1-2 min and washed 3-4 times in washing buffer at 40°C. The magnetic beads were resuspended to a desired concentration in RNase-free water and thereby to release the immobilized tRNA molecules by incubation at 70°C for 5 min. Accordingly, the isolated and purified tRNA molecules of SEQ ID NO: 201 to 225 were obtained.
EXAMPLE 2
Synthesis of RNA molecules
The inventors designed and synthesized RNA molecules having a length of about 19 to 22 bp based on the 25 isolated tRNA sequences in Example 1. In particular, the 26
2018101287 04 Sep 2018 tRNA sequences are considered to have at least 3 portions, namely a 5’-terminal portion (5’-t), a 3’-terminal portion (3’-t) and an anticodon portion. Each of the specifically designed RNA molecules contains any one of the portions. For instance, designed RNA molecules containing a 5’ terminal portion of the corresponding full5 length tRNA sequence are referred as 5’-t group RNA molecules; designed RNA molecules containing a 3’ terminal portion of the corresponding full-length tRNA sequence are referred as 3’-t group RNA molecules; designed RNA molecules containing an anticodon portion of the corresponding full-length tRNA sequence are referred as anticodon group RNA molecules. The RNA molecules having a sense 10 sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 and a complementary antisense sequence selected from SEQ ID NO: 101 to SEQ ID NO: 200, as shown in Table 2, were designed and synthesized by cleavage at different sites on the tRNA sequences in Table 1.
EXAMPLE 3
Cytotoxic effect of RNA molecules on cancer cells
A2780, Taxol-resistant A2780, HCT-8, Taxol-resistant HCT-8 and Taxol-resistant A549 cell lines were cultured in RPMI Medium 1640 medium containing 10% FBS and 1% penicillin/streptomycin. HepG2 and MCF-7 cell lines were cultured in Minimum 20 Essential medium containing 10% FBS and 1 % penicillin/streptomycin. All cell lines above were cultured at humidified atmosphere containing 5% CO2 at 37°C.
In the cytotoxicity assay, exponentially growing cells of each cancer cell line were plated in 96-well microplate at a density of 5000 cells per well in 100 pL of culture 25 medium and allowed to adhere for 24 h before treatment. Serial concentrations of
RNA molecules obtained in Example 1 and 2 in a mixture containing a gene delivery carrier, i.e. LipofectamineTM RNAiMAX Transfection Reagent (Thermo Fisher Scientific, U.S.A.) were then added to the cells. After treated for 48 h, MTT solution (50 pL per well, 1 mg/mL solution) was added to each well and incubated for 4 h at 30 37°C. Subsequently, 200 pL dimethyl sulfoxide (DMSO) were added and the optical densities of the resulting solutions were calorimetrically determined at 570 nm using a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA, U.S.A). Doseresponse curves were obtained, and the IC50 values were calculated by GraphPad Prism 5 (GraphPad, La Jolla, CA, USA). Each experiment was carried out for three 35 times. IC50 results were expressed as means ± standard deviation.
2018101287 04 Sep 2018
With reference to Fig. 3, A2780 cells, HepG2 cells and MCF-7 cells were treated with 25 nM RNA molecules of tRNAHis<GUG>, tRNAGIU<uuc>, tRNATrp<CCA>, tRNALeu<CAA>, tRNAAr9<ACG>, i.e. SEQ ID NO: 201 to 205, for 48 h before addition of MTT solution. The cell viability of these cells is compared to a control group and a RNAiMAX group where a transfection reagent was added to the cells. The results show that these RNA molecules are capable of inhibiting the growth and proliferation of ovarian cancer cells, liver cancer cells, and breast cancer cells, whereas the RNA molecules achieve more prominent effect on ovarian and liver cancer cells.
Fig. 4A shows the cytotoxic effect of tRNATrp(CCA), i.e. SEQ ID NO: 203, on A2780 cells. Different concentrations of tRNATrp(CCA) were used, i.e. 0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM and 25 nM, and compared to a control group and a RNAiMAX group. It is shown that the IC50 value of tRNATrp(CCA) on ovarian cells in particular A2780 cells is about 14.3 nM. A comparative example using Taxol was conducted. Fig.
4B show the cytotoxic effect of Taxol on A2780 cells.
Fig. 5A and Fig. 5B show the cytotoxic effect of RNA molecules synthesized in Example 2 on A2780 cells, in particular those having sense sequence of SEQ ID NO: 1 to 36. The results show that the RNA molecules designed and synthesized based on the tRNA sequences identified in Example 1 are also effective in inhibiting the growth and proliferation of cancer cells in particular ovarian cancer cells in this example. Further, Fig. 5C and 5D further demonstrated that the RNA molecules in Example 2 are also capable of inhibiting the growth and/or proliferation of Taxolresistant A2780 cells. In other words, RNA molecules having sense sequence of SEQ
ID NO: 1 to 36 and the complementary antisense sequence are useful in treating cancer which is resistant against Taxol, in particular Taxol-resistant ovarian cancer.
Fig. 5E show the cytotoxic effect of RNA molecules synthesized in Example 2 on HCT-8 cells, in particular those having a sense sequence of SEQ ID NO: 1 to 36 and a complementary antisense sequence. The results show that these RNA molecules are also effective in inhibiting the growth and proliferation of colorectal cancer cells. Further, Fig. 5F further demonstrated that the RNA molecules in Example 2 are also capable of inhibiting the growth and/or proliferation of Taxol-resistant HCT-8 cells. The results also show that the RNA molecules HC18, HC34, HC36, HC37 and HC39 are useful in treating cancer which is resistant against Taxol, in particular Taxol-resistant colorectal cancer.
2018101287 04 Sep 2018
The inventors then specifically determined the cytotoxic effect and IC50 of RNA molecule HC11 on A2780 cells, at different concentrations, i.e. 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM. As shown in Fig. 6A, the results are compared to a control group and a RNAiMAX group containing a transfecting agent. The results demonstrated that RNA molecule HC11 has a dose-dependent effect on inhibiting the growth and proliferation of ovarian cancer cells. The IC50 of it is 31 nM. A comparative example was conducted using Taxol with results presented in Fig. 6B.
Further, the inhibitory effect of HC11 against Taxol-resistant cancer cells was determined. Fig. 6C shows the cell viability of Taxol-resistant A2780T cells after treatment with HC11 at different concentrations, i.e. 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM and 200 nM, compared to a control group and a RNAiMAX group while Fig. 6D shows a comparative example using Taxol in the treatment. The results demonstrated that RNA molecule HC11 has a dose-dependent effect on inhibiting the growth and proliferation of Taxol-resistant ovarian cancer cells and its IC50 is 32.3 nM.
Meanwhile, Fig. 6E shows the cell viability of Taxol-resistant A549T cells after treatment with HC11 at different concentrations, and Fig. 6F shows the cell viability of
Taxol-resistant A549T cells after treatment with Taxol at different concentrations. The results demonstrated that RNA molecule HC11 has a dose-dependent effect on inhibiting the growth and proliferation of Taxol-resistant lung cancer cells with IC50 being 87.3 nM.
Similarly, the inventors specifically determined the cytotoxic effect and IC50 of RNA molecules HC36 and 37 on HCT-8 cells, at different concentrations, i.e. 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM. As shown in Fig. 6G and Fig. 6H, the results are compared to a control group and a RNAiMAX group containing a transfecting agent. The results demonstrated that RNA molecules HC36 and HC37 have dose-dependent effect on inhibiting the growth and proliferation of colorectal cancer cells. The IC50 of HC36, 37 is 8.2 and 9.3 nM. A comparative example was conducted using Taxol with results presented in Fig. 6I.
Based on the above results, it is found that the small tRNA molecules isolated or derived from Taxus chinensis (Pilger) Rehd. var. mairei are highly effective at
2018101287 04 Sep 2018 inhibiting growth and proliferation of cancer cells in vitro. The RNA molecules are also effective against Taxol-resistant cell lines.
EXAMPLE 4
In vivo antitumor effect of the RNA molecules
Animal model having xenograft cancer was set. Female BALB/c nude mice (6-8-week old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. and maintained at 25 °C with free access to food and water in a special pathogen-free laboratory of the animal environment facilities. The animal experiments were 10 performed in compliance with institutional animal care guidelines and according to committee-approved protocol. To generate tumor xenografts, A2780 cells (4.Οχ 106) were injected subcutaneously in 100 pL of 1640 medium through a 27-guage needle into the armpit of 8-week-old BALB/c nude mice. After 4-5 weeks after tumors had reached 60-70 mm3, the tumor-bearing nude mice were treated with synthesized tRF 15 with atelocollagen (Koken Co., Ltd., Tokyo, Japan). The concentration of atelocollagen was 1%, and tumor-adjacent injection was performed by one dose of HC11 or HC30 (RNA molecule of SEQ ID NO: 1 or SEQ ID NO: 9 ) (GenePharma Co., Ltd., Shanghai, China) at concentration of 2.4 mg/kg with atelocollagen once a week. A control group was set up in which vehicle was administered to the mice. A Taxol 20 group for administering 1 mg/kg Taxol to the mice was also set as a comparison. The entire treatment lasted for 28 days.
Tumor diameters were measured at maximum length and maximum width with digital calipers. And the tumor volume was calculated by the formula: 25 volume=(width)2xlength/2. The data were statistically analyzed using GraphPad Prism (GraphPad, La Jolla, CA, USA). The results are presented in Fig. 7A and 7B. According to the results, HC11 and HC30 are effective in inhibiting the growth of the tumor inside the mice, and maintaining a relative constant body weight. In other words, the RNA molecules of the present invention are effective in treating cancer cells both 30 in vivo and in vitro.

Claims (42)

1. A method of treating a subject suffering from cancer comprising a step of administering an effective amount of a RNA molecule to the subject, wherein the RNA
5 molecule is isolated or derived from a plant of the genus Taxus.
2. The method of claim 1, wherein the RNA molecule has a sequence length of from about 50 to 200 nucleotides or from about 10 to 30 base pairs.
10
3. The method of claim 1, wherein the RNA molecule comprises a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue thereof.
4. The method of claim 3, wherein the RNA molecule is a non-coding molecule. 15
5. The method of claim 4, wherein the RNA molecule is a transfer RNA molecule.
6. The method of claim 3, wherein the RNA molecule comprises a sequence selected from SEQ ID NO: 201 to SEQ ID NO: 225 or a functional variant or
20 homologue thereof.
7. The method of claim 3, wherein the RNA molecule is a double-stranded RNA molecule comprising a sense sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue therefore, and a complementary antisense
25 sequence.
8. The method of claim 7, wherein the RNA molecule further comprises 2 mer 3' overhangs.
30
9. The method of claim 7, wherein the sense sequence is selected from SEQ ID
NO: 1 to SEQ ID NO: 36 or a functional variant or homologue thereof.
10. The method of claim 3, wherein the RNA molecule comprises at least one modified nucleoside selected from inosine, 1-methyladenosine, 2-methyladenosine, 35 A^-methyladenosine, A^-isopentenyladenosine, 2'-O-methyladenosine, Λ/631
2018101287 04 Sep 2018 acetyladenosine, 1-methylinosine, pseudouridine, dihydrouridine, or 2-methylthio-/V6methyladenosine.
11. The method of claim 1, wherein the cancer is ovarian cancer, liver cancer, breast cancer, colorectal cancer, or lung cancer.
12. The method of claim 1, wherein the cancer is resistant against Taxol.
13. The method of claim 1, wherein the RNA molecule is isolated or derived from Taxus chinensis (Pilger) Rehd. var. mairei.
14. The method of claim 1, wherein the step of administering the RNA molecule to the subject comprises contacting cancer cells of the subject with the RNA molecule.
15. A method of inhibiting growth or proliferation of cancer cells comprising a step of contacting said cells with an effective amount of a RNA molecule isolated or derived from a plant of the genus Taxus.
16. The method of claim 15, wherein the RNA molecule has a sequence length of from about 50 to 200 nucleotides or from about 10 to 30 base pairs.
17. The method of claim 15, wherein the RNA molecule comprises a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue thereof.
18. The method of claim 17, wherein the RNA molecule is a non-coding molecule.
19. The method of claim 18, wherein the RNA molecule is a transfer RNA molecule.
20. The method of claim 17, wherein the RNA molecule comprises a sequence selected from SEQ ID NO: 201 to SEQ ID NO: 225 or a functional variant or homologue thereof.
21. The method of claim 17, wherein the RNA molecule is a double-stranded RNA molecule comprising a sense sequence selected from SEQ ID NO: 1 to SEQ ID NO:
2018101287 04 Sep 2018
100 or a functional variant or homologue therefore, and a complementary antisense sequence.
22. The method of claim 21, wherein the RNA molecule further comprises 2 mer 3' 5 overhangs.
23. The method of claim 21, wherein the sense sequence is selected from SEQ ID NO: 1 to SEQ ID NO: 36 or a functional variant or homologue thereof.
10
24. The method of claim 17, wherein the RNA molecule comprises at least one modified nucleoside selected from inosine, 1-methyladenosine, 2-methyladenosine, A^-methyladenosine, A^-isopentenyladenosine, 2'-O-methyladenosine, Λ/6acetyladenosine, 1-methylinosine, pseudouridine, dihydrouridine, or 2-methylthio-A/6methyladenosine.
25. The method of claim 15, wherein the cancer cells are ovarian cancer cells, liver cancer cells, breast cancer cells, colorectal cancer cells, or lung cancer cells.
26. The method of claim 15, wherein the cancer cells are resistant against Taxol.
27. The method of claim 15, wherein the RNA molecule is isolated or derived from Taxus chinensis (Pilger) Rehd. var. mairei.
28. The method of claim 15, wherein the RNA molecule is provided in a 25 composition comprising a gene delivery carrier.
29. A pharmaceutical composition for treating cancer comprising a RNA molecule and a pharmaceutically tolerable excipient, wherein the RNA molecule is isolated or derived from a plant of the genus Taxus.
30. The pharmaceutical composition of claim 29, wherein the RNA molecule has a sequence length of from about 50 to about 200 nucleotides or about 10 to 30 base pairs.
2018101287 04 Sep 2018
31. The pharmaceutical composition of claim 29, wherein the RNA molecule comprises at least one sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue thereof.
5
32. The pharmaceutical composition of claim 31, wherein the RNA molecule is a non-coding molecule.
33. The pharmaceutical composition of claim 32, wherein the RNA molecule is a transfer RNA molecule.
34. The pharmaceutical composition of claim 33, wherein the RNA molecule comprises a sequence selected from SEQ ID NO: 201 to SEQ ID NO: 225 or a functional variant or homologue thereof.
15
35. The pharmaceutical composition of claim 31, wherein the RNA molecule is a double-stranded RNA molecule comprising a sense sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue therefore, and a complementary antisense sequence.
20
36. The method of claim 35, wherein the RNA molecule further comprises 2 mer 3' overhangs.
37. The pharmaceutical composition of claim 35, wherein the sense sequence is selected from SEQ ID NO: 1 to SEQ ID NO: 36 or a functional variant or homologue
25 thereof.
38. The pharmaceutical composition of claim 31, wherein the RNA comprises at least one modified nucleoside selected from inosine, 1-methyladenosine, 2methyladenosine, /V-methyladenosine, /V-isopentenyladenosine, 2'-O-
30 methyladenosine, /V-acetyladenosine, 1-methylinosine, pseudouridine, dihydrouridine, or 2-methylthio-/\/6-methyladenosine.
39. The pharmaceutical composition of claim 29, wherein the RNA molecule is isolated or derived from Taxus chinensis (Pilger) Rehd. var. mairei.
2018101287 04 Sep 2018
40. A double-stranded RNA molecule comprising a sense sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100 or a functional variant or homologue therefore, a complementary antisense sequence, and optionally a 3’ overhang.
5
41. The double-stranded RNA molecule of claim 40 comprises at least one modified nucleoside selected from inosine, 1-methyladenosine, 2-methyladenosine, A^-methyladenosine, A^-isopentenyladenosine, 2'-O-methyladenosine, Λ/6acetyladenosine, 1-methylinosine, pseudouridine, dihydrouridine, or 2-methylthio-A/6methyladenosine.
42. A recombinant vector comprising the double-stranded RNA molecule of claim 40.
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