# Patent

## Title
Composition for treating cancer associated with HPV infection

## Abstract

The present invention relates to a composition for preventing or treating diseases associated with human papillomavirus (HPV), and more specifically, cancer associated with HPV, and even more specifically, cervical cancer. The nucleotide sequence of the present invention, the sequence in which the base thereof is modified, and a specific combination thereof can be useful in a composition for effectively treating diseases associated with HPV infection by greatly inhibiting the expression of the E6/E7 gene of HPV type 16 or 18.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS AND CLAIM OF PRIORITY
This application is a National Stage entry from International Application No. PCT/KR2013/006963, filed 1 Aug. 2013, which claims priority from Korean Patent Application No. 10-2012-0084820 filed Aug. 2, 2012, entire contents of which are incorporated herein by reference.
BACKGROUND
Technical Field
This patent application claims priority of Korean Patent Application No. 10-2012-0084820, filed on Aug. 2, 2012, the entire content of which is hereby incorporated by reference.
The present invention relates to a composition for gene therapy for a disease associated with HPV infection including cervical cancer.
Background Art
High-risk human papilloma virus (hereinafter, “HPV”) types 16 and 18 are major factors of cervical cancer and cervical dysplasia, and become causes of other genital cancers and a head and neck squamous cancer. Cervical cancer is one of the most general types of malignant tumors of women. Although the incidence of invasive cervical cancer has been slowly reduced, the invasive cervical cancer is a still most frequent cancer of women in developing countries, which holds 25% of female cancers. HPV is a small DNA virus having approximately 8,000 base sequences which causes benign or malignant tumors. So far, depending on a genomic difference, at least about 100 HPV subtypes have been identified, and genotypes of approximately 90 HPVs have been completely analyzed. Among these types, high-risk HPV types (e.g., HPV-16, 18, 31, 33, 35, 45, 51, 52, and 56) relate to almost 90% of cervical cancer. At least 50% of cervical cancer infected with HPV relate to HPV type 16, and followed by HPV type 18 (12%), HPV type 45 (8%), and HPV type 31 (5%). These HPVs encode 2 oncogenic proteins, which are, protein E6 and E7. Both proteins are involved in HPV-mediated cell immortalization and cell transformation. The oncogenic E6 protein binds to wild-type p53 tumor suppressor protein to thereby degrade p53 through an ubiquitin pathway. On the other hand, the E7 protein directly binds to Rb to thereby overphosphorylate Rb. At first, E6 forms a complex with an E6-associated protein (E6-AP) which is an E3 ubiquitin-protein ligase. Then, the E6/E6-AP complex binds to and ubiqutinate wild-type p53, and then interferes with p53-mediated cellular reaction to DNA damage. Mostly, the p53 tumor suppressor protein is regulated by Mdm2-mediated ubiquitination, however, in HPV-infected cervical cancer cells, degradation of p53 is completely changed to E6-mediated ubiquitination from Mdm2-mediated ubiquitination. Thus, unlike many other cancers, most cases of HPV-infected cervical cancer have the wild-type p53 gene. However, an expression level of the p53 protein is very low due to the consistent degradation by the E6 protein. Particularly, the HPV E6 protein has been significantly noticeable as a specific target for killing just cervical cancer cells. These strategies, targeting to E6 or the E6/E6-AP complex, include various treatment.
Examples include: use of a cellular toxin agent, an inhibitor to release zinc of the E6 oncogenic protein, an epitope peptide (mimotope) mimicking E6-AP, anti-E6 ribozyme, a peptide aptamer which is targeted to the E6 oncogenic protein of a virus, siRNA which is targeted to the E6 oncogenic protein of the virus, and a combined treatment thereof. Recently, it has been proven that siRNA selectively silences an intrinsic gene in animal cells, and as well as, selectively silences a viral gene in a disease caused by a virus. RNA interference (RNAi) due to transfection of siRNA has been emerged as a novel therapy for treating viral infection of the human. siRNA, which is targeted to E6 and E7 genes in HPV-infected cervical cancer cells, causes p53 and pRb accumulation which leads to apoptosis or cell senescence. For an HPV-16 infected cervical cancer cell line and an HPV-18 infected cell line, it has been found that RNAi, which is targeted to E6 and E7 oncogenes of viruses, selectively sciences expression of these proteins.
Meanwhile, efficacy of a nucleic acid having various modifications for nucleic acids (for example, in a base, a sugar and/or phosphate) is enhanced by inhibiting degradation caused by serum ribonuclease. Several examples describing sugar, base and phosphate modifications, which may be introduced to a nucleic acid, are known in the art. For example, an oligonucleotide is modified to enhance stability and/or enhance the biological activity through a modification by a nuclease-resistant group, for example, through 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, and 2′-H nucleotide base modification (see Eckstein et al., PCT Laid-open Publication WO 92/07065; document [Perrault et al., Nature 344:565-568, 1990]; document [Pieken et al., Science 253: 314-317, 1991]; document [Usman and Cedergren, Trends in Biochem. Sci. 17: 334-339, 1992]; Usman et al. PCT Laid-open Publication WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and document [Beigelman et al., J. Biol. Chem., 270:25702, 1995]; Beigelman et al., PCT Laid-open Publication WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., PCT Laid-open Publication WO 98/13526; Thompson et al., U.S. Patent Application No. 60/082,404 (filed on Apr. 20, 1998); document [Karpeisky et al., Tetrahedron Lett., 39:1131, 1998]; document [Earnshaw and Gait, Biopolymers(Nucleic acid Sciences) 48:39-55, 1998]; document [Verma and Eckstein, Annu. Rev. Biochem. 67:99-134, 1998]; and document [Burlina et al., Bioorg. Med. Chem. 5: 1999-2010, 1997]). Similar modifications may be used to modify the nucleic acid of the present invention.
In 1999, as a result of combined therapy of cisplatin-based chemotherapy and radiotherapy, survival rate of women who have a severe cervical cancer in local has been significantly improved. Currently, cisplatin is a DNA-damaging drug which is widely used to treat cancers including ovarian, cervical, head and neck, non-small cell lung cancers and so forth. More recently, a working mechanism of a medicine based on platinum has been investigated. However, it is still not fully understood about a process in cells including DNA repair, cell death, cell cycle trajectory, signaling of DNA damage, and regulation in absorption and secretion of a drug due to cisplatin treatment. In HPV-18 HeLa cells, the p53 protein is escaped from E6-mediated degradation and preferentially accumulated in nucleolus of a nucleus after cisplatin treatment. Also, HPV-16 SiHa cells recover p53 function by simultaneous radiotherapy and cisplatin treatment, thereby increasing radiosusceptibility.
Therefore, as a result of attempting to investigate an effective siRNA having a novel sequence by imparting a chemical modification to E6/E7-specific siRNA so that the siRNA may show the anti-cancer effect, alone or in a complex combination, or show a synergistic effect when performing combination therapy with conventional chemotherapy or radiotherapy, the present inventors have found that nucleotides listed on following Examples and Claims and particular combinations thereof reduce expression of relating proteins TP53 and E7, and HPV E6 mRNA, and induce cell death, and also experimentally proven that efficacy achieved when used alone or in combination with anti-cancer agents is much better than that of RNA, which does not have a base sequence residue modification.
Throughout the specification, numerous journals and patent documents are referenced, and the citation is indicated. Disclosures of cited journals and patent documents are incorporated herein in their entireties by reference to more clearly describe the level of the technical field to which the present invention belongs and features of the present invention.
SUMMARY
The present inventors continuously study and try to develop an efficient gene therapeutic agent for various diseases caused by human papilloma virus (HPV) infection. As a result, the present invention has been completed by finding that when using particular RNA for inhibiting expression, which is targeted to an E6/E7 gene of HPV type 16 or HPV type 18 virus, or a RNA sequence having a modification in a base of the RNA, HPV gene expression is efficiently inhibited, to thereby show an excellent therapeutic activity on diseases associated with HPV infection including a cervical cancer.
One object of the present invention is to provide a composition for preventing or treating a disease associated with HPV infection, more particularly, an HPV infection-associated cancer, and further more particularly a cervical cancer.
Another object of the present invention is to provide a method for preventing or treating a disease associated with HPV infection, more particularly, an HPV infection-associated cancer, and further more particularly a cervical cancer.
Other objects and benefits of the present invention become clear by appended detailed description of the invention, claims, and drawings.
According to one aspect of the present invention, the present invention provides a composition for treating or preventing a disease associated with human papilloma virus (HPV) infection, the composition including, as an active ingredient, one or more nucleotide sequences selected from the group consisting of sequences of SEQ ID NOs: 16, 22, 28, 34, 40, 66, 72, 84, 90, and 108, and antisense nucleotide sequences thereof.
The present inventors continuously study and try to develop an efficient gene therapeutic agent for various diseases caused by HPV infection. Consequently, it has been found that when using particular RNA for inhibiting expression, which is targeted to E6/E7 genes of HPV type 16 or HPV type 18 virus, or a RNA sequence having a modification in a base of the RNA, HPV gene expression is efficiently inhibited to thereby show an excellent therapeutic activity on diseases associated with HPV infection including cervical cancer.
According to the present invention, sequences of SEQ ID Nos: 16, 22, 28, 34, and 40 and sequences of SEQ ID Nos: 72, 84, 90, and 108 are RNA nucleic acid sequences for inhibiting expression, wherein sequences of SEQ ID Nos: 16, 22, 28, 34, and 40 are targeted to HPV type 16 virus; and sequences of SEQ ID Nos: 72, 84, 90, and 108 are targeted to HPV type 18 virus.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 show images of results which verify improvement in stability (FIG. 1a), an increase in an effect at the molecular level of a protein (FIG. 1b), and an increase in an effect at the molecular level of mRNA (FIG. 1c) according to a substitutional modification of a residue of a base sequence in siRNA of HPV 16, and 18 types.
FIG. 2 show images of results which verify an excellent effect of inducing cell senescence (FIG. 2a) and an excellent effect of killing cells (FIG. 2b) of siRNA having substituted residue in a base sequence of HPV 18 type.
FIG. 3 are images showing the effect of inducing cell senescence by combined treatment of the anti-cancer agent, cisplatin, and 426 siRNA having a substitution in a base sequence in a HeLa cervical cancer cell line infected with the HPV 18 type virus (FIG. 3a) and a microscopically observed result thereof (FIG. 3b).
FIG. 4 show images of results which verify a therapeutic effect of a pool of excellent siRNA having a substitution in a base sequence of HPV 16, and 18 types, and a synergetic effect by combined therapy with cisplatin. FIGS. 4a, 4b, and 4c respectively show a cell-proliferation inhibiting effect, a cell-killing effect, and an effect at the molecular level of a protein.
FIG. 5 show images of results which verify an off-target effect of siRNA of HPV 18 type in cells and animals. FIGS. 5a and 5b respectively show an effect of reducing IL-6 on cells and an effect of reducing INF-gamma in animals.
FIG. 6 shows an image of results which quantify 426 siRNA of HPV 18 type through stem-loop real-time PCT.
FIG. 7 shows an image of results which verify the fact that siRNAs in various types of liposomes show the same cell-killing effect on cells.
FIG. 8 are images showing a synergistic effect by combined treatment of an anti-cancer agent and a pool of siRNAs, which have a substitution in a base sequence and show the excellent effect, in an animal experiment. FIGS. 8a, 8b, and 8c respectively show a variation in a size of a tumor of a mouse, an image of the tumor of the mouse, and a variation in body weight of the mouse.



DETAILED DESCRIPTION
As used herein, the term “nucleotide” means a ribonucleotide present in a single strand or a double strand form, and includes a natural nucleotide analogue unless otherwise specifically indicated (see Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584(1990)).
As used herein, the term “inhibition of expression” means to lead decline in a function of a target gene, and preferably means that expression of the target gene become undetectable or resultantly exists at the meaningless level
According to a preferred embodiment of the present invention, the nucleotide sequence of the present invention is an RNA sequence having an ability to silence E6/E7 genes of HPV type 18 or HPV type 16 virus, and preferably, siRNA, shRNA, or an antisense oligonucleotide.
As used herein, the term “siRNA” means short double chain RNA which may induce RNA interference (RNAi) through cleavage of particular mRNA. The siRNA consist of a sense RNA strand having a homologous sequence with mRNA of a target gene, and an antisense RNA strand having a complementary sequence thereto. siRNA can inhibit expression of the target gene, and is thus provided as an efficient gene knock-down method or a gene therapy method.
siRNA is not limited such that siRNA having a double chain RNA part with RNA pairs is completely paired; rather includes a part which does not form a pair because of mismatch (i.e., a corresponding base is not complementary), bulge (i.e., there is no base corresponding to one-side chain), and so forth. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 17 to 23 bases. Both a blunt end and a cohesive end are available as an siRNA end structure, if it is possible to inhibit target gene expression by the RNAi effect. For the cohesive end structure, both a 3 end protrusion structure and a 5 end protrusion structure are available. The number of protruded bases is not limited. For example, the number of bases may become 1 to 8 bases, and preferably 2 to 6 bases. In addition, siRNA may include, for example, low-molecular weight RNA (e.g., natural RNA molecules such as tRNA, rRNA, and viral RNA, or synthetic RNA molecules) in a protrusion part of one end in the range where the effect of inhibiting target gene expression may be retained. In the end structure of siRNA, it is not necessary to have a cleavage structure in both sides, and the structure may be a stem-loop structure in which an end-portion of one side of double chain RNA is connected by linker RNA. A length of the linker is not particularly limited unless the length affects paring in a stem part.
As used herein, the term “small hairpin RNA (shRNA)” means 50 to 70 single-stranded nucleotides, and forms the stem-loop structure in vivo. In other word, shRNA is a RNA sequence to form a tight hairpin structure to inhibit gene expression through RNA interference. A double strand stem is formed by base-pairing of long RNA having 15 to 30 complementary nucleotides at both sides of a loop site having 5 to 10 nucleotides. For constitutive expression, shRNA is transduced into cells through a vector including U6 promoter, and mostly transferred to daughter cells to hereditary transmit inhibition of gene expression. The shRNA hairpin structure is cleaved by an intracellular mechanism to become siRNA, and then binds to an RNA-induced silencing complex (RISC). These RISCs bind to and cleave mRNA. shRNA is transcribed by RNA polymerase. According to the present invention, the nucleotide sequence of the present invention may form the shRNA structure having a double strand stem sequence at both sides of the loop site.
As used herein, the term “microRNA (miRNA)” means a single strand RNA molecule which regulates gene expression and includes 10 to 50 nucleotides in full-length, preferably 15 to nucleotides, and more preferably 17 to 25 nucleotides. miRNA is an oligonucleotide which is not expressed in cells and has a short stem-loop structure. miRNA is fully or partially homologous with at least one messenger RNA (mRNA), and inhibits target gene expression by complementarily binding to the mRNA.
As used herein, the term “antisense oligonucleotide” means RNA containing a nucleotide sequence complementary to a particular mRNA sequence, or a derivative thereof, and inhibits translation of mRNA into a protein by binding to the complementary sequence in mRNA. The antisense nucleotide sequence of the present invention means an RNA sequence that may be complementary to mRNA of a target gene to bind to mRNA of the target gene, and may inhibit translation of the target gene into mRNA, translocation into cytoplasm, maturation, or other essential activities for overall biological functions.
To enhance efficacy of the antisense oligonucleotide, a modification may be made at a position of one or more of bases, sugars or backbones (see De Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55, 1995). The oligonucleotide backbone may be modified with phosphorothioate, phosphotriester, methyl phosphonate, single-chain alkyl, cycloalkyl, single-chain heteroatomic, heterocyclic sugar sulfonate, and so forth. In addition, an antisense nucleic acid may include one or more substituted sugar moieties. The antisense oligonucleotide may include a modified base. Examples of modified bases include hypoxanthine, 6-methyl adenine, 5-methyl pyrimidine (particularly, 5-metyl cytosine), 5-hydroxymethyl cytosine (HMC), glycosyl HMC, gentobiosyl HMC, 2-aminoadenine, 2-tiouracil, 2-tiothymine, 5-bromouracil, 5-hydroxymethyl uracil, 8-azaguanin, 7-deazaguanin, N6(6-aminohexyl)adenine, 2,6-diaminopurine, 2-O-methyl uracil, 2-O-methylguanin, 2-fluorocytisine, and so forth.
According to a more preferred embodiment of the present invention, the nucleotide sequence of the present invention is an siRNA sequence.
According to another aspect of the present invention, the present invention provides a composition for treating or preventing a disease associated with HPV infection, the composition including, as an active ingredient, one or more nucleotide sequences selected from the group consisting of sequences of SEQ ID NOs: 1, 7, 12, 16, 22, 28, 34, 40, 46, 51, 56, 62, 66, 72, 78, 84, 90, 96, 102 and 108, and antisense nucleotide sequences thereof, which have a modified backbone or one or more modified bases. In other word, a nucleotide having the nucleotide sequences listed above, in which a backbone or a base is modified, may become the composition of the present invention for preventing or treating a disease associated with HPV infection.
Modifications of a backbone or a base to be applied to the nucleotide of the present invention may include any modification which is conventionally employed in the art to increase stability or the desired activity.
Preferably, the modified backbone of the present invention includes one or more modifications selected from the group consisting of alkylphosphonate, phosphorothioate, phosphorodithioate, al kylphosphonothioate, phosphoamidate, phosphate ester, carbamate, acetamidate, carboxymethyl ester, carbonate, and phosphate triester.
Preferably, the modified base of the present invention includes one or more modifications selected from the group consisting of methylation, glycosylation and halogenation. More preferably, the modified base of the present invention is a 2′-O methylated or a 2′-fluorinated base.
According to the present invention, when imparting a modification such as 2′-O methylation or 2′-fluorination to a particular position of RNA targeted to E6/E7 genes of HPV type 16 or HPV type 18 virus for inhibiting expression, comparing with an unmodified nucleic acid molecule, the present inventors have found that: efficiency of inhibiting target gene expression is remarkably increased; stability in human serum is increased; and half-life is considerably increased by at least two times in a pharmacokinetic experiment in animals.
2′-O methylation means that a hydroxyl group attached to carbon number two of ribose of an RNA molecule is methylated, and thus modified to a 2′-methoxy group; and 2′-fluorination means that the hydroxyl group attached to carbon number two of ribose of the RNA molecule is substituted with a fluoro group and thus modified to a 2′-fluoro group.
According to a preferred embodiment of the present invention, the 2′-o methylated base in the nucleotide of the present invention is U or G.
According to a preferred embodiment of the present invention, the 2′-fluorinated base in the nucleotide of the present invention is C.
According to a preferred embodiment of the present invention, the nucleotide sequence of the present invention having one or more of the 2′-O methylated or the 2′-fluorinated base is selected from the group consisting of sequences of SEQ ID NOs: 2, 3, 5, 6, 8, 10, 11, 13, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 49, 50, 52, 54, 55, 57, 58, 60, 61, 63, 65, 67, 68, 70, 71, 73, 74, 76, 77, 79, 80, 82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 97, 98, 100, 101, 103, 104, 106, 107, 109, 110, 112 and 113.
According to another embodiment of the present invention, the present invention provides a composition for preventing or treating a disease associated with HPV infection, the composition including, as an active ingredient, a nucleotide pool selected from the group consisting of: a pool having nucleotide sequences of SEQ ID Nos: 2, 4, 8, 9, 12, and 15; a pool having nucleotide sequences of SEQ ID Nos: 18, 21, 29, and 32; a pool having nucleotide sequences of SEQ ID Nos: 42, 45, 52, and 55; a pool having nucleotide sequences of SEQ ID Nos: 58, 59, 63 and 65; a pool having nucleotide sequences of SEQ ID Nos: 68, 71, 91 and 94; and a pool having nucleotide sequences of SEQ ID Nos: 98, 100, 109 and 112.
According to the present invention, the present inventors have found that when the nucleotide sequence of the present invention is used as a pool having particular combination, efficiency of inhibiting target gene expression is considerably increased, when compared with the case where a single sequence of RNA is used, so that a more outstanding therapeutic activity to a disease associated with HPV infection is achieved.
According to a preferred embodiment of the present invention, a disease associated with HPV infection to be treated by the composition of the present invention is selected from the group consisting of genital warts, vagina inflammation, pelvic inflammation and a cancer, and more preferably, a cancer treated by the composition of the present invention is selected from the group consisting of cervical cancer, vagina cancer, vulva cancer, anal cancer, penis cancer, tonsil cancer, pharynx cancer, larynx cancer, head and neck cancer and lung adenocarcinoma. Most preferably, the cancer to be treated by the composition of the present invention is cervical cancer.
The composition of the present invention may be prepared as a pharmaceutical composition including a pharmaceutically effective amount of the nucleic acid molecule of the present invention.
As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the activity or efficacy of treating, alleviating, or preventing arthritis, as described above, of the present invention.
A pharmaceutically acceptable carrier, which is included in the pharmaceutical composition of the present invention, is one typically used in preparation, and includes, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, cyclodextrin and a copolymer thereof, mineral oil, and so forth. The pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetening agent, a favoring agent, an emulsifier, a suspending agent, and a preserving agent besides the components above. A suitable pharmaceutically acceptable carrier and a preparation are described in Remington's Pharmaceutical Sciences (19th ed., 1995).
The pharmaceutical composition of the present invention may be orally or parenterally administered, and preferably parenterally administered. For parenteral administration, intravenous infusion, subcutaneous infusion, intramuscular infusion, peritoneal infusion, topical administration, transdermal administration, and intraarticular administration may be used.
Suitable administration dose of the pharmaceutical composition of the present invention may be differentially prescribed depending on various factors such as a method for formulation, a mode of administration, age, weight, sex, and disease states, dietary of patients, time of administration, a route of administration, a secretion rate and reaction susceptibility. A preferable administration dose of the pharmaceutical composition of the present invention is 0.0001 to 100 mg/kg per day.
When the pharmaceutical composition of the present invention is used as an anti-cancer agent, the composition may be used as combination with the anti-cancer composition typically used in the art. More specifically, the composition may be combinatorially administered with anti-cancer agents such as cisplatin or paclitaxel.
The pharmaceutical composition of the present invention is prepared in a unit dosage form by being formulated using a pharmaceutically acceptable carrier and/or excipient, or prepared by being incorporated into a multi-dose container, according to a method by which a person with ordinary skill in the technical field to which the present invention belongs could easily carry out. In this case, the formulation may be a form of a solution, suspension, or emulsion in an oil or an aqueous medium, or extract, powder, granule, tablet, or capsule form, and may further include a dispersing agent or a stabilizer.
According to more preferred embodiment of the present invention, the nucleic acid molecule of the present invention is included in a gene delivery system.
As used herein, the term “gene delivery system” means a mediator to introduce a desired target gene in subject cells to express. The ideal gene delivery system should be nontoxic to the human body, easily mass produced, and deliver efficiently the gene.
As used herein, the term “gene delivery” means delivering the gene into cells, and has the same meaning as cellular transduction of the gene. At the tissue level, the term gene delivery has the same meaning as spread of the gene. Thus, the gene delivery system of the present invention may be described as the gene transduction system and the gene spread system.
To prepare the gene delivery system of the present invention, the nucleotide sequence of the present invention is preferably present within a suitable expression construct. In the expression construct, it is preferable that the nucleotide sequence of the present invention is operatively linked to a promoter. As used herein, the term “operatively linked to” means a functional binding between a regulatory sequence of nucleic acid expression (for example, a promoter, a signal sequence, or an array at a transcription regulatory factor binding site) with other nucleic acid sequences, and the regulatory sequence thus regulates transcription and/or translation of the other nucleic acid sequences. In the present invention, a promoter, which binds to the nucleotide sequence of the present invention, may be operated preferably in animal cells, and more preferably in mammalian cells to regulate transcription of relaxin gene, and includes, but not limited to a promoter derived from mammalian virus and a promoter derived from a genome of mammalian cells such as mammalian cytomegalovirus (CMV) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, RSV promoter, EF1 alpha promoter, metallothionein promoter, beta-actin promoter, a promoter of human IL-2 gene, a promoter of human IFN gene, a promoter of human IL-4 gene, a promoter of human lymphotoxin gene, a promoter of human GM-CSF gene, and U6 promoter.
The gene delivery system of the present invention may be constructed in various forms which are (i) a naked recombinant DNA molecule, (ii) a plasmid, (iii) a virus vector, and (iv) a liposome or a noisome form including the naked recombinant DNA molecule or the plasmid.
The nucleotide sequence of the present invention may be applied to whole gene delivery system used for typical gene therapy. Preferably, the nucleotide sequence of the present invention may be applied to a plasmid, adenovirus (Lockett L J, et al., Clin. Cancer Res. 3:2075-2080(1997)), adeno associated virus (AAV, Lashford L S., et al., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), retrovirus (Gunzburg W H, et al., Retroviral vectors. Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), lentivirus (Wang G. et al., J. Clin. Invest. 104(11):R55-62(1999)), herpes simplex virus (Chamber R., et al., Proc. Natl. Act. Sci USA 92:1411-1415(1995)), vaccinia virus (Puhlmann M. et al., Human Gene Therapy 10:649-657(1999)), a liposome (Metho s in Molecular Biology, Vol 199, S. C. Basu and M. Basu (Eds.), Human Press 2002) or a niosome.
Most preferably, the nucleotide sequence of the present invention is delivered by using a cationic liposome.
According to another aspect of the present invention, the present invention provides a method for treating or preventing a disease associated with HPV infection, the method including administering, to a subject, a pharmaceutical composition including: (a) a pharmaceutically effective amount of one or more nucleotide sequences selected from the group consisting of sequences of SEQ ID Nos: 16, 22, 28, 34, 40, 66, 72, 84, 90, and 108 and antisense nucleotide sequences thereof; and (b) a pharmaceutically acceptable carrier.
According to another aspect of the present invention, the present invention provides a method for treating or preventing a disease associated with HPV infection, the method including administering, to a subject, a pharmaceutical composition including: (a) a pharmaceutically effective amount of one or more nucleotide sequences selected from the group consisting of sequences of SEQ ID Nos: 1, 7, 12, 16, 22, 28, 34, 40, 46, 51, 56, 62, 66, 72, 78, 84, 90, 96, 102 and 108, and antisense nucleotide sequences thereof which have a modified backbone or one or more modified bases; and (b) a pharmaceutically acceptable carrier.
According to another aspect of the present invention, the present invention provides a method for preventing or treating a disease associated with HPV infection, the method including administering, to a subject, a pharmaceutical composition including: (a) a pharmaceutically effective amount of nucleotide pool selected from the group consisting of: a pool having nucleotide sequences of SEQ ID Nos: 2, 4, 8, 9, 12, and 15; a pool having nucleotide sequences of SEQ ID Nos: 18, 21, 29, and 32; a pool having nucleotide sequences of SEQ ID Nos: 42, 45, 52, and 55; a pool having nucleotide sequences of SEQ ID Nos: 58, 59, 63 and 65; a pool having nucleotide sequences of SEQ ID Nos: 68, 71, 91 and 94; and a pool having nucleotide sequences of SEQ ID Nos: 98, 100, 109 and 112; and (b) a pharmaceutically acceptable carrier.
Since the method of the present invention uses the present composition described above, the features common to both the composition and the method are not described herein to avoid excessive complexity in the specification.
Advantageous Effects
The features and benefits of the present invention are summarized as follows:
(a) The present invention is to provide a composition for preventing or treating a disease associated with human papilloma virus (HPV) infection, more particularly, an HPV infection-associated cancer, and further more particularly a cervical cancer;
(b) The nucleotide sequence of the present invention, a sequence having a modification in a base of the nucleotide sequence, and particular combination thereof significantly inhibits expression of E6/E7 genes of HPV type 16 or HPV type 18 viruses, and is thus usefully employed as a composition or a method for efficiently treating a disease associated with HPV infection.
Hereinafter, the present invention will be described in more detail with reference to examples. These examples are provided to only specifically describe the present invention, and it will be obvious to a person skilled in the art that the scope of the present invention is not limited to the examples according to the essential features of the present invention.
EXAMPLE
Experiment Method
Construction of siRNA of HOV 16, and 18 Types
siRNAs in Tables 1 and 2 below are obtained from Bioneer corporation (Korea) through customized production. In the table below, the underlined siRNA sequence indicates a nucleotide substituted with a 2′-O-Me modified nucleotide in which a methyl group is bound to a residue of the base, and the thick italicized sequence indicates a base substituted with 2′-F modified nucleotide in which a hydroxyl group of a residue of the base is substituted with fluoro.







TABLE 1







siRNA to HPV 16 type









SEQ. ID.




No.
Sequence
Note





sequence
5′-GCA AAG ACA UCU GGA CAA A-3′
HPV 16 Type


1

siRNA 366


sequence
5′-GCA AAG ACA UCU GGA CAA A-3′



2




sequence
5′-GCA AAG ACA UCU GGA CAA A-3′



3




sequence
5′-UUU GUC CAG AUG UCU UUG C-3′



4




sequence
5′-UUU GUC CAG AUG UCU UUG C-3′



5




sequence
5′-UUU GUC CAG AUG UCU UUG C-3′



6




 


sequence
5′-UCA AGA ACA CGU AGA GAA A-3′
HPV 16 Type


7

siRNA 448


sequence
5′-UCA AGA ACA CGU AGA GAA A-3′



8




sequence
5′-UUU CUC UAC GUG UUC UUG A-3′



9




sequence
5′-UUU CUC UAC GUG UUC UUG A-3′



10




sequence
5′-UUU CUC UAC GUG UUC UUG A-3′



11




 


sequence
5′-GAC CGG UCG AUG UAU GUC UUG-3′
HPV 16 Type


12

siRNA 497


sequence
5′-GAC CGG UCG AUG UAU GUC UUG-3′



13




sequence
5′-AGA CAU ACA UCG ACC GGU CCA-3′



14




sequence
5′-AGA CAU ACA UCG ACC GGU CCA-3′



15




 


sequence
5′-GGA GCG ACC CAG AAA GTT A-3′
HPV 16 Type


16

siRNA 39


sequence
5′-GGA GCG ACC CAG AAA GTT A-3′



17




sequence
5′-GGA G G A AG AAA GTT A-3′



18




sequence
5′-UAA CUU UCU GGG UCG CUC C-3′



19




sequence
5′-UAA CUU UCU GGG UCG CUC C-3′



20




sequence
5′-UAA CUU UCU GGG UCG CUC C-3′



21




 


sequence
5′-CAG AAA GTT ACC ACA GTT A-3′
HPV 16 Type


22

siRNA 48


sequence
5′-CAG AAA GTT ACC ACA GTT A-3′



23




sequence
5′- AG AAA GTT A  ACA GTT A-3′



24




sequence
5′-UAA CUG UGG UAA CUU UCU G-3′



25




sequence
5′-UAA CUG UGG UAA CUU UCU G-3′



26




sequence
5′-UAA CUG UGG UAA CUU UCU G-3′



27




 


sequence
5′-GCA CAG AGC TGC AAA CAA C-3′
HPV 16 Type


28

siRNA 68


sequence
5′-GCA CAG AGC TGC AAA CAA C-3′



29




sequence
5′-G A CAG AG  TG  AAA  AA C-3′



30




sequence
5′-GUU GUU UGC AGC UCU GUG C-3′



31




sequence
5′-GUU GUU UGC AGC UCU GUG C-3′



32




sequence
5′-GUU GUU UGC AGC UCU GUG C-3′



33




 


sequence
5′-GCA AAC AAC TAT ACA TGA T-3′
HPV 16 Type


34

siRNA 78


sequence
5′-GCA AAC AAC TAT ACA TGA T-3′



35




sequence
5′-G A AA  AA  TAT A A TGA T-3′



36




sequence
5′-AUC AUG UAU AGU UGU UUG C-3′



37




sequence
5′-AUC AUG UAU AGU UGU UUG C-3′



38




sequence
5′-AUC AUG UAU AGU UGU UUG C-3′



39




 


sequence
5′-AGC AAA GAC ATC TGG ACA A-3′
HPV 16 Type


40

siRNA 365


sequence
5′-AGC AAA GAC ATC TGG ACA A-3′



41




sequence
5′-AG  AAA GA  AT  TGG A A A-3′



42




sequence
5′-UUG UCC AGA UGU CUU UGC U-3′



43




sequence
5′-UUG UCC AGA UGU CUU UGC U-3′



44




sequence
5′-UUG UCC AGA UGU CUU UGC U-3′



45




 


sequence
5′-CAC CUA CAU UGC AUG AAU AUA-3′
HPV 16 Type


46

siRNA 573


sequence
5′-CAC CUA CAU UGC AUG AAU AUA-3′



47




sequence
5′-UAU AUU CAU GCA AUG UAG GUG-3′



48




sequence
5′-UAU AUU CAU GCA AUG UAG GUG-3′



49




sequence
5′-UAU AUU CAU GCA AUG UAG GUG-3′



50




 


sequence
5′-CUU CGG UUG UGC GUA CAA AGC-3′
HPV 16 Type


51

siRNA 792


sequence
5′-CUU CGG UUG UGC GUA CAA AGC-3′



52




sequence
5′-GCU UUG UAC GCA CAA CCG AAG-3′



53




sequence
5′-GCU UUG UAC GCA CAA CCG AAG-3′



54




sequence
5′-GCU UUG UAC GCA CAA CCG AAG-3′



55
















TABLE 2







siRNA to HPV 18 type









SEQ. ID.




No.
Sequence
Note





sequence
5′-CAA CCG AGC ACG ACA GGA A-3′
HPV 18 Type


56

siRNA 426


sequence
5′- AA  CG AG  A G A A GGA A-3′



57




sequence
5′-CAA CCG AGC ACG ACA GGA A-3′



58




sequence
5′-UUC CUG UCG UGC UCG GUU G-3′



59




sequence
5′-UUC CUG UCG UGC UCG GUU G-3′



60




sequence
5′-UUC CUG UCG UGC UCG GUU G-3′



61




 


sequence
5′-CCA ACG ACG CAG AGA AAC A-3′
HPV 18 Type


62

siRNA 450


sequence
5′-CCA ACG ACG CAG AGA AAC A-3′ 



63




sequence
5′-UGU UUC UCU GCG UCG UUG G-3′



64




sequence
5′-UGU UUC UCU GCG UCG UUG G-3′



65




 


sequence
5′-ACT GCA AGA CAT AGA AAT A-3′
HPV 18 Type


66

siRNA 72


sequence
5′-ACT GCA AGA CAT AGA AAT A-3′



67




sequence
5′-A T G A AGA  AT AGA AAT A-3′



68




sequence
5′-UAU UUC UAU GUC UUG CAG U-3′



69




sequence
5′-UAU UUC UAU GUC UUG CAG U-3′



70




sequence
5′-UAU UUC UAU GUC UUG CAG U-3′



71




 


sequence
5′-GTA TAT TGC AAG ACA GTA T-3′
HPV 18 Type


72

siRNA 97


sequence
5′-GTA TAT TGC AAG ACA GTA T-3′



73




sequence
5′-GTA TAT TG  AAG A A GTA T-3′



74




sequence
5′-AUA CUG UCU UGC AAU AUA C-3′



75




sequence
5′-AUA CUG UCU UGC AAU AUA C-3′



76




sequence
5′-AUA CUG UCU UGC AAU AUA C-3′



77




 


sequence
5′-GCA AGA CAG TAT TGG AAC T-3′
HPV 18 Type


78

siRNA 103


sequence
5′-GCA AGA CAG TAT TGG AAC T-3′



79




sequence
5′-G A AGA  AG TAT TGG AA  T-3′



80




sequence
5′-AGU UCC AAU ACU GUC UUG C-3′



81




sequence
5′-AGU UCC AAU ACU GUC UUG C-3′



82




sequence
5′-AGU UCC AAU ACU GUC UUG C-3′



83




 


sequence
5′-ATT GGA ACT TAC AGA GGT A-3′
HPV 18 Type


84

siRNA 113


sequence
5′-ATT GGA ACT TAC AGA GGT A-3′



85




sequence
5′-ATT GGA A T TA  AGA GGT A-3′



86




sequence
5′-UAC CUC UGU AAG UUC CAA U-3′



87




sequence
5′-UAC CUC UGU AAG UUC CAA U-3′



88




sequence
5′-UAC CUC UGU AAG UUC CAA U-3′



89




 


sequence
5′-CTC CAA CGA CGC AGA GAA A-3′
HPV 18 Type


90

siRNA 448


sequence
5′-CTC CAA CGA CGC AGA GAA A-3′



91




sequence
5′-CT  CAA  GA  G  AGA GAA A-3′



92




sequence
5′-UUU CUC UGC GUC GUU GGA G-3′



93




sequence
5′-UUU CUC UGC GUC GUU GGA G-3′



94




sequence
5′-UUU CUC UGC GUC GUU GGA G-3′



95




 


sequence
5′-ACG CAG AGA AAC ACA AGT A-3′
HPV 18 Type


96

siRNA 456


sequence
5′-ACG CAG AGA AAC ACA AGT A-3′



97




sequence
5′-ACG CAG AGA AAC ACA AGT A-3′



98




sequence
5′-UAC UUG UGU UUC UCU GCG U-3′



99




sequence
5′-UAC UUG UGU UUC UCU GCG U-3′



100




sequence
5′-UAC UUG UGU UUC UCU GCG U-3′



101




 


sequence
5′-GCA GAG AAA CAC AAG TAT A-3′
HPV 18 Type


102

siRNA 458


sequence
5′-GCA GAG AAA CAC AAG TAT A-3′



103




sequence
5′-G A GAG AAA  A  AAG TAT A-3′



104




sequence
5′-UAU ACU UGU GUU UCU CUG C-3′



105




sequence
5′-UAU ACU UGU GUU UCU CUG C-3′



106




sequence




107
5′-UAU ACU UGU GUU UCU CUG C-3′



 


sequence
5′-CAG AGA AAC ACA AGT ATA A-3′
HPV 18 Type


108

siRNA 459


sequence
5′-CAG AGA AAC ACA AGT ATA A-3′



109




sequence
5′- AG AGA AA  A A AGT ATA A-3′



110




sequence
5′-UUA UACUUG UGU UUC UCU G- 3′



111




sequence
5′-UUA UACUUG UGU UUC UCU G- 3′



112




sequence
5′-UUA UACUUG UGU UUC UCU G- 3′



113









Evaluation of Stability of siRNA
siRNA was mixed with 10% fetal bovine serum or 10% human serum while staying at 37, and samples were taken in a time based manner. Then, samples were quick frozen and stored at −70. Collected samples were subjected to electrophoresis for one and a half hours on 12% polyacrylamide gel at 50 V followed by Et-Br staining for UV measurement.
Cell Culture and Transduction of siRNA
A cervical cancer cell, a HeLa cervical cancer cell line (ATCC CCL-2) infected with HPV 18 type virus, or SiHa (ATCC HTB-35) or CaSki (ATCC CRL-1550) cervical cancer cell line infected with HPV 16 type virus was seeded on a 6-well plate in the cell number of 2×105 or 1.6×105, and respectively cultured in RPMI1640 or DMEM medium having 10% fetal bovine serum added thereto for 24 hours under the condition of 37, and 5% CO2. After culturing for 24 hours in the medium to attach cells to a surface of the culture plate, unmodified siRNA as a control and an siRNA oligonucleotide modified by the method described above as an experimental group (20 nM for each), were transduced by using DharmaFECT 1 (Dharmacon, USA), and the resultant was cultured for 24 hours.
Anti-Cancer Agent Treatment
A HeLa or Caski cell line, which was seeded in a 6-well plate in 2×105 cells or 1.5×105 cells and then cultured for a day by the method as described above, was transduced with siRNA. Then, each transduced cell line was treated with cisplatin (CDDP) in a final concentration of 2.5 uM, and cultured.
Cell Senescence-Associated β-Galactosidase (SA-β-gal) Activity Measurement
By the method as described above, a HeLa or Caski cell line was transduced with siRNA, alone or in combination with an anti-cancer agent, and then cultured for a day. By using the cell senescence assay kit (BioVision, USA), cells were washed with PBS and treated with SA-β-gal staining solution for 12 hours at 37. Cells stained in blue were observed by using a general optical microscope with the magnification of 100 to 200 times.
Measurement of Cell Death by Using Flow Cytometry
By the method as described above, a HeLA or Caski cell line was transduced with siRNA, alone or in combination with an anti-cancer agent, and then cultured for a day. The cells were stained with and reacted to Annexin V and propidium iodide (PI) reagents for 30 minutes at room temperature by using cell death assay kit (BD, USA), and thereafter cell death was evaluated by using a flow cytometry.
Investigation of Influence of siRNA Treatment
By the method as described above, a HeLA or Caski cell line was transduced with siRNA, alone or in combination with an anti-cancer agent, and then cultured for a day. To observe a change in a protein, cells were disrupted by adding RIPA cell lysis buffer [150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 5 mM EDTA, 0.1% SDS, 0.5% deoxycholate and 1% NP-40]. Then, variation in protein level was observed through the general western-blot method. Anti-TP53, anti-E7, and anti-actin mouse antibody were purchased from Santa Cruz (USA), diluted to 1:1000, and used. The goat anti-mouse IgG HRP conjugated antibody was purchased from Jackson Laboratories (USA), diluted to 1:3000, and used.
Further, to observe variation in mRNA level, cells were disrupted by using a Trizol solution (Invitrogen, USA), and RNA was detected passing through ethanol purification to thereby observe variation in mRNA level through the general real-time polymerase chain reaction (PCR) method.
Investigation of Influence of Off-Target Effect of siRNA
A HeLA or Caski cell line was seeded on a 6-well plate, and respectively cultured for 24 hours in RPMI1640 or DMEM medium having 10% fetal bovine serum under the condition of 37, and 5% CO2. β-gal siRNA, which was a positive control, and siRNA were transduced and cultured, and then the medium was collected to perform the general IL-6 (BD, USA) ELISA method.
A mouse at the age of 6 weeks was intravenously injected with β-gal siRNA as a positive control, siRNA as a negative control, and siRNA, and the reaction was proceeded for 6 hours. Thereafter, blood was collected from the mouse, and serum was separated to perform INF-gamma (BD, USA) ELISA.
Stem-Loop Real-Time PCR to Quantify siRNA
A rat weighing about 260 to 300 g (at the age of 4 weeks) was intravenously injected with siRNA, and then blood of the rat was collected to separate plasma. The separated plasma was diluted in 0.25% triton X-100 buffer. cDNA was synthesized by using Taqman microRNA Reverse Transcription kit (Applied Biosystem, USA), and quantified by the real-time PCR method to detect siRNA.
Animal Test of siRNA
A female nude mouse was xenografted with 5×106 of HeLa cells of HPV 18 type, and generation of cancer cells was evaluated 10 days later. Then, 3 mg/kg of siRNA to be used was intravenously injected to tails at the 2-3 day interval. Cisplatin (2 mg/kg) and paclitaxel (4 mg/kg) were repeatedly injected 9 times by an intraperitoneal injection at 3-4 day interval. The size of a tumor was measured at 2-3 day interval.
Experimental Result
Variation in siRNA Treatment Concentration and Treatment Number
For a Caski or HeLa cell line infected with HPV 16 or 18 type, when performing siRNA transduction and an anti-cancer agent treatment, alone or in combination, in conventional technique, efficacy of siRNA was showed in successive treatment for long period at a high concentration (100 nM), while the modified siRNA of the present invention exhibited excellent efficacy in single treatment for short period at a low concentration (20 nM).
Stability Test of siRNA having Substituted Residue
siRNAs of combination 51 to 54, obtained by the method described above, were mixed with 10% human serum. Then, each siRNA was taken in a time-based manner at the temperature of 37, and stored at −70. Thereafter, gel electrophoresis was performed on 12% polyacrylamide gel. The resultant was stained with Et-Br, and measured with UV. Consequently, as shown in FIG. 1a, siRNA of combination 51 having an unsubstituted residue in a base sequence was disappeared within two hours, while siRNAs of combination 52 to 54 having substituted residue in a base sequence were remained at least 4 hours, indicating a considerable increase in stability. In particular, combination 54 showed the most outstanding stability which lasts over 24 hours.
Effect of siRNA having Substituted Residue at the Molecular Level
By the method described above, a HeLa cell line was transduced with siRNAs of combination 44 to 50, mock and GFP siRNA, and then variation in TP 53, and E7 protein levels was evaluated, while using actin as a housekeeping gene, wherein siRNAs of combination 44 to 50 were siRNAs of HPV 18 type, and mock and GFP were controls. As shown in FIG. 1b, variation in TP53 and E7 protein level of combination 44 was compared with that of combination 45 to 50, wherein combination 44 has an unsubstituted residue in a base sequence, and combination 45 to 50 has a substituted residue in a base sequence. It was proven that efficacy of combination 48 to increase TP53 protein expression and to reduce E7 protein expression was superior to other sequences.
Further, for siRNA 497 of HPV 16 type, a Caski cell line was transduced with combination 12 to 15 by the method described above, and then mRNA was extracted to evaluate mRNA expression levels of E6 and P21. Consequently, as shown in FIG. 1c, it has been proven that combination 13 reduced by 60% or more of E6 mRNA and increased by 1100% or more of p21 mRNA with respect to the control, wherein, combination 13 has a substituted residue in a base sequence. It has been also evaluated that combination 13 was superior to other sequences comparing with combination 12 which has an unsubstituted residue in a base sequence.
Cell Senescence-Inducing Effect of siRNA having Substituted Residue
SA β-Gal activity of a HeLA or Caski cell line, which was treated by the method as described above, was measured. Consequently, in FIG. 2a, in the cases where HPV 18E6/E7 siRNA and combination 51 were transduced, the SA-β Gal activity was incased by about 10 to 20 times, while the SA-β Gal activity for combination 54 was increased by 50 times or more comparing with that of the control siRNA, wherein HPV 18E6/E7 siRNA was used in the previous invention; combination 51 consisted of 450 siRNA having an unsubstituted residue in a base sequence; and combination 54 consisted of siRNA having a substituted residue in a base sequence. The result showed that the cell senescence effect of combination 54 having substation in a base sequence was considerably better than that of combination 51 having no substitution of a base sequence.
Cell-Killing Effect of siRNA having Substituted Residue
By the method described above, a cell-killing effect was measured by using a flow cytometry. As a result obtained by evaluating the cell-killing effect by comparing HPV 18E6/E7, 18E6 siRNA, combination 48, and combination 54 (which have a substituted residue in a base sequence) with the control, it has been proven that HPV 18E6/E7 and 18E6 siRNA groups showed cell-killing effect of only 15 to 20%, while siRNA groups having a substituted residue showed about 60% or more of cell-killing effect, with respect to the control (FIG. 2b). The result showed that siRNA sequences having substitution in a base sequence showed more significant cancer cell-killing effect than typical siRNA.
Effect of Combined Treatment of Cisplatin and siRNA having Substituted Residue
As a result of measuring SA β-Gal activity by treating a HeLa cell line with cisplatin and combination 48 having a substituted residue in a base sequence by the method as described above, as shown in FIGS. 3a and 3b, cells were stained in blue in cell lines respectively treated with cisplatin and combination 48 alone, showing SA β-Gal activity. However, almost of cells were stained in dark blue which indicates strong SA β-Gal activity for the combined treatment group of combination 48 and cisplatin. The result showed that the combined treatment group exhibited much superior cell senescence effect to the mono-treatment group of siRNA or cisplatin.
Effect of Inhibiting Cell Proliferation by Mono-Treatment or Combined Treatment of siRNA Pool
By the method described above, HPV 16 type Caski cell lines were respectively treated with 20 mM of siRNA combination 2, 9, and 13 alone, wherein the siRNA combination 2, 9, and 13 has substitution in a base sequence of HPV 16 type, and HPV 16 type Caski cell lines were transduced with: the pool of combination 2 and combination 9 (10 nM, for each); the pool combination 2 and combination 13 (10 nM, for each); the pool of combination 9 and combination 13 (10 nM, for each); and pool of combination 2, combination 9, and combination 13 (7 nM, for each). Then, cell number was measured 24 hours later. Consequently, as shown FIG. 4a, cell proliferation was reduced in the mono treatment groups of each siRNA having substitution in a base sequence, and the siRNA pool treatment group in a similar manner. Particularly, although each siRNA was treated in an amount of 7 nM, the pool of 3 types of siRNA showed the effect equivalent to the case where 20 nM of each siRNA was treated.
Combination of the siRNA pool used herein was shown in Table 3 below, and siRNA pool shown in Table 4 below includes a mixture of two or more of combination having particularly higher cell proliferation inhibiting effects.







TABLE 3







Combination of siRNA










Number of





combination
Sense
Antisense
note





combination1
sequence 1
sequence 4
HPV 16 type


combination2
sequence 2
sequence 4
siRNA 366


combination3
sequence 2
sequence 5


combination4
sequence 2
sequence 6


combination5
sequence 3
sequence 4


combination6
sequence 3
sequence 5


combination7
sequence 3
sequence 6


combination8
sequence 7
sequence 9
HPV 16 type


combination9
sequence 8
sequence 9
siRNA 448


combination10
sequence 8
sequence 10


combination11
sequence 8
sequence 11


combination12
sequence 12
sequence 14
HPV 16 type


combination13
sequence 12
sequence 15
siRNA 497


combination14
sequence 13
sequence 14


combination15
sequence 13
sequence 15


combination16
sequence 16
sequence 19
HPV 16 Type


combination17
sequence 17
sequence 20
siRNA 39


combination18
sequence 18
sequence 21


combination19
sequence 18
sequence 21


combination20
sequence 22
sequence 25
HPV 16 Type


combination21
sequence 23
sequence 25
siRNA 48


combination22
sequence 24
sequence 26


combination23
sequence 24
sequence 27


combination24
sequence 28
sequence 31
HPV 16 Type


combination25
sequence 29
sequence 32
siRNA 68


combination26
sequence 30
sequence 32


combination27
sequence 30
sequence 33


combination28
sequence 34
sequence 34
HPV 16 Type


combination29
sequence 35
sequence 37
siRNA 78


combination30
sequence 36
sequence 38


combination31
sequence 36
sequence 39


combination32
sequence 40
sequence 43
HPV 16 Type


combination33
sequence 41
sequence 44
siRNA 365


combination34
sequence 42
sequence 44


combination35
sequence 42
sequence 45


combination36
sequence 46
sequence 48
HPV 16 Type


combination37
sequence 46
sequence 49
siRNA 573


combination38
sequence 47
sequence 48


combination39
sequence 47
sequence 50


combination40
sequence 51
sequence 53
HPV 16 Type


combination41
sequence 51
sequence 55
siRNA 792


combination42
sequence 52
sequence 54


combination43
sequence 52
sequence 55


combination44
sequence 56
sequence 59
HPV 18 type


combination45
sequence 57
sequence 59
siRNA 426


combination46
sequence 57
sequence 60


combination47
sequence 57
sequence 61


combination48
sequence 58
sequence 59


combination49
sequence 58
sequence 60


combination50
sequence 58
sequence 61


combination51
sequence 62
sequence 64
HPV 18 type


combination52
sequence 62
sequence 65
siRNA 450


combination53
sequence 63
sequence 64


combination54
sequence 63
sequence 65


combination55
sequence 66
sequence 69
HPV 18 Type


combination56
sequence 67
sequence 70
siRNA 72


combination57
sequence 67
sequence 69


combination58
sequence 68
sequence 71


combination59
sequence 72
sequence 76
HPV 18 Type


combination60
sequence 73
sequence 75
siRNA 97


combination61
sequence 73
sequence 77


combination62
sequence 74
sequence 77


combination63
sequence 78
sequence 83
HPV 18 Type


combination64
sequence 79
sequence 82
siRNA 103


combination65
sequence 80
sequence 81


combination66
sequence 80
sequence 82


combination67
sequence 84
sequence 89
HPV 18 Type


combination68
sequence 85
sequence 87
siRNA 113


combination69
sequence 86
sequence 88


combination70
sequence 86
sequence 89


combination71
sequence 90
sequence 95
HPV 18 Type


combination72
sequence 91
sequence 94
siRNA 448


combination73
sequence 92
sequence 93


combination74
sequence 92
sequence 95


combination75
sequence 96
sequence 99
HPV 18 Type


combination76
sequence 97
sequence 99
siRNA 456


combination77
sequence 98
sequence 100


combination78
sequence 98
sequence 101


combination79
sequence 102
sequence 107
HPV 18 Type


combination80
sequence 103
sequence 106
siRNA 458


combination81
sequence 103
sequence 107


combination82
sequence 104
sequence 105


combination83
sequence 108
sequence 113
HPV 18 Type


combination84
sequence 109
sequence 112
siRNA 459


combination85
sequence 110
sequence 112


combination86
sequence 110
sequence 113
















TABLE 4







siRNA Pool














Number



Number


of com-


of pool
Sense
Antisense
bination
Note














SP1
sequence 2
sequence 4
2
HPV 16 type



sequence 8
sequence 9
9
siRNAs 366/



sequence 12
sequence 15
13
448/497


SP2
sequence 18
sequence 21
18
HPV16 type



sequence 29
sequence 32
25
siRNAs 39/68


SP3
sequence 42
sequence 45
35
HPV16 type



sequence 52
sequence 55
43
siRNAs 365/792


SP4
sequence 58
sequence 59
48
HPV 18 type



sequence 63
sequence 65
54
siRNAs 426/450


SP5
sequence 68
sequence 71
58
HPV18 type



sequence 91
sequence 94
72
siRNAs 72/448


SP6
sequence 98
sequence 100
77
HPV18 type



sequence 109
sequence 112
84
siRNAs 456/459









Cell-Killing Effect of Mono-Treatment and Combined Treatment of siRNA Pool
By the method described above, an HPV 18 type HeLa cell line was treated with combination 48 and combination 54 (20 nm, for each) wherein, combination 48 and combination 20 have siRNA of HPV 18 type having substation in a base sequence, and transduced with the 10 nM of the SP4 pool, which is the pool of siRNA of 5 nM of combination 48 and 5 nM of combination 54. After 24 hours, the cell line was stained with Annexin V and propidium iodide, and cell-killing effect was measured by using a flow cytometry. Consequently, as shown in FIG. 4b, both the mono-treatment groups of siRNA, which have substitution in a base sequence, and the siRNA pool (SP4) showed the effect of killing 80% or more of cells. As a result, it has been proven that 20 nM of the siRNA mono-treatment group showed the cell-killing effect similar to that of 10 nM of the siRNA pool, indicating that the siRNA pool was better.
Effect of siRNA Pool in Mono- and Combined Treatment at Molecular Level
By the method described above, in a HPV 16 type CaSki cell line, the western-bolt method was used to compare a TP53 protein expression level for a combined treatment group of cisplatin and combination 2, 9, 13; a mono-treatment group of cisplatin; a combined treatment group of cisplatin and combination 2 and 9; a combined treatment group of cisplatin and combination 2 and 13; a combined treatment group of cisplatin and combination 9 and 13; and a combined treatment group of cisplatin and pool SP1 (a pool of combination 2, combination 9, and combination 13), wherein combination 2, 9, and 13 have substitution in a base sequence. Consequently, as shown in FIG. 4C, among combined treatment groups of siRNA pools and cisplatin, the highest increase in a TP53 protein expression level was shown in the pool SP1 which was treated with low concentration of 7 nM.
The result showed that since the pool selectively consisted of siRNA which was competent and efficient while mimicking features of naturally-occurring siRNA pool, it is possible to mix and use in a concentration lower than the concentration of the typical treatment, and reduce off-target effect.
Off-Target Effect of siRNA
By the method described above, HPV 18 type HeLa cell line was transduced with β-gal siRNA as a positive control, combination 44 having an unsubstituted residue, and combination 48 having a substituted residue in a base sequence. Then, an immune response experiment of IL-6 was performed. Consequently, as shown in FIG. 5a, immune response of IL-6 was increased in the positive control and combination 44, while immune response of IL-6 was reduced to 1⁄2 level of the positive control for combination 48 having a substituted residue in a base sequence.
Further, a mouse at the age of 6 weeks was intravenously injected with β-gal siRNA and HPV 18 type siRNA combination 44 and 48 which arise immune response, and reacted for 6 hours to perform an immune response experiment of INF-gamma (FIG. 5b). Although immune response was observed in the positive control β-gal siRNA and combination 44 which was expressed by an increase in the INF-gamma level, immune response was not observed in combination 48 since INF-gamma in combination 48 showed a similar level to that of the negative control and did not increased.
Thus, it has been found that immune response was reduced in the siRNA treatment group having a substituted residue comparing with that siRNA having an unsubstituted residue does.
Pharmacokinetic Experiment of siRNA having Substituted Residue
By the method described above, a rat was intravenously injected with HPV 18 type siRNA combination 44 and combination 48, and blood was collected in a time-based manner. Then, plasma was separated to quantify siRNA through stem-loop real-time PCR method. Consequently, as shown in FIG. 6a, half-life of combination 48 was at least twice longer than that of combination 44 meaning that combination 48, which has a substituted residue in a base sequence, is more stable in vivo.
Effect of siRNA in Various Types of Liposome
By the method described above, a HPV 18 type HeLa cell line was transduced with siRNA by using commercially available Dharmafect (Dharmacon), Oligofectamine and Lipofectamine 2000 (Invitrgen) drug delivery systems and a cationic liposome prepared by the present inventors. After 24 hours, cell number was measured to evaluate the effect of inhibiting cell proliferation. Consequently, as shown in FIG. 7a, siRNA was effectively delivered to a cell line infected with HPV in various drug delivery systems.
Effect of Combined Treatment of siRNA Pool and Anti-Cancer Agent
By the method described above, cancer cells were xenografted to a mouse. After 10 days, generation of cancer cells was evaluated. Then, siRNA and an anti-cancer agent were repeatedly injected 9 times, and the size of a tumor was measured at 2-3 day interval. Consequently, as shown in FIG. 8a, the combined treatment group of the anti-cancer drug and SP4 (a pool of combination 48 and 54) showed the significantly outstanding therapeutic effect than the combined treatment group of the anti-cancer drug and the pool of combination 44 and 51. It has been found that SP4 having a substituted residue in a base sequence showed better efficacy and effect than the siRNA pool of combination 44 and 51 having a unsubstituted residue in a base sequence. Moreover, as shown in FIG. 8b, when compared a size of tumors of a mouse of the anti-cancer agent administration group of cisplatin and paclitaxel with that of the combined treatment group of the anti-cancer agent and SP4 pool on day 17, it has been proven that there are large differences in the size and state. Also, as shown in FIG. 8c, a result obtained by observing amounts of variation in body weight of the mouse on day 9 and 28 showed no reduction in body weight caused by toxicity. Thus, it has been determined that there was no side effect caused by toxicity of siRNA.
Hitherto, specific features of the present invention are described in detail. However, it would be apparent to a person skilled in the art that the specific description is preferable embodiment only, and the scope of the invention is not limited thereto. Therefore, substantial scope of the present invention would be defined by accompanying claims and equivalents thereof.

## Claims

1. A method for treating or preventing a disease caused by HPV infection, the method comprising administering, to a subject, a pharmaceutical composition including: (a) a pharmaceutically effective amount of one or more nucleotide pairs selected from the group consisting of: (i) a nucleotide pair of: a nucleotide comprising SEQ ID NO:1, and a nucleotide comprising a sequence antisense to SEQ ID NO:1, (ii) a nucleotide pair of: a nucleotide comprising SEQ ID NO:7, and a nucleotide comprising a sequence antisense to SEQ ID NO:7, (iii) a nucleotide pair of: a nucleotide comprising SEQ ID NO:12, and a nucleotide comprising a sequence antisense to SEQ ID NO:12, (iv) a nucleotide pair of: a nucleotide comprising SEQ ID NO:56, and a nucleotide comprising a sequence antisense to SEQ ID NO:56, and (v) a nucleotide pair of: a nucleotide comprising SEQ ID NO:62, and a nucleotide comprising a sequence antisense to SEQ ID NO:62, wherein the nucleotide pair includes one or more modified bases which are selected from the group consisting of 2′-O methylated U, 2′-O methylated G, and 2′-fluorinated C and (b) a pharmaceutically acceptable carrier.

2. The method of claim 1, wherein, the nucleotide pair is siRNA or shRNA.

3. The method of claim 2, wherein, the nucleotide is siRNA.