Patent Grant
11866704
This application claims priority to, and the benefit of, Chinese Patent Application No. 202010083971.2 filed on Feb. 10, 2020. The entire contents of the foregoing application are hereby incorporated by reference for all purposes.
This application contains a sequence listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 25, 2022, is named “M006_092_NPRUS_Sequence_list_revised.txt” and is 83 kilobytes in size.
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.
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.
Escherichia coli (Migula) Castellani & Chalmers, a species from the genus Escherichia that belong to the family of Enterobacteriaceae. It is a famous gut microbiota mainly distributed in the colon of human beings and animals, which possess almost 0.1% of gut microorganisms. Non-pathogenic Escherichia coli as a part of normal bacteria in human gut intestinal can produce vitamin K, as well as avoiding the progression of other pathogenic strains, which is beneficial to the human beings. Nevertheless, there still remains a need to derive effective molecules from various sources such as human gut microbiota for treatments.
According to the limitations of current techniques, through lots of experiments and investigations, the inventors successfully separated and purified tRNA-derived fragments from bacteria of the genus Escherichia for cancer prevention and/or treatment, especially for the double-stranded RNA molecules containing antisense from SEQ ID NO: 1 to 3 and sense from SEQ ID NO: 4 to 6, which provides a novel and effective method for preventing and/or treating cancer.
In a first aspect, the invention provides a group of double-stranded RNA molecules comprising an antisense sequence selected from SEQ ID NO: 7 to SEQ ID NO: 100 and a sense sequence selected from SEQ ID NO: 101 to SEQ ID NO: 194, or a functional variant or homologue therefore.
Preferably, the said double-stranded RNA molecules or a functional variant or homologue therefore comprising an antisense sequence selected from SEQ ID NO: 47, 48, 89, 90, 91 and 92, and a sense sequence selected from SEQ ID NO: 141, 142, 183, 184, 185 and 186.
In a further aspect, the antisense sequence of said double-stranded RNA molecules or a functional variant or homologue therefore is shown as SEQ ID NO: 47, 48, 89, 90, 91 and 92, and the sense sequence of said double-stranded RNA molecules or a functional variant or homologue therefore is shown as SEQ ID NO: 141, 142, 183, 184, 185 and 186.
Still further, the antisense sequence of said double-stranded RNA molecules or a functional variant or homologue therefore is shown as SEQ ID NO: 89, and the sense sequence of said double-stranded RNA molecules or a functional variant or homologue therefore is shown as SEQ ID NO: 183.
Preferably, the said double-stranded RNA molecules or a functional variant or homologue therefore comprising a 3′ overhang.
Preferably, the said double-stranded RNA molecules or a functional variant or homologue therefore comprising one or more preferably modified nucleotides. The said nucleotides comprising m1A, m2A, m5A, m7A, m2G, m6A, m22G, Um, i6A, ms2i6A, t6A, m6t6A, s2C, s2U, s4U, ac4C, f5C, acp3U, mo5U, cmo5U, mcmo5U, mcm5U, mcm5Um, mcm5s2U, nm5s2U, mnm5U, mnm5s2U, ncm5U, ncm5Um, cmnm5U, cmnm5Um, cmnm5s2U, tm5U and tm5s2U. Preferably, said chemical modifications comprising one or more selected from m1A, m7G, m6A, Gm, Cm, Am, Um, m22G, s4U and cmo5U. Preferably, the said chemical modified nucleotides comprising uridine or guanosine. In a further aspect, the said chemical modified nucleotides comprising s4U and/or Gm. Still further, the said double-stranded RNA molecules or a functional variant or homologue therefore comprising an antisense sequence selected from SEQ ID NO: 1, 2 and 3, and a sense sequence selected from SEQ ID NO: 4, 5 and 6. Most preferably, the said double-stranded RNA molecules or a functional variant or homologue therefore is shown as SEQ ID NO: 1, 2 and 3, and a sense sequence is shown as SEQ ID NO: 4, 5 and 6.
In a further aspect, the invention provides to a pharmaceutical composition for cancer prevention and/or treatment. The pharmaceutical composition comprises the said double-stranded RNA molecules or a functional variant or homologue therefore and pharmaceutically tolerable carrier, diluent and/or excipient.
Preferably, the said pharmaceutical composition comprises the said nucleic acid stabilizer.
In a further aspect, the invention provides a delivery system for cancer prevention and/or treatment, which comprises the said double-stranded RNA molecules or a functional variant or homologue therefore and pharmaceutically tolerable carrier, diluent and/or excipient.
In another aspect, the invention provides the use of the said double-stranded RNA molecules or a functional variant or homologue therefore in preparation of pharmaceutical composition for cancer prevention and/or treatment.
Correspondingly, the invention provides a method for cancer prevention and treatment, said method comprising administrating an effective amount of said double-stranded RNA molecules or a functional variant or homologue therefore to objects for cancer prevention and/or treatment.
In the mentioned pharmaceutical composition, delivery system, use or method, said cancer prevention and/or treatment can be inhibiting growth, proliferation or migration of cancer cells.
According to an embodiment, in the mentioned pharmaceutical composition, delivery system, use or method, said cancer prevention and/or treatment is colorectal cancer prevention and/or treatment. Preferably, said cancer prevention and/or treatment is inhibiting colorectal cancer cells. Preferably, cancer prevention and/or treatment is fluorouracil-resistant cancer.
The inventors have found that non-coding RNA molecules isolated from a bacteria of the genus Escherichia, particularly transfer RNA molecules, and RNA molecules derived from Escherichia are particularly useful in treatment of cancer. The RNA molecules with a sequence length of about 10 to 200 nucleotides and their homologue double-stranded RNA molecules with a sequence length of about 10 to 30 nucleotides are highly effective in inhibiting growth and proliferation of cancer cells in vitro and exhibit an antitumor effect in vivo. Said RNA molecules are also effective against fluorouracil-resistant cell lines. Further, the pharmaceutical composition comprising the RNA molecule that is isolated or derived from a bacteria of the genus Escherichia 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 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.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one skilled in the art to which the invention belongs.
As used herein and in the claims, “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 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 and in the claims, the singular forms of “a” and “an” also include the corresponding plurals unless the context clearly dictates 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 synthesized according to the sequences disclosed in the present invention, and preferably the RNA molecule is isolated or derived from a bacteria of the genus Escherichia. 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: 195 to SEQ ID NO: 241 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 another embodiment, the RNA molecule comprises at least one nucleotide having one or more chemical modifications. In some embodiments, the one or more chemical modifications is selected from the group consisting of 1-methyl, 2-methyl, 5-methyl, 7-methyl, N2 methyl, N6 methyl, N2,N2 dimethyl, 2′-O-methyl, N6-isopentenyl, 2-methylthio-N6-isopentenyl, N6 threonide carbamoyl, N6-methyl-N6-threosylcarbamoyl, 2-thio, 4-thio, N4 acetyl, 5-formyl, 3-(3-amino-3-carboxypropyl), 5-methoxy, 5-oxoacetic acid, 5-oxoacetate methyl ester, 5-methoxycarbonylmethyl, 5-methoxycarbonylmethyl-2′-O-methyl, 5-methoxycarbonylmethyl-2-thio, 5-aminomethyl-2-thio, 5-methylaminomethyl, 5-methylaminomethyl-2-thio, 5-aminoformylmethyl, 5-aminoformylmethyl-2′-O-methyl, 5-carboxymethyl aminomethyl, 5-carbamoylmethyl-2′-O-methyl, 5-carboxymethylaminomethyl-2-methyl, 5-taurine, 5,2′-O-dimethyl, and 5-tauromethyl-2-thio.
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 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.
Unless otherwise indicated, the term “thereof” in the term “functional variant or homologue thereof” as used herein in the claims refers to each and every member of the entire group(s) or list(s) preceding the term in the claim. For example, in a claim that claims “an antisense sequence selected from one of SEQ ID NO: 7 to SEQ ID NO: 100 and a sense sequence selected from one of SEQ ID NO: 101 to SEQ ID NO: 194; or a functional variant or homologue thereof”, the group of double-stranded RNA molecules covered by the claim includes functional variants or homologs of all the sequences from SEQ ID NO: 7 to SEQ ID NO: 100, and functional variants or homologs of all the sequences from SEQ ID NO: 101 to SEQ ID NO: 194.
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 double-stranded RNA molecule of the invention comprises an antisense sequence selected from SEQ ID NO: 7 to SEQ ID NO: 100 or a functional variant or homologue thereof; or the RNA molecule comprises a sense sequence selected from SEQ ID NO: 101 to SEQ ID NO: 194 or a functional variant or homologue thereof; or the RNA molecule consists of a sequence selected from SEQ ID NO: 7 to SEQ ID NO: 100 or SEQ ID NO: 101 to SEQ ID NO: 194 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.
In an alternative embodiment, the RNA molecule or a functional variant or homologue thereof comprises a sequence selected from SEQ ID NO: 195 to SEQ ID NO: 241, in particular SEQ ID NO: 218 or SEQ ID NO: 237; or consists of a sequence selected from SEQ ID NO: 195 to SEQ ID NO:241, in particular SEQ ID NO: 218 to SEQ ID NO: 237. Preferably, the RNA molecule is a double-stranded RNA molecule having an antisense sequence selected from SEQ ID NO: 7 to SEQ ID NO: 100 or a functional variant or homologue thereof, and a complementary sense sequence. The antisense sequence is complementary to the sense sequence and the antisense sequence is preferably derived from SEQ ID NO: 195 to SEQ ID NO: 241 or functional variant or homologue thereof. Preferably, said double-stranded RNA molecule comprises an antisense sequence selected from SEQ ID NO: 47, 48, 89, 90, 91 or 92, and said double-stranded RNA molecule comprises an sense sequence selected from SEQ ID NO: 141, 142, 183, 184, 185 or 186; Preferably, said double-stranded RNA molecule comprises an antisense sequence shown as SEQ ID NO: 89, and said double-stranded RNA molecule comprises a sense sequence shown as SEQ ID NO: 183. In particular, RNA molecule is chemically modified double-stranded RNA molecule or functional variant or homologue thereof, and comprises an antisense sequence selected from SEQ ID NO: 1 to SEQ ID NO: 3, and comprises a complementary sense sequence selected from SEQ ID NO: 4 to SEQ ID NO: 6 in accordance with an example embodiment. The inventors unexpectedly found that the double-stranded RNA molecules of the present invention are particularly useful in treatment of cancer such as fluorouracil-resistant cancer as described in detail below.
The RNA molecule of the present invention is preferably isolated or derived from the bacteria of the genus Escherichia. The bacteria of the genus Escherichia include and only include Escherichia coli (Migula) Castellani & Chalmers. In an example embodiment, RNA molecule is isolated or derived from Escherichia coli (Migula) Castellani & Chalmers.
In more detail, the preferred sequences of the RNA molecules and double-stranded RNA molecules of the present invention are listed in Tables 1 and 3 below. In an embodiment, RNA molecules of SEQ ID NO: 195 to 241 as shown in Table 1 are isolated from a bacteria of genus Escherichia in particular from Escherichia coli (Migula) Castellani & Chalmers. These sequences are obtained by purification of RNA from Escherichia coli (Migula) Castellani & Chalmers. One possible approach to obtain the RNA molecules from a particular bacteria Escherichia co/i (Migula) Castellani & Chalmers 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 the scope of the present invention.
The antisense sequences of SEQ ID NO: 7 to SEQ ID NO: 100 and the sense sequences of SEQ ID NO: 101 to SEQ ID NO: 194 as shown in Table 3 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 Escherichia coli (Migula) Castellani & Chalmers. Said derived sequence fragments are classified into 2 groups, namely a 5′-tRFs, and a 3′-tRFs. The 5′-t group RNA molecules contain a 2-35 nucleotides cleaved at 5′ terminal portion, D loop, D stem loop, anticodon loop or anticodon stem loop of the corresponding full-length tRNA molecules isolated from the bacteria; and the 3′-t group RNA molecules contain a 2-35 nucleotides cleaved at 3′-CCA terminal portion, T loop, T stem loop, anticodon loop, anticodon stem loop of the corresponding full-length tRNA molecules isolated from the bacteria. In another embodiment, tRF obtained from tRNA-Cys(GCA) comprises 22 nucleotides long 5′-tRFs “GGCGCGUUAACAAAGCGGUUAU”, which corresponds to SEQ ID No: 7 and 22 nucleotides long 5′-tRFs “UCGACUCCGGAACGCGCCUCCA”, which corresponds to SEQ ID No: 8.
Each of the sense sequences together with the corresponding antisense sequence form a double-stranded RNA molecule. As shown in Table 3, the sense sequence of SEQ ID NO: 101 and the antisense sequence of SEQ ID NO: 7 form a double-stranded RNA molecule with a length of 22 base pairs, and the resultant RNA molecule is denoted as EC for easy reference.
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 bacteria; and the 3′-t group RNA molecules contain a 3′ terminal portion of the corresponding full-length RNA molecules isolated from the bacteria. In another embodiment, RNA molecules may contain the anticodon loop portion of the corresponding full-length RNA molecules isolated from the gut microorganisms and referred as anticodon group RNA molecules. The antisense sequences of SEQ ID NO: 7 to SEQ ID NO: 100 can be generated by cleavage at different sites on the full-length RNA molecules SEQ ID NO: 195 to 241.
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.
The inventors unexpectedly found that the natural chemical modifications of RNA sequence derived from EC83 in Table 3 can enhance its inhibition effects on proliferation of colorectal cancer cells. The said antisense sequences of SEQ ID NO: 1 to SEQ ID NO: 3 and the sense sequences of SEQ ID NO: 4 to SEQ ID NO: 6 as shown in Table 4 are artificially synthesized in accordance with the present invention.
The inventors unexpectedly found that the RNA molecules isolated or derived from a bacteria of genus Escherichia in particular Escherichia coli (Migula) Castellani & Chalmers are effective against cancer cells, in particular they are capable of inhibiting the growth, 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 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 colorectal 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 fluorouracil, i.e. can be used to treat cancer which is resistant against fluorouracil. Specifically, the RNA molecules of the present invention can be used to treat fluorouracil-resistant colorectal 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 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 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 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.
The RNA molecule of the present invention may be administered in form of a pharmaceutical composition comprising the RNA molecule and at least one 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 fluorouracil. 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 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 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 double-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 Lipofectamine RNAiMAX 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 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 as Lipofectamine® RNAiMAX transfection reagent for delivering the RNA molecule to 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 bacteria of the genus Escherichia as described above, in particular from Escherichia coli (Migula) Castellani & Chalmers.
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 bacteria of the genus Escherichia or comprises a sequence selected from SEQ ID NO: 195 to SEQ ID NO: 241 or a functional variant or homologue thereof. The cancer cells are as defined above. Preferably, the cancer cells are colorectal cancer cells. The cancer cells may be resistant against currently existing cancer drugs such as but are not limited to fluorouracil.
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, 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: 7 to SEQ ID NO: 100 or a functional variant or homologue thereof; or the RNA molecule comprises SEQ ID NO: 101 to SEQ ID NO: 194 or a functional variant or homologue thereof, or the RNA molecule consists of a sequence selected from SEQ ID NO: 7 to SEQ ID NO: 100 and/or SEQ ID NO: 101 to SEQ ID NO: 194 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 an antisense sequence selected from SEQ ID NO: 7 to SEQ ID NO: 100 or a functional variant or homologue thereof. Preferably, said double-stranded RNA molecule or a functional variant or homologue thereof comprises an antisense sequence selected from SEQ ID NO: 7 to SEQ ID NO: 100 and a complementary sense sequence selected from SEQ ID NO: 101 to SEQ ID NO: 194. Preferably, said double-stranded RNA molecule or a functional variant or homologue thereof comprises an antisense sequence as shown in SEQ ID NO: 47, 48, 89, 90, 91 or 92, and said double-stranded RNA molecule or a functional variant or homologue thereof comprises a sense sequence as shown in SEQ ID NO: 141, 142, 183, 184, 185 or 186. Preferably, said double-stranded RNA molecule or a functional variant or homologue thereof comprises an antisense sequence as shown in SEQ ID NO: 89, and said double-stranded RNA molecule or a functional variant or homologue thereof comprises a sense sequence as shown in SEQ ID NO: 183. In particular, in an embodiment, RNA molecule or a functional variant or homologue thereof is chemically modified double-stranded RNA molecules, comprising an antisense sequence selected from SEQ ID NO: 1 to SEQ ID NO: 3, and comprising a complementary sense sequence selected from SEQ ID NO: 4 to SEQ ID NO: 6. 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.
The present invention pertains to a double-stranded RNA molecule as described above, i.e. comprising an antisense sequence selected from SEQ ID NO: 7 to SEQ ID NO: 100 or a functional variant or homologue thereof, and a complementary sense sequence. In particular, the double-stranded RNA molecule consists of an antisense sequence selected from SEQ ID NO: 7 to SEQ ID NO: 100 or a functional variant or homologue thereof, a complementary sense sequence selected from SEQ ID NO: 101 to SEQ ID NO: 194, and optionally a 3′ overhang.
Example embodiments of the double-stranded RNA molecule are presented in Table 3. The double-stranded RNA may be subject to modification and therefore may carry at least one modified nucleoside selected form inosine, 1-methyladenosine, 2-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, 2′-O-methyladenosine, N6-acetyladenosine, 1-methylinosine, pseudouridine, dihydrouridine, or 2-methylthio-N6-methyladenosine.
In addition to the above, the present invention pertains to tRNA-half cleaved from tRNA molecule in Table 1 using specific method such as S1 nuclease or other enzymes or reagents to probably cleave tRNA into tRNA-half. The tRNA-half may further being chemically modified, and therefore may carry at least one modified nucleoside selected form inosine, 1-methyladenosine, 2-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, 2′-O-methyladenosine, N6-acetyladenosine, 1-methylinosine, pseudouridine, dihydrouridine, or 2-methylthio-N6-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: 6 or a functional variant or homologue thereof. In an embodiment, the 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 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 bacteria of the genus Escherichia, or in particular a RNA molecule comprising a sequence selected from SEQ ID NO: 1 to 6. 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 fluorouracil-resistant cell lines.
The invention is now described in the following non-limiting examples.
Escherichia coli MRE 600 total transfer ribonucleic acid was purchased from Roche (Basel, Switzerland). MicroRNA marker and low range ssRNA ladder were purchased from New England BioLabs (Massachusetts, U.S.A.). Diethylpyrocarbonate (DEPC)—treated water, S1 nuclease, RNase T1 and polyacrylamide containing a ratio of Acrylamide/Bis (19:1, w/w), tris/borate/EDTA (TBE), ammonium persulphate (APS) and tetramethylethylenediamine (TEMED), mirVana™ miRNA isolation kit, SYBR gold nucleic acid gel stain and gel loading buffer II were purchased from Thermo Fisher Scientific (U.S.A.). Guanidinium thiocyanate, triethylammonium acetate, hexafluoro-2-propanol and fluorouracil (5-FU) were purchased from Sigma (Missouri, U.S.A.). Ethanol was purchased from Anaqua Chemicals Supply Inc. Ltd. (U.S.A.). Deionized water was prepared by a Millipore Milli-Q Plus system (Millipore, U.S.A.). HCT-8 human ileocecal colorectal adenocarcinoma cell line and its 5-FU-resistant strain (HCT-8/5-FU), LoVo colorectal adenocarcinoma cell line and its 5-FU-resistant strain (LoVo/5-FU) were purchased from American Type Culture Collection (ATCC, U.S.A.). Opti-MEM medium, RPMI 1640 medium, 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) were purchased from Gibco (New Zealand). F-12K medium was purchased from Thermo (U.S.A.), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was purchased from Sigma (St Louis, MO, U.S.A.).
Isolation of RNA molecules from mixed total tRNA of Escherichia coli Weighed powders of Escherichia coli MRE600 total tRNA were dissolved in DEPC-treated water. RNA concentration of the solutions was determined by NanoDrop (Thermo, U.S.A.). 100 μg of Escherichia coli total tRNA was directly injected into high-performance liquid weak-anion chromatography coupled with a diode array detector for separation of six fractions using the chromatographic conditions as follows: Column: TSKgel DNA-STAT column (4.6×100 i.d., 5 μm, Tosoh, Tokyo, Japan); The flow rate was set as 0.2 mL/min; The column was equilibrated with 20 mM Tris buffer (pH=8.5). Gradient elution with (A) 20 mM Tris buffer (pH=8.5) and (B) 20 mM Tris buffer+1 M NaCl (pH=8.5) was 0-120 min, 53%-63% B. The obtained six fractions were freeze-dried using a Speed-Vac system RVC 2-18 (Marin Christ, Germany). Powders of RNA and inorganic salts were then dissolved with DEPC-treated water and desalted by mirVana™ miRNA Isolation Kit.
Subsequently, fraction 2 and fraction 3 were separated by high-performance liquid ion-pair chromatography coupled with a diode array detector. The chromatographic conditions are as follows: Column: DNAPac RP column (3.0×100 i.d., 4 μm, Thermo); The flow rate was set as 0.2 mL/min; The column was equilibrated with 100 mM triethylammonium acetate (pH=7.0). Gradient elution with (A) 100 mM triethylammonium acetate (pH=7.0) and (B) 25% acetonitrile in A was 0-5 min, 30%-37% B; 5-25 min, 37%-45% B; 25-35 min, 45%-100% B; 35-45 min, 100% B.
Further, to characterize the tRNAs by mass spectrometry. The inventors employed ultra-high performance liquid chromatography coupled with quadruple time-of-flight mass technique (UHPLC-QTOF-MS) to quantitative analyze the accurate sequence information and chemical modifications of digested products of purified tRNAs. tRNA would be cleaved to several oligonucleotide fragments in length of 2-15 nt with terminal of guanine 3′-phosphoric acid. In negative mode of ESI source, oligonucleotides would have molecular ion peak with multiple charges. Charge numbers depend on the length of nucleotides, which means that nucleotide with longer length would carry more charges. The sequence information and chemical modifications are determined through collision-induced dissociation (CID) analysis of the rule and products information of tRNA-RNase T1 digestions. At the same impact voltage, the stronger the excimer ion peak intensity is, the greater the fragment response intensity is. In addition, the molecular ion peaks with more charges are easier to be cleaved, and the more fragment information is generated. Due to the complexity of CID profiles of oligonucleotide fragments longer than 8 nt, multiple excimer ions and the corresponding optimal collision energy were selected in the process of sequence analysis. CID cleavage of oligonucleotides is most easily cleaved at the phosphodiester bond and the junction between base and ribose, resulting in a series of characteristic fragments, mainly a-B, c, y and w type ions.
The purified RNA was freeze-dried and redissolved with RNase-treated water. Each 1 μg of purified RNA was mixed with 50 units of RNase T1 and mixed with ammonium acetate (220 mM). After incubation in water bath at 37° C. for 1.5 h, the mixed solutions were incubated at 70° C. for 10 minutes to terminate the reaction. After centrifugation at 10000×g for 1 min, the supernatant was collected for UHPLC-MS analysis.
UHPLC-MS/MS was performed on an Agilent UHPLC 1290 system (Agilent Technologies, Santa Clara, CA, USA), equipped with a vacuum degasser, a quaternary pump, an autosampler, a diode array detector and an Agilent ultrahigh definition 6545 Q-TOF mass spectrometer. Separation was carried out on an ACQUITY UPLC OST C18 Column (2.1×100 mm i.d., 1.7 μm, Waters, Massachusetts, U.S.A.) at 60° C. The flow rate was set at 0.2 mL/min and sample injection volume was 20 μL. Gradient elution with (A) 100 mM hexafluoro-2-propanol (HFIP)+15 mM trimethylamine (TEA) and (B) 50% MeOH in A was 0-1.5 min, 2% B, 1.5-8.3 min, 2%-28% B, 8.3-16.5 min, 28%-34% B, followed by washing with 80% B and equilibration with 2% B. ESI conditions were as follows: gas temperature 320° C., spray voltage 3.5 kV, sheath gas flow and temperature were set as 12 L/min and 350° C., respectively.
atheoretical monoisotopic mass
bDifference = (Deconvoluted mass) − (Calculated mass)
Further, the inventors employed specific S1 nuclease to prepare tRNA-half molecule. Each 500 ng of purified tRNA molecule was dissolved with DEPC-treated RNase-free water and mixed with 8 units of S1 nuclease, followed by added with 2 μL of 5× reaction buffer solutions and mixed with deionized water to 20 μL. After incubation in water bath at 25° C. for 40 min, the mixed solutions were with 0.5 μL of 0.5 M EDTA solution to terminate the reaction. After centrifugation at 10000×g for 1 min, the supernatant was collected and analyzed by urea denatured polyacrylamide gel electrophoresis.
The inventors designed and synthesized RNA molecules having a length of about 22 bp based on the 147 isolated tRNA sequences in database. In particular, the 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 full-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 sequence selected from SEQ ID NO: 101 to SEQ ID NO: 194 and a complementary antisense sequence selected from SEQ ID NO: 7 to SEQ ID NO: 100, as shown in Table 3, were designed and synthesized by cleavage at different sites on the tRNA sequences in Table 1. The RNA molecules having an antisense sequence selected from SEQ ID NO: 1 to SEQ ID NO: 3 and a complementary sense sequence selected from SEQ ID NO: 4 to SEQ ID NO: 6, as shown in Table 4, were designed and synthesized by cleavage at different sites on the EC83 sequence in Table 3.
HCT-8, fluorouracil-resistant HCT-8, fluorouracil-resistant LoVo cell lines were cultured in RPMI Medium 1640 medium containing 10% FBS and 1% penicillin/streptomycin. LoVo cell line was cultured in F-12K 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 μL of culture medium and allowed to adhere for 24 h before treatment. Serial concentrations of RNA molecules obtained in Example 1 to 4 in a mixture containing a gene delivery carrier, i.e. Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific, U.S.A.) were then added to the cells. After treated for 48 h, MTT solution (50 μL per well, 1 mg/mL solution) was added to each well and incubated for 4 h at 37° C. Subsequently, 200 μL 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). Dose-response 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 times. IC50 results were expressed as means±standard deviation.
With reference to Figure. 6A, HCT-8 cells were treated with 50 nM RNA molecules of tRNA-Val(UAC), tRNA-Leu(CAG), 5′-tRNA-Val(UAC) half, 3′-tRNA-Val(UAC) half, 5′-tRNA-Leu(CAG) half, 3′-tRNA-Leu(CAG) half, EC41 mimic, EC42 mimic, EC85 mimic and EC86 mimic for 48 h before addition of MTT solution. The cell viability of these cells is compared to a control group and a RNAiMAX group. A comparative example using fluorouracil was conducted.
The inventors then specifically determined the cytotoxic effect and IC50 of RNA molecule EC83 mimic and different chemically modified RNA molecules EC83-M1, EC83-M2 and EC83-M3 mimic on HCT-8 and LoVo cells, at different concentrations, i.e. 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM. As shown in
In the clonogenic assay, HCT-8 or LoVo cells were plated in 6-well microplate at a density of 1000 cells per well in 2 mL of culture medium and allowed to adhere for 24 h before treatment. Single concentration of RNA molecules obtained in Example 1 to 4 in a mixture containing a gene delivery carrier, i.e. Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific, U.S.A.) were then added to the cells. After treated for 48 h, discarded the drug solutions and replaced with 2 mL of fresh culture medium. After culture 14 days, discarded the culture medium and the cells were fixated with 4% paraformaldehyde fix solution for 20 min, followed by stained with crystal violet for 10 min and then washed by deionized water. The 6-well microplate was photographed and the number of colonies with more than 50 individual cells were counted using ImageJ software (U.S.A.). Each experiment was carried out for three times. Results were expressed as means±standard deviation.
With reference to
In the wound-healing assay, HCT-8 or LoVo cells were plated in 6-well microplate at a density of 500,000 cells per well in 2 mL of culture medium and allowed to adhere for 24 h before treatment. Serial concentrations of RNA molecules obtained in Example 1 to 4 in a mixture containing a gene delivery carrier, i.e. Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific, U.S.A.) were then added to the cells. After treated for 48 h, discarded the drug solutions and replaced with 2 mL of fresh culture medium. After culture 14 days, discarded the culture medium and the cells were fixated with 4% paraformaldehyde fix solution for 20 min, followed by stained with crystal violet for 10 min and then washed by deionized water. The 6-well microplate was photographed and the number of colonies with more than 50 individual cells were counted using ImageJ software (U.S.A.). Each experiment was carried out for three times. Results were expressed as means±standard deviation. A 1 mL bacteria-free pipette tip was used to make a cross-scratch at the bottom of 6-well microplate. The cells in the scratch were washed away using phosphor buffer solution and the 6-well microplate was photographed under a microscope. Single concentration of RNA molecules obtained in Example 1 to 4 in a mixture containing a gene delivery carrier, i.e. Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific, U.S.A.) were then added to the cells. The cells were then photographed under microscope at 24 and 48 h. ImageJ software was applied to quantify the area of wound created. Each experiment was carried out for three times. Results were expressed as means±standard deviation.
With reference to
The implementation is further described with reference to the following numbered embodiments.
Embodiment 1: A double-stranded RNA molecule comprising an antisense sequence selected from one of SEQ ID NO: 7 to SEQ ID NO: 100, and a sense sequence selected from one of SEQ ID NO: 101 to SEQ ID NO: 194; or a functional variant or homologue thereof.
Embodiment 2: The double-stranded RNA molecule of embodiment 1, wherein the antisense sequence is selected from one of SEQ ID NO: 47, 48, 89, 90, 91 and 92, and the sense sequence is selected from one of SEQ ID NO: 141, 142, 183, 184, 185 and 186; or a functional variant or homologue thereof.
Embodiment 3: The double-stranded RNA molecule of embodiment 1, wherein the antisense sequence is SEQ ID NO: 47, 48, 89, 90, 91 and 92, and the sense sequence is SEQ ID NO: 141, 142, 183, 184, 185 and 186; or a functional variant or homologue thereof.
Embodiment 4: The double-stranded RNA molecule of embodiment 1, wherein the antisense sequence is SEQ ID NO: 89, and the sense sequence is SEQ ID NO: 183; or a functional variant or homologue thereof.
Embodiment 5: The double-stranded RNA molecule of embodiment 1 comprising a 3′ overhang; or a functional variant or homologue thereof.
Embodiment 6: The double-stranded RNA molecule of embodiment 1, wherein the antisense sequence and/or the sense sequence comprises one or more chemically modified nucleotides; wherein the one or more chemically modified nucleotides are selected from the group consisting of m1A, m2A, m5A, m7A, m2G, m6A, m22G, Um, i6A, ms2i6A, t6A, m6t6A, s2C, s2U, s4U, ac4C, f5C, acp3U, mo5U, cmo5U, mcmo5U, mcm5U, mcm5Um, mcm5s2U, nm5s2U, mnm5U, mnm5s2U, ncm5U, ncm5Um, cmnm5U, cmnm5Um, cmnm5s2U, tm5U and tm5s2U; or a functional variant or homologue thereof.
Embodiment 7: The double-stranded RNA molecule of embodiment 1, wherein the antisense sequence and/or the sense sequence comprises one or more chemically modified nucleotides; wherein the one or more chemically modified nucleotides are selected from the group consisting of m1A, m7G, m6A, Gm, Cm, Am, Um, m22G, s4U and cmo5U; or a functional variant or homologue thereof.
Embodiment 8: The double-stranded RNA molecule of embodiment 7, wherein the one or more chemically modified nucleotides are s4U and/or Gm; or a functional variant or homologue thereof.
Embodiment 9: The double-stranded RNA molecule of embodiment 1, wherein the antisense sequence and/or the sense sequence comprises at least one nucleotide having one or more chemical modifications; wherein the nucleotide is selected from the group consisting of adenosine, guanosine, cytidine and uridine; or a functional variant or homologue thereof; and the one or more chemical modifications is selected from the group consisting of 1-methyl, 2-methyl, 5-methyl, 7-methyl, N2 methyl, N6 methyl, N2,N2 dimethyl, 2′-O-methyl, N6-isopentenyl, 2-methylthio-N6-isopentenyl, N6 threonide carbamoyl, N6-methyl-N6-threosylcarbamoyl, 2-thio, 4-thio, N4 acetyl, 5-formyl, 3-(3-amino-3-carboxypropyl), 5-methoxy, 5-oxoacetic acid, 5-oxoacetate methyl ester, 5-methoxycarbonylmethyl, 5-methoxycarbonylmethyl-2′-O-methyl, 5-methoxycarbonylmethyl-2-thio, 5-aminomethyl-2-thio, 5-methylaminomethyl, 5-methylaminomethyl-2-thio, 5-aminoformylmethyl, 5-aminoformylmethyl-2′-O-methyl, 5-carboxymethyl aminomethyl, 5-carbamoylmethyl-2′-O-methyl, 5-carboxymethylaminomethyl-2-methyl, 5-taurine, 5,2′-O-dimethyl, and 5-tauromethyl-2-thio.
Embodiment 10: The double-stranded RNA molecule of embodiment 9, wherein the one or more chemical modifications is selected from the group consisting of 1-methyl, 7-methyl, N6 methyl, 2′-O-methyl, 5,2′-O-dimethyl, 4-thio, and 5-oxoacetic acid.
Embodiment 11: The double-stranded RNA molecule of embodiment 9, wherein the nucleotide is uridine and/or guanosine; or a functional variant or homologue thereof.
Embodiment 12: A double-stranded RNA molecule comprising an antisense sequence selected from the group consisting of SEQ ID NO: 1, 2 and 3, and a sense sequence selected from the group consisting of SEQ ID NO: 4, 5 and 6; or a functional variant or homologue thereof.
Embodiment 13: The double-stranded RNA molecule embodiment 12, wherein the antisense sequence is SEQ ID NO: 1, 2 or 3, and the sense sequence is SEQ ID NO: 4, 5 or 6; or a functional variant or homologue thereof.
Embodiment 14: A pharmaceutical composition comprising the double-stranded RNA molecule of embodiment 1 or a functional variant or homolog thereof, and a pharmaceutically tolerable carrier, diluent and/or excipient.
Embodiment 15: The pharmaceutical composition of embodiment 14, wherein the pharmaceutical composition further comprises a nucleic acid stabilizer.
Embodiment 16: The pharmaceutical composition of embodiment 14, wherein the pharmaceutical composition is used for preventing and/or treating cancer, wherein the cancer is colorectal cancer or fluorouracil resistant cancer.
Embodiment 17: A method of preventing and/or treating cancer in a subject in need thereof, comprising administering an effective amount of the double-stranded RNA molecule of embodiment 1 to the subject.
Embodiment 18: The method of embodiment 17, wherein the cancer is colorectal cancer or fluorouracil resistant cancer.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010083971 .2 | Feb 2020 | CN | national |
| Number | Date | Country |
|---|---|---|
| WO-03106631 | Dec 2003 | WO |
| WO-2004046375 | Jun 2004 | WO |
| Entry |
|---|
| Ahmed et al. PLoS One 6(8): e23443, pp. 1-8 (Year: 2011). |
| Mlotshwa, S., et al.(2015). A novel chemopreventive strategy based on therapeutic microRNAs produced in plants. Cell research, 25(4), 521-524. |
| Goodarzi, H., et al.(2015). Endogenous tRNA-derived fragments suppress breast cancer progression via YBX1 displacement. Cell, 161(4), 790-802. |
| Number | Date | Country | |
|---|---|---|---|
| 20210254066 A1 | Aug 2021 | US |
