Nucleic acid lipid particle vaccine encapsulating HPV mRNA

Abstract

To provide a vaccine for preventing and / or treating infections with human papillomavirus.SOLUTION: The present invention relates to a lipid particle encapsulating a nucleic acid capable of expressing the E6 and E7 antigens of human papillomavirus, wherein the lipid comprises a cationic lipid represented by general formula (Ia) or a pharmaceutically acceptable salt thereof, wherein R1 and R2 each independently represent a C1-C3 alkyl group; L1 represents a C17-C19 alkenyl group which may have one or a plurality of C2-C4 alkanoyloxy groups; L2 represents a C10-C19 alkyl group which may have one or a plurality of C2-C4 alkanoyloxy groups, or a C10-C19 alkenyl group which may have one or a plurality of C2-C4 alkanoyloxy groups; and p is 3 or 4.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a nucleic acid-lipid particle vaccine encapsulating HPV mRNA.

Background Art

[0002] Human papillomavirus (HPV) is a virus having a circular double-stranded DNA without an envelope membrane as its genome, and there are around 200 genotypes (Non-Patent Document 1). Among them, there are genotypes that transform infected cells into cancer cells. In particular, genotypes 16 and 18, which have been proven to be correlated with cancer development typified by cervical cancer, are classified as high-risk types (Non-Patent Document 2).

[0003] The HPV genome encodes eight viral proteins, which are classified into early genes (E1, E2, E4, E5, E6, E7) and late genes (L1, L2) according to the expression time in the viral life cycle. Early genes control viral replication and the transformation of infected cells into cancer cells, and L1 and L2 are structural proteins that form the outer shell (capsid) of viral particles (Non-Patent Document 3).

[0004] HPV infects keratinocyte progenitor cells present in the basal layer of squamous epithelium. HPV infection is initiated by the adsorption of the L1 protein that constitutes the capsid to heparan sulfate proteoglycan present on the surface of the host cell membrane (Non-Patent Document 4). Since neutralizing antibodies responsible for HPV infection defense target the L1 protein, currently marketed preventive vaccines contain the VLP (virus-like particle) antigen of the L1 protein as an active ingredient. Also, all three existing preventive vaccines contain the L1 VLP antigens of genotypes 16 and 18. In any of the vaccines, in populations of young people naive to HPV infection, the preventive effect against HPV genotypes 16 and 18 is 95% or more, but no therapeutic effect on cervical cancer and cervical dysplasia, which is a precancerous state, is recognized (Non-Patent Document 5).

[0005] HPV-infected cells cause abnormalities in the cell cycle due to the oncogenic proteins E6 and E7. This is because the functions of p53 and pRb, which are responsible for the cell cycle and apoptosis induction, are inhibited by E6 and E7 (Non-Patent Documents 6 and 7). The regions important for the oncogenic activities of E6 and E7 have been clarified, and when using E6 and E7 as vaccine antigens, it is possible to enhance safety by inserting inactivating mutations (Non-Patent Documents 8-10).

[0006] Host defense immunity against HPV infection is the induction of neutralizing antibodies and the induction of cytotoxic T cells (CTLs) and helper T cells. In particular, the non-structural proteins of E6 and E7 are target antigens for CTL induction and have been attracting attention as vaccine antigens for the treatment of cervical cancer and cervical dysplasia caused by HPV infection (Non-Patent Document 11).

[0007] Patent Document 1 describes the E6 and E7 fusion antigen gene sequences of HPV genotypes 6, 11, 16, 18, 31, 33, 39, 45, 52, and 58. In the gene sequences described in this document, an IgE leader sequence is added to the N-terminus of E6, and a furin peptidase cleavage site is inserted between the translation region sequences of E6 and E7. In addition, mutations are inserted into the p53-binding region of E6 and the pRb-binding region of E7 to inactivate the oncogenic activities of E6 and E7. This gene sequence is introduced into a mammalian expression plasmid, and the efficacy evaluation in a mouse model is carried out as a DNA gene vaccine against HPV. Immunization is intramuscular administration to the thigh of a mouse using the electroporation method.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

[0009] Patent Document 1 JP 2016-512553 Summary of the Invention Problems to be Solved by the Invention

[0010] An object of the present invention is to provide a vaccine for preventing and / or treating infection by human papillomavirus. Means for Solving the Problems

[0011] The inventors of the present invention administered lipid particles encapsulating mRNA encoding the E6-E7 antigen of human papillomavirus to mice transplanted with cancer cells, and found that a cancer regression effect was observed, thus completing the present invention.

[0012] The gist of the present invention is as follows. (1) A lipid particle encapsulating a nucleic acid capable of expressing the E6 antigen and E7 antigen of human papillomavirus, wherein the lipid contains a cationic lipid represented by the general formula (Ia) or a pharmaceutically acceptable salt thereof. [Chemical formula] In the formula, R 1 and R 2 each independently represents a C1-C3 alkyl group; L 1 represents a C 17 -C 19 alkenyl group which may have one or more C2-C4 alkanoyloxy groups; L 2 represents a C 10 -C 19 alkyl group which may have one or more C2-C4 alkanoyloxy groups, or a C 10 -C 19 alkenyl group which may have one or more C2-C4 alkanoyloxy groups; p is 3 or 4. (2) The particle according to (1), wherein R 1 and R 2 in the general formula (Ia) are both methyl groups. (3) The particle according to (1) or (2), wherein p in the general formula (Ia) is 3. (4) The particle according to any one of (1) to (3), wherein L 1 in the general formula (Ia) is a C 17 -C 19 alkenyl group which may have one or more acetoxy groups. (5) L in the general formula (Ia) 2which may have one or more acetoxy groups, C 10 -C 12 alkyl group, or C which may have one or more acetoxy groups 10 -C 19 alkenyl group, the particle according to any one of (1) to (4). (6) L in the general formula (Ia) 2 which may have one or more acetoxy groups, C 10 -C 12 alkyl group, or C which may have one or more acetoxy groups 17 -C 19 alkenyl group, the particle according to any one of (1) to (4). (7) L in the general formula (Ia) 1 is (R)-11-acetoxy-cis-8-heptadecenyl group, cis-8-heptadecenyl group, or (8Z,11Z)-heptadecadienyl group, the particle according to any one of (1) to (6). (8) L in the general formula (Ia) 2 is decyl group, cis-7-decenyl group, dodecyl group, or (R)-11-acetoxy-cis-8-heptadecenyl group, the particle according to any one of (1) to (7). (9) The cationic lipid has the following structural formula:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0013] According to the present invention, it becomes possible to prevent and / or treat infections caused by human papillomavirus. Further, according to the present invention, it becomes possible to prevent and / or treat diseases (such as cervical cancer and cervical dysplasia) caused by infections with human papillomavirus. Further, the particles of the present invention have excellent properties in terms of metabolic stability, in vitro activity, in vivo activity, onset of drug efficacy, duration of drug efficacy, physical stability, drug interaction, safety, etc., and are useful as a medicament for treating or preventing the above diseases. This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2019-207001, which is the basis of the priority of the present application.

Brief Description of the Drawings

[0014]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Mode for Carrying Out the Invention

[0015] Hereinafter, embodiments of the present invention will be described in more detail.

[0016] The present invention provides lipid particles encapsulating a nucleic acid capable of expressing E6 antigen and E7 antigen of human papillomavirus, wherein the lipid contains a cationic lipid represented by the general formula (Ia) or a pharmaceutically acceptable salt thereof.

Chemical formula

[0017] R in general formula (Ia) 1 and R 2 independently represent a C1-C3 alkyl group, preferably both are methyl groups.

[0018] p in general formula (Ia) is 3 or 4, preferably 3.

[0019] L in general formula (Ia) 1 represents a C 17 -C 19 alkenyl group which may have one or more C2-C4 alkanoyloxy groups, preferably a C 17 -C 19 alkenyl group which may have one or more acetoxy groups. As L 1 , specifically, (R)-11-acetyloxy-cis-8-heptadecenyl group, cis-8-heptadecenyl group, or (8Z,11Z)-heptadecadienyl group etc. can be exemplified.

[0020] L in general formula (Ia) 2 represents a C 10 -C 19 alkyl group which may have one or more C2-C4 alkanoyloxy groups, or a C 10 -C19 represents an alkenyl group, preferably a C which may have one or more acetoxy groups 10 -C 12 alkyl group, or a C which may have one or more acetoxy groups 10 -C 19 alkenyl group. Alternatively, L in the general formula (Ia) 2 is preferably a C which may have one or more acetoxy groups 10 -C 12 alkyl group, or a C which may have one or more acetoxy groups 17 -C 19 alkenyl group. Specifically, examples of L 2 include a decyl group, a cis-7-decenyl group, a dodecyl group, or an (R)-11-acyloxy-cis-8-heptadecenyl group, etc.

[0021] Specific examples of the cationic lipid that constitutes the particles of the present invention include the following structural formulas:

Chemical formula

Chemical formula

Chemical formula

[0022] The cationic lipid represented by the general formula (Ia) may be a single compound or a combination of two or more compounds.

[0023] A method for producing the cationic lipid represented by the general formula (Ia) is described in the pamphlet of International Publication No. 2015 / 005253.

[0024] The lipid of the present invention may further contain an amphiphilic lipid, sterols, and a PEG lipid.

[0025] The amphiphilic lipid is a lipid having an affinity for both polar and nonpolar solvents. Specifically, examples thereof include distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, and combinations thereof. The amphiphilic lipid preferably used in the particles of the present invention is distearoyl phosphatidylcholine and / or dioleoyl phosphatidylethanolamine, and more preferably distearoyl phosphatidylcholine.

[0026] The sterols are sterols having a hydroxy group, and specific examples thereof include cholesterol.

[0027] The PEG lipid is a PEG-modified lipid. Specifically, examples thereof include 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine, and combinations thereof. Preferably, it is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol.

[0028] The lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is not particularly limited, but in molar amounts, the amphiphilic lipid is 22.5% or less, sterols are 15 - 55%, cationic lipid is 40 - 65%, PEG lipid is 1 - 5%, and the ratio of the total lipid weight to the nucleic acid weight is preferably 15 - 30. It is more preferably that the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, the amphiphilic lipid is 5 - 22.5%, sterols are 15 - 55%, cationic lipid is 40 - 65%, PEG lipid is 1 - 5%, and the ratio of the total lipid weight to the nucleic acid weight is 15 - 30. It is even more preferably that the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, the amphiphilic lipid is 10 - 22.5%, sterols are 15 - 55%, cationic lipid is 40 - 65%, PEG lipid is 1 - 5%, and the ratio of the total lipid weight to the nucleic acid weight is 15 - 30. In the above lipid composition, the proportion of the PEG lipid is more preferably 1 - 3% in molar amounts, even more preferably 1 - 2% in molar amounts, and particularly preferably 1.5 - 2% in molar amounts. Also, in the above lipid composition, the ratio of the total lipid weight to the nucleic acid weight is more preferably 15 - 25, even more preferably 15 - 22.5, and particularly preferably 17.5 - 22.5.

[0029] When using 3-dimethylaminopropyl (9Z,12Z)-octacosa-19,22-dien-11-yl carbonate or (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)penta-35-triaconta-9,26-dien-7,29-diyl diacetate as the cationic lipid, the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is not particularly limited, but in molar amounts, it is preferably that the amphiphilic lipid is 15% or less, sterols are 20 - 55%, the cationic lipid is 40 - 65%, and the PEG lipid is 1 - 5%. It is more preferably that the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, the amphiphilic lipid is 5 - 15%, sterols are 35 - 50%, the cationic lipid is 40 - 55%, and the PEG lipid is 1 - 3%. It is even more preferably that the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, the amphiphilic lipid is 10 - 15%, sterols are 35 - 45%, the cationic lipid is 40 - 50%, and the PEG lipid is 1 - 2%. It is even more preferably that the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, the amphiphilic lipid is 10 - 15%, sterols are 35 - 45%, the cationic lipid is 45 - 50%, and the PEG lipid is 1.5 - 2%. It is particularly preferably that the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, the amphiphilic lipid is 12.5%, sterols are 41%, the cationic lipid is 45%, and the PEG lipid is 1.5%. In the above lipid composition, the ratio of the total lipid weight to the nucleic acid weight is preferably 15 - 30, more preferably 15 - 25, even more preferably 15 - 22.5, and particularly preferably 17.5 - 22.5.

[0030] When using (7R,9Z)-18-({[3-(dimethylamino)propyl]oxy}carbonyl)oxy octacosa-9-en-7-yl acetate as the cationic lipid, the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is not particularly limited, but in terms of molar amount, it is preferably 12.5 to 22.5% for the amphiphilic lipid, 15 to 45% for sterols, 40 to 65% for the cationic lipid, and 1 to 5% for the PEG lipid. More preferably, the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is 15 to 22.5% for the amphiphilic lipid, 15 to 40% for sterols, 40 to 60% for the cationic lipid, and 1 to 3% for the PEG lipid in terms of molar amount. Even more preferably, the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is 15 to 22.5% for the amphiphilic lipid, 15 to 40% for sterols, 45 to 60% for the cationic lipid, and 1 to 2% for the PEG lipid in terms of molar amount. Even more preferably, the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is 17.5 to 22.5% for the amphiphilic lipid, 15 to 40% for sterols, 45 to 60% for the cationic lipid, and 1 to 2% for the PEG lipid in terms of molar amount. In the above lipid composition, the ratio of the total lipid weight to the nucleic acid weight is preferably 15 to 30, more preferably 15 to 25, even more preferably 15 to 22.5, and particularly preferably 17.5 to 22.5.

[0031] As specific lipid combinations in the present invention, phosphatidylcholine such as distearoylphosphatidylcholine, dioleoylphosphatidylcholine, or dioleoylphosphatidylethanolamine as amphiphilic lipids, cholesterol as sterols, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentahectaconta-9,26-diene-7,29-diyl diacetate, 3-dimethylaminopropyl (9Z,12Z)-octacosa-19,22-diene-11-yl carbonate, or (7R,9Z)-18-({[3-(dimethylamino)propyl)oxy]carbonyl}oxy)octacosa-9-ene-7-yl acetate as cationic lipids, and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol or N-[methoxy poly(ethylene glycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine as PEG lipids may be used in combination. Further, a lipid combination using distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine as amphiphilic lipids, cholesterol as sterols, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentahectaconta-9,26-diene-7,29-diyl diacetate or (7R,9Z)-18-({[3-(dimethylamino)propyl)oxy]carbonyl}oxy)octacosa-9-ene-7-yl acetate as cationic lipids, and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol as PEG lipids is preferred. More preferably, as specific lipid combinations in the present invention, distearoylphosphatidylcholine as amphiphilic lipids, cholesterol as sterols, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentahectaconta-9,26-diene-7,29-diyl diacetate or (7R,9Z)-18-({[3-(dimethylamino)propyl)oxy]carbonyl}oxy)octacosa-9-ene-7-yl acetate as cationic lipids, and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol as PEG lipids.

[0032] In the present invention, the nucleic acid encapsulated in the lipid particles is capable of expressing the E6 antigen and the E7 antigen of human papillomavirus. The E6 antigen and the E7 antigen of human papillomavirus expressed by the nucleic acid encapsulated in the lipid particles may be a fusion protein of the two, and a protease cleavage sequence may be included between the E6 antigen and the E7 antigen. The genotype of human papillomavirus is not particularly limited, and examples thereof include HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 have been proven to be correlated with the onset of cancers typified by cervical cancer.

[0033] The amino acid sequence of the E6 antigen of HPV type 16 is shown in SEQ ID NO: 8. The nucleic acid encapsulated in the lipid particles may preferably be one encoding the E6 antigen of HPV type 16, which consists of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequence of SEQ ID NO: 8.

[0034] The amino acid sequence of the E7 antigen of HPV type 16 is shown in SEQ ID NO: 9. The nucleic acid encapsulated in the lipid particles may preferably be one encoding the E7 antigen of HPV type 16, which consists of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequence of SEQ ID NO: 9.

[0035] The amino acid sequence of the E6 antigen of HPV type 18 is shown in SEQ ID NO: 14. The nucleic acid encapsulated in the lipid particles may preferably be one encoding the E6 antigen of HPV type 18, which consists of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequence of SEQ ID NO: 14.

[0036] The amino acid sequence of the E7 antigen of HPV type 18 is shown in SEQ ID NO: 15. The nucleic acid encapsulated in the lipid particle preferably encodes the E7 antigen of HPV type 18, which consists of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequence of SEQ ID NO: 15.

[0037] The amino acid sequence of the protease cleavage sequence (Furin Cleavage site) is shown in SEQ ID NO: 16. The protease cleavage sequence may be any sequence that can be cleaved by Furin protein, for example, a sequence represented by R-X-K / R-R (where R represents arginine, K represents lysine, and X represents any amino acid) (J. Biol. Chem. 1992, 267, 16396; J. Biol. Chem. 1991, 266, 12127).

[0038] The amino acid sequence of the fusion protein of the E6 and E7 antigens of HPV type 16 is shown in SEQ ID NO: 17. The nucleic acid encapsulated in the lipid particle preferably encodes the fusion protein of the E6 and E7 antigens of HPV type 16, which consists of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequence of SEQ ID NO: 17.

[0039] The amino acid sequence of the fusion protein of the E6 and E7 antigens of HPV type 18 is shown in SEQ ID NO: 18. The nucleic acid encapsulated in the lipid particle preferably encodes the fusion protein of the E6 and E7 antigens of HPV type 18, which consists of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequence of SEQ ID NO: 18.

[0040] The identity of an amino acid sequence is a quantification of the ratio of amino acid matches to the full-length sequence, considering corresponding amino acids that are exactly the same as the same amino acid. The sequence identity in the present invention is calculated using GENETYX-SV / RC (manufactured by Genetics Co., Ltd.), which is a sequence analysis software, and this algorithm is commonly used in the relevant technical field. The amino acids encoded by the nucleic acid encapsulated in the lipid particles of the present invention may have amino acid mutations (substitutions), deletions, insertions, and / or additions as long as they maintain a certain level of identity with SEQ ID NOs: 8, 9, 14-18.

[0041] The amino acids encoded by the nucleic acid encapsulated in the lipid particles of the present invention maintain the above-described sequence identity, and in the amino acid sequences of SEQ ID NOs: 8, 9, 14-18, at several positions (preferably 5 or fewer positions, more preferably 3, 2, or 1 position), several amino acids (preferably 10 or fewer, more preferably 7 or fewer, even more preferably 5, 4, 3, 2, or 1 amino acid) per position may be substituted, deleted, inserted, and / or added.

[0042] Nucleic acids capable of expressing the E6 antigen and E7 antigen of human papillomavirus (e.g., HPV type 16, HPV type 18) are preferably mRNAs containing a cap structure (Cap), a 5'untranslated region (5'-UTR), a leader sequence, a translation region of E6, a protease cleavage site (Furin Cleavage site), a translation region of E7, a 3'untranslated region (3'-UTR), and a polyA tail (polyA). The cap structure (Cap) is a site present at the 5' end of many eukaryotic mRNAs and has a 7-methylguanosine structure. Examples of the cap structure include cap0, cap1, cap2, ARCA (Anti-Reverse Cap Analog), etc., and the cap structure is as shown by the following structural formula.

Chemical formula

Chemical Structure

Chemical Structure

Chemical Structure

[0043] The nucleic acid encapsulated in the lipid particle may be in any form as long as it is a nucleic acid capable of expressing the E6 antigen and E7 antigen of the human papillomavirus. For example, single-stranded DNA, single-stranded RNA (e.g., mRNA), single-stranded polynucleotide mixed with DNA and RNA, double-stranded DNA, double-stranded RNA, DNA-RNA hybrid polynucleotide, double-stranded polynucleotide consisting of two polynucleotides mixed with DNA and RNA, etc. may be mentioned, and preferably mRNA.

[0044] The nucleotides constituting the nucleic acid encapsulated in the lipid particles may be natural or modified nucleotides, but it is preferable to contain at least one modified nucleotide.

[0045] The modified nucleotide may be one in which any part of the base, sugar, and phosphodiester bond is modified. The modification site may be one or two or more.

[0046] Examples of base modifications include 5-methylation, 5-fluorination, N4-methylation of cytosine, 5-methylation (thymine), 5-fluorination of uracil, N6-methylation of adenine, N2-methylation of guanine, and the like.

[0047] An example of sugar modification can be 2'-O-methylation of D-ribofuranose.

[0048] An example of modification of the phosphodiester bond can be a phosphorothioate bond.

[0049] The modified nucleotide preferably has a modified base part. For example, it may be a pyrimidine nucleotide substituted at the 5-position or pseudouridine which may be substituted at the 1-position. Specifically, 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, 1-alkylpseudouridine can be exemplified. Further, the 1-alkylpseudouridine may be 1-(C1-C6 alkyl)pseudouridine, preferably 1-methylpseudouridine or 1-ethylpseudouridine. More preferably, the modified nucleotides include 5-methylcytidine, 5-methyluridine, and 1-methylpseudouridine. Particularly preferably, the modified nucleotides include a combination of 5-methylcytidine and 5-methyluridine, or a combination of 5-methylcytidine and 1-methylpseudouridine.

[0050] Nucleic acids capable of expressing the E6 antigen and E7 antigen of human papillomavirus (e.g., HPV type 16, HPV type 18) of the present invention can be produced by an in vitro transcription reaction from DNA having a desired nucleotide sequence. Enzymes, buffers, and nucleoside-5'-triphosphate mixtures (adenosine-5'-triphosphate (ATP), guanosine-5'-triphosphate (GTP), cytidine-5'-triphosphate (CTP), and uridine-5'-triphosphate (UTP)) required for in vitro transcription are commercially available (AmpliScribe T7 High Yield Transcription Kit (Epicentre), mMESSAGE mMACHINE T7 Ultra Kit (Life thechnologies), etc.). DNA used for producing single-stranded RNA is cloned DNA, for example, plasmid DNA or DNA fragments. Plasmid DNA or DNA fragments may be commercially available ones, or can be produced by methods generally known in the art (e.g., the methods described in Sambrook, J. et al., Molecular Cloning a Laboratory Manual second edition (1989), Rashtchian, A., Current Opinion in Biotechnology, 1995, 6(1), 30-36, Gibson D. G. et al., Science, 2008, 319(5867), 1215-1220, etc.).

[0051] In order to obtain mRNA with improved stability and / or safety, in an in vitro transcription reaction, some or all of the unmodified nucleotides in the mRNA can be replaced with modified nucleotides by replacing some or all of the unmodified nucleoside-5'-triphosphates with modified nucleoside-5'-triphosphates (Kormann, M., Nature Biotechnology, 2011, 29, 154-157.).

[0052] To obtain mRNA with improved stability and / or safety, a cap structure (the above Cap0 structure) can be introduced at the 5'-end of mRNA by a method using a capping enzyme after an in vitro transcription reaction. Further, Cap0 in the mRNA can be converted to Cap1 by a method of allowing 2'-O-methyltransferase to act on the mRNA having Cap0. As the capping enzyme and 2'-O-methyltransferase, commercially available products can be used (for example, Vaccinia Capping System, M2080; mRNA Cap 2'-O-Methyltransferase, M0366, both manufactured by New England Biolab). When using commercially available products, mRNA having a cap structure can be produced according to the protocol attached to the product.

[0053] The cap structure at the 5'-end of mRNA can also be introduced by a method different from using an enzyme. For example, by adding ARCA or CleanCap to an in vitro transcription reaction, a cap analog structure of ARCA or a Cap1 structure derived from CleanCap can be introduced into mRNA. As ARCA and CleanCap, commercially available products can be used (ARCA, N-7003; CleanCap Reagent AG, N-7113, both manufactured by TriLink BioTechnologies). When using commercially available products, mRNA having a cap structure can be produced according to the protocol attached to the product.

[0054] The nucleic acid-encapsulating lipid particles of the present invention can be produced by methods such as the thin film method, reverse phase evaporation method, ethanol injection method, ether injection method, dehydration-rehydration method, surfactant dialysis method, hydration method, freeze-thaw method, etc. For example, nucleic acid-encapsulating lipid particles can be produced by the method described in International Publication No. 2015 / 005253. The nucleic acid-encapsulating lipid particles of the present invention can also be produced by mixing a nucleic acid solution and a lipid solution in a microchannel. For example, using NanoAssemblr (registered trademark) of Precision Nanosystems and manufacturing according to the method described in the attached protocol.

[0055] The particles of the present invention preferably have an average particle diameter of 30 nm to 300 nm, more preferably 30 to 200 nm, and even more preferably 30 to 100 nm. The average particle diameter can be obtained by measuring the volume average particle diameter based on the principle of dynamic light scattering using an instrument such as Zeta Potential / Particle Sizer NICOMP (registered trademark) 380ZLS (PARTICLE SIZING SYSTEMS).

[0056] The particles of the present invention can be used to produce a composition for preventing and / or treating diseases caused by human papillomavirus infection (cervical cancer, cervical dysplasia, anal cancer, oropharyngeal cancer, condyloma acuminata). The infection is preferably caused by human papillomavirus types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 6, 11, more preferably by HPV16 type and / or HPV18 type, and even more preferably by HPV16 type.

[0057] Using the particles of the present invention, the E6 antigen and E7 antigen of human papillomavirus can be expressed in vivo or in vitro. Therefore, the present invention provides a method for expressing the E6 antigen and E7 antigen of human papillomavirus in vitro, which includes introducing a composition containing the above particles into cells. In addition, the present invention also provides a method for expressing the E6 antigen and E7 antigen of human papillomavirus in vivo, which includes administering a composition containing the above particles to a mammal. By expressing the E6 antigen and E7 antigen of human papillomavirus in vivo, an immune response against human papillomavirus can be induced. As a result, human papillomavirus infection can be prevented and / or treated. Therefore, the present invention provides a method for inducing an immune response against human papillomavirus, which includes administering a composition containing the above particles to a mammal. In addition, the present invention provides a method for preventing and / or treating human papillomavirus infection, which includes administering a composition containing the above particles to a mammal.

[0058] The particles of the present invention can be used as a medicine and also as a reagent for experiments. The particles of the present invention are usually added to carriers such as water, buffer solution, and physiological saline, and this formulation (composition) can be introduced into cells (in vitro) or administered to mammals (in vivo). When administered to a mammal, the carrier is preferably a pharmaceutically acceptable carrier (for example, physiological saline). In addition, the particles of the present invention can be formulated into dosage forms such as creams, pastes, ointments, gels, and lotions using fats, fatty oils, lanolin, petrolatum, paraffin, waxes, resins, plastics, glycols, higher alcohols, glycerin, water, emulsifiers, suspending agents, etc. as base raw materials.

[0059] The particles of the present invention can be administered to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, pigs, monkeys, cats, dogs, horses, goats, sheep, and cows by oral administration or parenteral administration methods such as intramuscular administration, intravenous administration, rectal administration, transdermal administration, transmucosal administration, subcutaneous administration, and intradermal administration.

[0060] When administering the particles of the present invention to humans, for example, the dosage may be about 0.001 to 1 mg, preferably 0.01 to 0.2 mg per adult per administration, and it may be administered intramuscularly, subcutaneously, intradermally, by intravenous drip injection, or by intravenous injection once or several times. However, the dosage and the number of administrations can be appropriately changed depending on the type of disease, symptoms, age, administration method, etc.

[0061] When used as an experimental reagent, the particles of the present invention can be introduced into cells that express the E6 antigen and E7 antigen of human papillomavirus (for example, HEK293 cells and their derivative cells (HEK293T cells, FreeStyle 293 cells, and Expi293 cells), CHO cells, C2C12 mouse myoblasts, immortalized mouse dendritic cells (MutuDC1940)), and the E6 antigen and E7 antigen of human papillomavirus can be expressed in vitro. The expression of the E6 antigen and E7 antigen of human papillomavirus can be analyzed by detecting the E6 antigen and E7 antigen proteins of human papillomavirus in the sample by Western blotting or by detecting specific peptide fragments of the E6 antigen and E7 antigen of human papillomavirus by mass spectrometry.

[0062] In the present invention, "treatment" means the recovery, remission, alleviation, and / or delay of the progression of clinical symptoms of diseases that occur in patients with infectious diseases caused by viruses or bacteria, or diseases caused by such infections (for example, precancerous lesions and cancers).

[0063] In the present invention, "prevention" means reducing the incidence of diseases caused by infectious diseases caused by viruses or bacteria. Prevention includes reducing the risk of progression of diseases caused by infectious diseases caused by viruses or bacteria, or reducing the severity of those diseases. Since the particles of the present invention induce a defensive immune response, they are effective for the prevention and / or treatment of the above diseases.

Examples

[0064] Hereinafter, the present invention will be specifically described by way of examples. These examples are for explaining the present invention and do not limit the scope of the present invention. [Example 1] Preparation of HPV16 E6-E7 fusion2 mRNA -001 (1) Preparation of template DNA for in vitro transcription (IVT) of HPV16 E6-E7 fusion2 A plasmid was constructed to prepare template DNA for in vitro transcription (IVT). A DNA fragment (SEQ ID NO: 1) containing a sequence in which GCTAGC (NheI site), T7 promoter sequence, 5'-UTR sequence of human β-globin, KOZAK sequence, IgE leader sequence-HPV type 16 E6-Furin Cleavage site-HPV type 16 E7 translation region, 3'-UTR sequence of human β-globin, polyA tail, and ACTAGT (SpeI site) were ligated in sequence was introduced into a plasmid (pMA-HPV16_fusion2).

[0065] (2) Linearization of template DNA To the Nuclease-free water (2200 μL, Thermo Fisher catalog # AM9937) in which the plasmid (250 μg) obtained in Example 1-(1) was dissolved, 10× CutSmart Buffer (250 μL, New England Biolabs catalog # B7204S) and SpeI-HF (30 μL, New England Biolabs catalog # R3133L) were added, incubated at 37°C for 2 hours, and then incubated at 65°C for 20 minutes. 7.5 M ammonium acetate (1250 μL) and ethanol (7500 μL) were mixed and left standing at -20°C overnight. After centrifugation (4°C, 4000×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, after centrifugation (4°C, 4000×g, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was prepared into a 500 μg / mL solution with TE-Buffer.

[0066] (3) Preparation of HPV16 E6-E7 fusion2 mRNA -001 by in vitro transcription 500 μg / mL template DNA (200 μL) obtained in Example 1-(2), 100 mM CleanCap AG (200 μL, TriLink catalog # T-7113), 100 mM ATP (200 μL, Hongene catalog # R1331), 100 mM GTP (200 μL, Hongene catalog # R2331), 100 mM 5-Me-CTP (200 μL, Hongene catalog # R3-029), 100 mM 5-methyluridine triphosphate (200 μL), Nuclease-free water (1600 μL, Thermo Fisher catalog # AM9937), T7 Transcription 5× buffer (800 μL, Promega catalog # P140X), Enzyme mix, T7 RNA Polymerase (400 μL, Promega catalog # P137X) were mixed and incubated at 37°C for 4 hours. RQ1 RNase-Free DNase (100 μL, Promega catalog # M6101) was mixed and incubated at 37°C for 15 minutes. 8 M LiCl solution (2000 μL, Sigma-Aldrich catalog # L7026) was mixed and left standing at -20°C overnight. After centrifugation (4°C, 4000×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (4°C, 4000×g, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in Nuclease-free water and purified using the RNeasy Maxi kit (Qiagen catalog # 75162) according to the attached manual. The obtained eluate (10.3 mL, 17013 μg in terms of UV) was mixed with Nuclease-free water (247 μL), the buffer (1550 μL) and enzyme (3403 μL) of rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001), incubated at 37°C for 1 hour, and then incubated at 75°C for 15 minutes.8 M LiCl solution (7750 μL) was mixed and left standing overnight at -20°C. After centrifugation (4°C, 4000×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (4°C, 4000×g, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in Nuclease-free water and then purified by reverse-phase high-performance liquid chromatography (two Chromolith Semi-Prep (Merck catalog # 1.52016.0001) connected in series, 5% acetonitrile, 400 mM triethylamine acetate (pH 7.0) / 25% acetonitrile, 400 mM triethylamine acetate (pH 7.0), 80°C) to obtain the target mRNA. The obtained mRNA has the sequence of SEQ ID NO: 2. It was analyzed by Experion RNA StdSens (BIO-RAD catalog # 7007103JA) and confirmed to be of the desired length.

[0067] [Example 2] Preparation of HPV16 E6-E7 fusion10 mRNA -001 (1) Preparation of template DNA for IVT of HPV16 E6-E7 fusion10 A plasmid was constructed to prepare the template DNA used for the IVT template. A DNA fragment (SEQ ID NO: 3) containing a sequence in which GCTAGC (NheI site), T7 promoter sequence, 5'-UTR sequence of β-globin, KOZAK sequence, IgE leader sequence-HPV16 type E6-Furin Cleavage site-HPV16 type E7 translation region, 3'-UTR sequence of β-globin, polyA tail, and ACTAGT (SpeI site) were ligated in sequence was introduced into a plasmid (pMA-HPV16_fusion10).

[0068] (2) Preparation of HPV16 E6-E7 fusion10 mRNA -001 by in vitro transcription Instead of the plasmid obtained in Example 1-(1), the plasmid obtained in Example 2-(1) was used, and mRNA was obtained in the same manner as in Example 1-(2) and (3).

[0069] The obtained mRNA has the sequence of SEQ ID NO: 4. It was analyzed by Experion RNA StdSens and confirmed to be of the desired length.

[0070] [Example 3] Preparation of HPV16 E6-E7 fusion10 mRNA -002 Instead of the plasmid obtained in Example 1-(1), the plasmid obtained in Example 2-(1) was used, and template DNA was obtained in the same manner as in Example 1-(2). Then, instead of the template DNA in Example 1-(3), the obtained template DNA was used, and 100 mM Pseudo-UTP (Hongene catalog # R5-022) was used instead of 100 mM 5-Me-UTP, and mRNA was obtained in the same manner as in Example 1-(3).

[0071] The obtained mRNA has the sequence of SEQ ID NO: 4. It was analyzed by Experion RNA StdSens and confirmed to be of the desired length.

[0072] [Example 4] Preparation of HPV16 E6-E7 fusion10 mRNA -003 Instead of the plasmid obtained in Example 1-(1), the plasmid obtained in Example 2-(1) was used, and template DNA was obtained in the same manner as in Example 1-(2). Then, instead of the template DNA in Example 1-(3), the obtained template DNA was used, and 100 mM CTP (Hongene catalog # R3331) was used instead of 100 mM 5-Me-CTP, and 100 mM N1-methylpseudouridine-5’-triphosphate (TriLink catalog # N-1081) was used instead of 100 mM 5-Me-UTP, and mRNA was obtained in the same manner as in Example 1-(3).

[0073] The obtained mRNA has the sequence of SEQ ID NO: 4. It was analyzed by Experion RNA StdSens and confirmed to be of the desired length.

[0074] [Example 5] Preparation of HPV16 E6-E7 fusion10 mRNA -004 Instead of using the plasmid obtained in Example 1-(1), the plasmid obtained in Example 2-(1) was used to obtain template DNA in the same manner as in Example 1-(2). Subsequently, instead of the template DNA in Example 1-(3), the obtained template DNA was used, and 100 mM CTP (Hongene catalog # R3331) was used instead of 100 mM 5-Me-CTP to obtain mRNA in the same manner as in Example 1-(3).

[0075] The obtained mRNA has the sequence of SEQ ID NO: 4. It was analyzed by Experion RNA StdSens and confirmed to be of the desired length.

[0076] [Example 6] Preparation of HPV16 E6-E7 fusion10 opt2 mRNA -001 (1) Preparation of template DNA for IVT of HPV16 E6-E7 fusion10 opt2 A plasmid was constructed to create the template DNA used for the IVT template. A DNA fragment (SEQ ID NO: 5) containing a sequence in which GCTAGC (NheI site), T7 promoter sequence, 5'-UTR sequence of β-globin, Kozak sequence, IgE leader sequence-HPV type 16 E6-Furin Cleavage site-HPV type 16 E7 translation region, 3'-UTR sequence of β-globin, polyA tail, and ACTAGT (SpeI site) were ligated in sequence was introduced to prepare a plasmid (pMA-HPV16_fusion10_opt2).

[0077] (2) Linearization of Template DNA The plasmid (250 μg) obtained in Example 6-(1) was dissolved in Nuclease-free water (2200 μL, Thermo Fisher catalog # AM9937), and 10× CutSmart Buffer (250 μL, New England Biolabs catalog # B7204S) and SpeI-HF (30 μL, New England Biolabs catalog # R3133L) were added. After incubation at 37°C for 2 hours, the mixture was incubated at 65°C for 20 minutes. 7.5 M ammonium acetate (1250 μL) and ethanol (7500 μL) were mixed and left standing at -20°C overnight. After centrifugation (4°C, 4000×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (4°C, 4000×g, 10 minutes), the supernatant was discarded and the pellet was air-dried. The resulting residue was prepared as a 500 μg / mL solution in TE-Buffer.

[0078] (3) Preparation of HPV16 E6-E7 fusion10 opt2 mRNA -001 by in vitro transcription 500 μg / mL template DNA (100 μL) obtained in Example 6-(2), 100 mM ATP (150 μL, Hongene catalog # R1331), 100 mM GTP (150 μL, Hongene catalog # R2331), 100 mM CTP (150 μL, Hongene catalog # R3331), 100 mM N1-methylpseudouridine-5’-triphosphate (150 μL, Hongene catalog # R5-027), Nuclease-free water (700 μL, Thermo Fisher catalog # AM9937), T7 Transcription 5× buffer (400 μL, Promega catalog # P140X), Enzyme mix, T7 RNA Polymerase (200 μL, Promega catalog # P137X) were mixed and incubated at 37°C for 4 hours. RQ1 RNase-Free DNase (50 μL, Promega catalog # M6101) was mixed and incubated at 37°C for 15 minutes. 8 M LiCl solution (1000 μL, Sigma-Aldrich catalog # L7026) was mixed and left standing at -20°C overnight. After centrifugation (4°C, 4000×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (4°C, 4000×g, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in Nuclease-free water. To this solution (962 μL, 5400 μg in terms of UV), Nuclease-free water (2818 μL) was added, heated at 70°C for 20 minutes, and then cooled on ice for 10 minutes.Add 540 μL of 10× capping buffer (500 mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM MgCl2, 50 mM DTT), 20 mM GTP (270 μL, diluted with nuclease-free water from 100 mM GTP, Hongene catalog # R2331), 20 mM SAM (270 μL, diluted with nuclease-free water from 32 mM SAM, New England Biolabs catalog # B9003S), and Vaccinia Capping Enzyme (540 μL, Hongene catalog # ON-028). After incubation at 37°C for 4 hours, add 10× capping buffer (90 μL), 20 mM SAM (270 μL), and 2’-O-Methyltransferase (540 μL, Hongene catalog # ON-014), and incubate at 37°C for 4 hours. Mix with 8 M LiCl solution (6300 μL) and let stand at -20°C overnight. After centrifugation (4°C, 4000×g, 30 minutes), discard the supernatant, add 70% ethanol, and after centrifugation (4°C, 4000×g, 10 minutes), discard the supernatant and air-dry. Dissolve the obtained residue in nuclease-free water and purify by reverse-phase high-performance liquid chromatography (two connected Chromolith Semi-Prep (Merck catalog # 1.52016.0001), 5% acetonitrile, 400 mM triethylamine acetate (pH 7.0) / 25% acetonitrile, 400 mM triethylamine acetate (pH 7.0), 80°C) to obtain the target mRNA.

[0079] The obtained mRNA has the sequence of SEQ ID NO: 6. It was analyzed by Experion RNA StdSens and confirmed to be of the desired length.

[0080] (4) Preparation of HPV16 E6-E7 fusion10 opt2 mRNA-001 by in vitro transcription 500 μg / mL template DNA (200 μL) obtained in Example 6-(2), 100 mM ATP (300 μL, Hongene catalog # R1331), 100 mM GTP (300 μL, Hongene catalog # R2331), 100 mM CTP (300 μL, Hongene catalog # R3331), 100 mM N1-methylpseudouridine-5’-triphosphate (300 μL, Hongene catalog # R5-027), Nuclease-free water (1400 μL, APPLIED-BIO catalog # AM9937), 800 μL of T7 Transcription 5× buffer, (400 mM HEPES-KOH (pH 7.5), 80 mM MgCl2, 10 mM spermidine, 200 mM DTT), and 400 μL of Enzyme mix, T7 RNA Polymerase (Promega catalog # P137X) were mixed and incubated at 37°C for 8 hours. 100 μL of RQ1 RNase-Free DNase (Promega catalog # M6101) was mixed and incubated at 37°C for 15 minutes. 2000 μL of 8 M LiCl solution (Sigma-Aldrich catalog # L7026) was mixed and left standing at -20°C overnight. After centrifugation (4°C, 4000×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (4°C, 4000×g, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in Nuclease-free water. To this solution (1590 μL, 6000 μg in terms of UV), Nuclease-free water (2610 μL) was added, heated at 70°C for 10 minutes, and then cooled on ice for 10 minutes.Add 600 μL of 10× capping buffer (500 mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM MgCl2, 50 mM DTT), 20 mM GTP (300 μL, diluted from 100 mM GTP, Hongene catalog # R2331 with nuclease-free water for use), 20 mM SAM (300 μL, S-adenosyl-L-methionine disulfate tosylate, OX-CHEM catalog # AX8250818 dissolved in 0.005 M sulfuric acid, 10% ethanol solution for use), and Vaccinia Capping Enzyme (600 μL, Hongene catalog # ON-028). After incubation at 37°C for 4 hours, add 100 μL of 10× capping buffer, 300 μL of 20 mM SAM, and 2’-O-Methyltransferase (600 μL, Hongene catalog # ON-014), and incubate at 37°C for 4 hours. Mix with 7000 μL of 8 M LiCl solution and let stand at -20°C overnight. After centrifugation (4°C, 4000×g, 30 minutes), discard the supernatant, add 70% ethanol, and after centrifugation (4°C, 4000×g, 10 minutes), discard the supernatant and air-dry. Dissolve the obtained residue in nuclease-free water and purify by reverse-phase high-performance liquid chromatography (Chromolith Performance (Merck catalog # 1.02129.0001), 5% acetonitrile, 400 mM triethylamine acetate (pH 7.0) / 25% acetonitrile, 400 mM triethylamine acetate (pH 7.0), 45°C) to obtain the target mRNA.

[0081] The obtained mRNA has the sequence of SEQ ID NO: 6. It was analyzed by LabChip GX Touch HT mRNA StdSens (PerkinElmer catalog # CLS960010) to confirm that it is of the desired length.

[0082] [Example 7] Preparation of HPV16 E6-E7 fusion10 opt2 mRNA -002 Instead of 100 mM CTP in Example 6-(3), 100 mM 5-Me-CTP was used, and instead of 100 mM N1-methylpseudouridine-5’-triphosphate, 100 mM 5-methyluridine triphosphate was used. mRNA was obtained in the same manner as in Example 6-(3). The obtained mRNA has the sequence of SEQ ID NO: 6. It was analyzed by Experion RNA StdSens and confirmed to be of the desired length.

[0083] [Example 8] Preparation of HPV mRNA-encapsulated nucleic acid lipid particles described in Example 1 (1) Preparation of mRNA-encapsulated nucleic acid lipid particles 1,2-Distearoyl-sn-glycero-3-phosphocholine (hereinafter referred to as DSPC, NOF CORPORATION), cholesterol (hereinafter referred to as Chol, Sigma-Aldrich, Inc.), (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)penta- triaconta-9,26-dien-7,29-diyl of diacetic acid (the compound described in Example 23 of WO2015 / 005253) (hereinafter referred to as LP), and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol with a polyethylene glycol molecular weight of about 2000 (1,2-Dimyristoyl-sn-Glycero-3-Methoxypolyethylene Glycol, hereinafter referred to as PEG-DMG, NOF CORPORATION) were dissolved in ethanol at a total lipid concentration of 5 mM at a molar ratio of DSPC:Chol:LP:PEG-DMG = 10:43.5:45:1.5.

[0084] On the other hand, the HPV16 fusion2 mRNA-001 obtained in Example 1 was prepared at 51.8 μg / mL in a citrate buffer (20 mM Citrate Buffer, pH 4.0).

[0085] The above lipid solution and mRNA solution were mixed in a microchannel using NanoAssemblr BenchTop (Precision Nanosystems Inc.) so that their volume ratio was 1:3 to obtain a crude dispersion of nucleic acid-lipid particles. The dispersion of nucleic acid-lipid particles was dialyzed (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) against about 25 to 50 volumes of phosphate buffer (pH 7.4) for 12 - 18 hours to remove ethanol, thereby obtaining a dispersion of purified mRNA-encapsulated nucleic acid-lipid particles.

[0086] Incidentally, LP was synthesized according to the method described in Example 23 of WO2015005253.

[0087] (2) Characterization of mRNA-encapsulated nucleic acid lipid particles The characteristics of the dispersion containing the nucleic acid-lipid particles prepared in (1) were evaluated. The methods for each characteristic evaluation will be described.

[0088] (2-1) Encapsulation efficiency of mRNA The encapsulation efficiency of mRNA was measured using the Quant-iT RiboGreen RNA Assay kit (Invitrogen) according to the attached instructions. That is, in the presence and absence of 0.015% Triton X-100 surfactant, the mRNA in the dispersion of nucleic acid-lipid particles was quantified, and the encapsulation efficiency was calculated by the following formula. {[Amount of mRNA in the presence of surfactant] - [Amount of mRNA in the absence of surfactant]} / [Amount of mRNA in the presence of surfactant]} x 100 (%)

[0089] (2-2) Ratio of mRNA to lipid The amount of mRNA in the dispersion of nucleic acid-lipid particles was measured by reverse-phase chromatography (System: Agilent 1100 series, Column: Bioshell A400 Protein C4 (10 cm × 4.6 mm, 3.4 μm) (SUPELCO), Buffer A: 0.1 M triethylamine acetate (pH 7.0), Buffer B: acetonitrile, (B%): 5 - 50% (0 - 15 min), Flow Rate: 1 mL / min, Temperature: 70 °C, Detection: 260 nm).

[0090] The amount of each lipid in the dispersion of nucleic acid-lipid particles was measured by reverse-phase chromatography (System: DIONEX UltiMate 3000, Column: XSelect CSH (50 mm × 3 mm, 5 μm) (Thermo Fisher Scientific), Buffer A: 0.2% formic acid, Buffer B: 0.2% formic acid, methanol, (B%): 75 - 95% (0 - 15 min), 95% (15 - 17 min), Flow Rate: 0.4 mL / min, Temperature: 50 °C, Detection: Corona CAD (Charged Aerosol Detector)). The ratio of the total lipid mass to the mRNA was calculated by the following formula.

[0091] [Total lipid concentration] / [mRNA concentration] (wt / wt)

[0092] (2-3) Average particle size The particle size of the nucleic acid-lipid particles was measured using a Zeta Potential / Particle Sizer NICOMPTM 380ZLS (PARTICLE SIZING SYSTEMS). The average particle size in the table represents the volume-average particle size, and ± below represents the deviation.

[0093] The results are shown in Table 1.

[0094] [Examples 9-13] Preparation of HPV mRNA-encapsulated nucleic acid lipid particles In the same manner as in Example 8, the preparation and property evaluation of the mRNA-encapsulated nucleic acid lipid particles described in Examples 2, 3, 4, 5, and 6 were carried out. However, the molar ratio of the constituent lipid composition was set to DSPC:Chol:LP:PEG-DMG = 12.5:41:45:1.5. The results are shown in Table 1.

[0095] [Examples 14-17] Preparation of HPV mRNA-encapsulated nucleic acid lipid particles In the same manner as in Example 8, the preparation and property evaluation of the mRNA-encapsulated nucleic acid lipid particles described in Examples 2, 4, 6, and 7 were carried out. However, the molar ratio of the constituent lipid composition was set to DSPC:Chol:LP:PEG-DMG = 12.5:41:45:1.5. The results are shown in Table 1.

[0096] [Example 18] Preparation of HPV mRNA-encapsulated nucleic acid lipid particles described in Example 2 In the same manner as in Example 8, the preparation and property evaluation of the mRNA-encapsulated nucleic acid lipid particles described in Example 2 were carried out. However, the molar ratio of the constituent lipid composition was set to DSPC:Chol:LP:PEG-DMG = 12.5:41:45:1.5, and the ratio of the total lipid weight to the mRNA weight was set to 25. The results are shown in Table 1.

[0097] [Example 19] Preparation of HPV mRNA-encapsulated nucleic acid lipid particles described in Example 2 In the same manner as in Example 8, the preparation and property evaluation of the mRNA-encapsulated nucleic acid lipid particles described in Example 2 were carried out. However, the molar ratio of the constituent lipid composition was set to DSPC:Chol:LP:PEG-DMG = 12.5:41:45:1.5, and the ratio of the total lipid weight to the mRNA weight was set to 30. The results are shown in Table 1.

[0098] [Example 20] Preparation of nucleic acid-lipid particles encapsulating HPV mRNA described in Example 2 In the same manner as in Example 8, the preparation and property evaluation of the mRNA-encapsulated nucleic acid lipid particles described in Example 2 were carried out. The results are shown in Table 1.

[0099] [Example 21] Preparation of nucleic acid-lipid particles encapsulating OVA mRNA Preparation of nucleic acid-lipid particles encapsulating mRNA In the same manner as in Example 8-(1), nucleic acid-lipid particles encapsulating mRNA having the translation region of OVA (Ovalbumin) described in SEQ ID NO: 7 were prepared. However, instead of PEG-DMG, N-[methoxy poly(ethylene glycol)2000 carbamyl]-1,2-dimyristyloxypropyl-3-amine with a polyethylene glycol molecular weight of about 2000 (hereinafter referred to as PEG-C-DMA), the compound 12) described in Journal of Controlled Release 112 (2006) 280-290 was used, and the constituent lipid composition was set to a molar ratio of DSPC:Chol:LP:PEG-C-DMA = 10:38.5:50:1.5. (2) Characterization of nucleic acid-lipid particles encapsulating mRNA (1) The characteristics of the dispersion containing the nucleic acid-lipid particles prepared were evaluated. The methods for each characteristic evaluation will be described. (2-1) Encapsulation efficiency of mRNA The encapsulation efficiency of mRNA was measured according to the attached document using the Quant-iT RiboGreen RNA Assay kit (Invitrogen). That is, in the presence and absence of 0.015% Triton X-100 surfactant, the mRNA in the dispersion of the nucleic acid-lipid particles was quantified, and the encapsulation efficiency was calculated by the following formula. {[Amount of mRNA in the presence of surfactant] - [Amount of mRNA in the absence of surfactant]} / [Amount of mRNA in the presence of surfactant]} x 100 (%) (2-2) Ratio of mRNA to lipid The amount of mRNA in the dispersion of the nucleic acid-lipid particles was taken as the "amount of mRNA in the presence of surfactant" in (2-1). The amount of phospholipid in the dispersion of the nucleic acid-lipid particles was measured according to the attached document using Phospholipid C-Test Wako (FUJIFILM Wako Pure Chemical Corporation). That is, in the presence of 2% Triton X-100 surfactant, the amount of phospholipid in the sample was measured. The amounts of cholesterol and LP in the dispersion of nucleic acid-lipid particles were measured by reverse-phase chromatography (System: DIONEX UltiMate 3000, Column: Chromolith Performance RP-18 endcapped 100-3 monolithic HPLC-column (Merck, Cat.#: 1.52001.0001), Buffer A: 0.01% trifluoroacetic acid, Buffer B: 0.01% trifluoroacetic acid, methanol, (B%): 82-97% (0-17 min), Flow Rate: 2 mL / min, Temperature: 50 °C, Detection: Corona CAD (Charged Aerosol Detector)). The total lipid amount was calculated from the measured values of phospholipids, cholesterol, and LP and the composition ratios of the lipid components constituting the nucleic acid-lipid particles. The ratio of the total lipid amount to mRNA was calculated by the following formula. [Total lipid concentration] / [mRNA concentration] (wt / wt) (2-3) Average particle size The particle size of the nucleic acid-lipid particles was measured using a Zeta Potential / Particle Sizer NICOMPTM 380ZLS (PARTICLE SIZING SYSTEMS). The average particle size in the table represents the volume-average particle size, and ± below represents the deviation. The results are shown in Table 2.

[0100] [Example 22] Preparation of nucleic acid-lipid particles encapsulating OVA mRNA In the same manner as in Example 21, preparation and property evaluation of nucleic acid-lipid particles encapsulating mRNA having the translation region of OVA (Ovalbumin) described in SEQ ID NO: 7 were carried out. However, the molar ratio of the constituent lipid composition was DSPC:Chol:LP:PEG-C-DMAG = 10:35:50:5. The results are shown in Table 2.

[0101] [Example 23] Preparation of nucleic acid-lipid particles encapsulating OVA mRNA In the same manner as in Example 21, preparation and property evaluation of nucleic acid-lipid particles encapsulating mRNA having the translation region of OVA (Ovalbumin) described in SEQ ID NO: 7 were carried out. However, the molar ratio of the constituent lipid composition was DSPC:Chol:LP:PEG-C-DMA = 10:23.5:65:1.5. The results are shown in Table 2.

[0102] [Example 24] Preparation of nucleic acid-lipid particles encapsulating OVA mRNA In the same manner as in Example 21, preparation and property evaluation of nucleic acid-lipid particles encapsulating mRNA having the translation region of OVA (Ovalbumin) described in SEQ ID NO: 7 were carried out. However, the molar ratio of the constituent lipid composition was DSPC:Chol:LP:PEG-C-DMA = 10:48.5:40:1.5. The results are shown in Table 2.

[0103] [Example 25] Preparation of nucleic acid-lipid particles encapsulating OVA mRNA In the same manner as in Example 21, preparation and property evaluation of nucleic acid-lipid particles encapsulating mRNA having the translation region of OVA (Ovalbumin) described in SEQ ID NO: 7 were carried out. However, the molar ratio of the constituent lipid composition was DSPC:Chol:LP:PEG-C-DMA = 5:43.5:50:1.5. The results are shown in Table 2.

[0104] [Example 26] Preparation of nucleic acid-lipid particles encapsulating OVA mRNA In the same manner as in Example 21, preparation and property evaluation of nucleic acid-lipid particles encapsulating mRNA having the translation region of OVA (Ovalbumin) described in SEQ ID NO: 7 were carried out. However, the molar ratio of the constituent lipid composition was DSPC:Chol:LP:PEG-C-DMA = 15:33.5:50:1.5. The results are shown in Table 2.

[0105] [Example 27] Preparation of nucleic acid-lipid particles encapsulating OVA mRNA In the same manner as in Example 21, preparation and property evaluation of nucleic acid-lipid particles encapsulating mRNA having the translation region of OVA (Ovalbumin) described in SEQ ID NO: 7 were carried out. However, the molar ratio of the constituent lipid composition was Chol:LP:PEG-C-DMA = 53.5:45:1.5. The results are shown in Table 2.

[0106] [Example 28] Preparation of nucleic acid-lipid particles encapsulating HPV mRNA described in Example 4 Using the same method as in Example 8, the preparation and property evaluation of the mRNA-encapsulated nucleic acid lipid particles described in Example 4 were carried out. However, instead of DSPC, dioleoylphosphatidylcholine (1,2-Dioleoyl-sn-glycero-3-phosphocholine; hereinafter referred to as DOPC, NOF CORPORATION) was used, and the constituent lipid composition was set to a molar ratio of DOPC:Chol:LP:PEG-DMG = 10:43.5:45:1.5. The results are shown in Table 3.

[0107] [Example 29] Preparation of nucleic acid-lipid particles encapsulating HPV mRNA described in Example 4 Using the same method as in Example 8, the preparation and property evaluation of the mRNA-encapsulated nucleic acid lipid particles described in Example 4 were carried out. However, instead of DSPC, DOPC was used, and the constituent lipid composition was set to a molar ratio of DOPC:Chol:LP:PEG-DMG = 15:38.5:45:1.5. The results are shown in Table 3.

[0108] [Example 30] Preparation of nucleic acid-lipid particles encapsulating HPV mRNA described in Example 4 Using the same method as in Example 8, the preparation and property evaluation of the mRNA-encapsulated nucleic acid lipid particles described in Example 4 were carried out. However, instead of DSPC, dioleoylphosphatidylethanolamine (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine; hereinafter referred to as DOPE, NOF CORPORATION) was used, and the constituent lipid composition was set to a molar ratio of DOPE:Chol:LP:PEG-DMG = 10:43.5:45:1.5. The results are shown in Table 3.

[0109] [Example 31] Preparation of nucleic acid-lipid particles encapsulating HPV mRNA described in Example 4 Using the same method as in Example 8, the preparation and property evaluation of the mRNA-encapsulated nucleic acid lipid particles described in Example 4 were carried out. However, instead of DSPC, DOPE was used, and the constituent lipid composition was set to a molar ratio of DOPE:Chol:LP:PEG-DMG = 15:38.5:45:1.5. The results are shown in Table 3.

[0110] [Example 32] Preparation of nucleic acid-lipid particles encapsulating HPV mRNA described in Example 4 Using the same method as in Example 8, the preparation and property evaluation of the mRNA-encapsulating nucleic acid lipid particles described in Example 4 were carried out. However, the molar ratio of the constituent lipid composition was DSPC:Chol:LP:PEG-DMG = 12.5:41:45:1.5. The results are shown in Table 3.

[0111] [Example 33] Preparation of HPV18 E6-E7 fusion1 opt1 mRNA -001 (1) Preparation of template DNA for in vitro transcription (IVT) of HPV18 E6-E7 fusion1 opt1 A plasmid was constructed to prepare the template DNA used for in vitro transcription (IVT). A DNA fragment (SEQ ID NO: 10) containing the sequence of GCTAGC (NheI site), T7 promoter sequence, 5'-UTR sequence of human β-globin, Kozak sequence, IgE leader sequence-HPV type 18 E6-Furin Cleavage site-HPV type 18 E7 ORF, 3'-UTR sequence of human β-globin, polyA tail, and ACTAGT (SpeI site) linked in sequence was introduced into a plasmid (pMA-HPV18_fusion1_opt1). (2) Linearization of template DNA To the nuclease-free water (3080 μL, Thermo Fisher catalog # AM9937) in which the plasmid (350 μg) obtained in Example 33-(1) was dissolved, 10× CutSmart Buffer (350 μL, New England Biolabs catalog # B7204S) and SpeI-HF (70 μL, New England Biolabs catalog # R3133L) were added. After incubation at 37°C for 2 hours, it was incubated at 65°C for 20 minutes. 7.5 M ammonium acetate (1750 μL) and ethanol (10500 μL) were mixed and left standing at -80°C overnight. After centrifugation (-10°C, 4000×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (-10°C, 4000×g, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was prepared as a 500 μg / mL solution in TE-Buffer. (3) Preparation of HPV18 E6-E7 fusion1 opt1 mRNA -001 by in vitro transcription500 μg / mL template DNA (150 μL) obtained in Example 33-(2), 100 mM CleanCap AG (150 μL, TriLink catalog # T-7113), 100 mM ATP (150 μL, Hongene catalog # R1331), 100 mM GTP (150 μL, Hongene catalog # R2331), 100 mM CTP (150 μL, Hongene catalog # R3331), 100 mM N1-Me-Pseudo UTP (150 μL, Hongene catalog # R5-027), Nuclease-free water (1200 μL, Thermo Fisher catalog # AM9937), T7 Transcription 5× buffer (600 μL, Promega catalog # P140X), Enzyme mix, T7 RNA Polymerase (300 μL, Promega catalog # P137X) were mixed and incubated at 37°C for 4 hours. RQ1 RNase-Free DNase (75 μL, Promega catalog # M6101) was mixed and incubated at 37°C for 15 minutes. 8 M LiCl solution (1500 μL, Sigma-Aldrich catalog # L7026) was mixed and left standing at -20°C overnight. After centrifugation (4°C, 4000×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (4°C, 4000×g, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in Nuclease-free water and purified using the RNeasy Maxi kit (Qiagen catalog # 75162) according to the attached manual. A part of the obtained eluate (11.0 mL, 9813 μg in terms of UV) was mixed with Nuclease-free water (537 μL), buffer (1500 μL) and enzyme (1963 μL) of rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001), incubated at 37°C for 30 minutes, and then incubated at 75°C for 3 minutes.8 M LiCl solution (15000 μL) was mixed and left standing at -20 °C for 3 hours. After centrifugation (-8 °C, 4000 × g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (-8 °C, 4000 × g, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in Nuclease-free water and then purified by reverse-phase high-performance liquid chromatography (YMC Triart-C8 5 μm 10 × 150 mm (YMC # TO12S05-1510WT), 5% acetonitrile, 400 mM triethylamine acetate (pH 7.0) / 25% acetonitrile, 400 mM triethylamine acetate (pH 7.0), 75 °C) to obtain the target mRNA. The obtained mRNA has the sequence of SEQ ID NO: 11. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) to confirm that it was of the desired length.

[0112] [Example 34] Preparation of HPV18 E6-E7 fusion1 opt1 mRNA -002 Instead of 100 mM CTP in Example 33-(3), 100 mM 5-Me-CTP (Hongene catalog # R3-029) was used, and instead of 100 mM N1-Me-Pseudo UTP, 100 mM 5-methyluridine triphosphate was used, and mRNA was obtained in the same manner as in Example 33-(3). The obtained mRNA has the sequence of SEQ ID NO: 11. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) to confirm that it was of the desired length.

[0113] [Example 35] Preparation of HPV18 E6-E7 fusion1 opt2 mRNA -001 (1) Preparation of template DNA for IVT of HPV18 E6-E7 fusion1 opt2 Plasmids were constructed to prepare template DNA for IVT templates. A DNA fragment (SEQ ID NO: 12) containing a sequence in which GCTAGC (NheI site), T7 promoter sequence, 5'-UTR sequence of β-globin, Kozak sequence, IgE leader sequence-HPV type 18 E6-Furin Cleavage site-HPV type 18 E7 ORF, 3'-UTR sequence of β-globin, polyA tail, and ACTAGT (SpeI site) were ligated in sequence was introduced into a plasmid (pMA-HPV18_fusion1_opt2). (2) Linearization of template DNA To the Nuclease-free water (3520 μL, Thermo Fisher catalog # AM9937) in which the plasmid (400 μg) obtained in Example 35-(1) was dissolved, 10× CutSmart Buffer (400 μL, New England Biolabs catalog # B7204S) and SpeI-HF (80 μL, New England Biolabs catalog # R3133L) were added, incubated at 37°C for 2 hours, and then incubated at 65°C for 20 minutes. 7.5 M ammonium acetate (2000 μL) and ethanol (12000 μL) were mixed and left standing at -20°C overnight. After centrifugation (4°C, 4000×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, after centrifugation (4°C, 4000×g, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was prepared as a 500 μg / mL solution in TE-Buffer. (3) Preparation of HPV18 E6-E7 fusion1 opt2 mRNA -001 by in vitro transcription 500 μg / mL template DNA (150 μL) obtained in Example 35-(2), 100 mM ATP (225 μL, Hongene catalog # R1331), 100 mM GTP (225 μL, Hongene catalog # R2331), 100 mM CTP (225 μL, Hongene catalog # R3331), 100 mM N1-Me-Pseudo UTP (225 μL, Hongene catalog # R5-027), Nuclease-free water (1050 μL, Thermo Fisher catalog # AM9937), T7 Transcription 5× buffer (600 μL, Promega catalog # P140X), Enzyme mix, T7 RNA Polymerase (300 μL, Promega catalog # P137X) were mixed and incubated at 37°C for 4 hours. RQ1 RNase-Free DNase (75 μL, Promega catalog # M6101) was mixed and incubated at 37°C for 15 minutes. 8 M LiCl solution (1500 μL, Sigma-Aldrich catalog # L7026) was mixed and left standing at -20°C overnight. After centrifugation (4°C, 4000×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (4°C, 4000×g, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in Nuclease-free water. To a part of this solution (2470 μL, 7500 μg in terms of UV), Nuclease-free water (2780 μL) was added, heated at 70°C for 10 minutes, and then cooled under ice for 5 minutes.Add 750 μL of 10× capping buffer (500 mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM MgCl2, 50 mM DTT), 20 mM GTP (375 μL, diluted with nuclease-free water from 100 mM GTP, Hongene catalog # R2331), 20 mM SAM (375 μL, diluted with nuclease-free water from 32 mM SAM, New England Biolabs catalog # B9003S), and Vaccinia Capping Enzyme (750 μL, Hongene catalog # ON-028). After incubation at 37°C for 4 hours, add 125 μL of 10× capping buffer, 375 μL of 20 mM SAM, and 2’-O-Methyltransferase (750 μL, Hongene catalog # ON-014), and incubate at 37°C for 4 hours. Mix with 8750 μL of 8 M LiCl solution and let stand at -20°C overnight. After centrifugation (4°C, 4000×g, 30 minutes), discard the supernatant, add 70% ethanol, and after centrifugation (4°C, 4000×g, 10 minutes), discard the supernatant and air dry. Dissolve the obtained residue in nuclease-free water and purify by reverse-phase high-performance liquid chromatography (YMC Triart-C8 5μm 10×150mm (YMC # TO12S05-1510WT), 5% acetonitrile, 400 mM triethylamine acetate (pH7.0) / 25% acetonitrile, 400 mM triethylamine acetate (pH 7.0), 75°C) to obtain the target mRNA. The obtained mRNA has the sequence of SEQ ID NO: 13. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) to confirm that it was the desired length.

[0114] [Example 36] Preparation of HPV18 E6-E7 fusion1 opt2 mRNA -002 Instead of 100 mM CTP in Example 35-(3), 100 mM 5-Me-CTP (Hongene catalog # R3-029) was used, and instead of 100 mM N1-Me-Pseudo UTP, 100 mM 5-methyluridine triphosphate was used, and mRNA was obtained in the same manner as in Example 35-(3). The obtained mRNA has the sequence of SEQ ID NO: 13. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) to confirm that it was the desired length.

[0115] [Examples 37 - 40] Preparation of nucleic acid-lipid particles encapsulating HPV mRNA (1) Preparation of nucleic acid-lipid particles encapsulating mRNA Instead of the mRNA of Example 1, the mRNAs of Examples 33 to 36 were each used, and the preparation and property evaluation of mRNA-encapsulated nucleic acid lipid particles were carried out in the same manner as in Example 8 according to the lipid molar ratios described in Table 4. The results are shown in Table 4.

[0116] [Examples 41 - 52] Preparation of nucleic acid-lipid particles encapsulating HPV mRNA (1) Preparation of nucleic acid-lipid particles encapsulating mRNA The preparation of mRNA-encapsulated nucleic acid lipid particles described in Example 4 was carried out in the same manner as in Example 8. However, instead of (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentahectaconta-9,26-dien-7,29-diyl diacetate, acetate (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacosa-9-en-7-yl (the compound described in Example 28 of WO2015 / 005253) (hereinafter referred to as LP2) was used, and the constituent lipid composition was set to the molar ratio described in Table 5. Incidentally, LP2 was synthesized according to the method described in Example 28 of WO2015005253. (2) Characterization of nucleic acid-lipid particles encapsulating mRNA The property evaluation of mRNA-encapsulated nucleic acid lipid particles was carried out in the same manner as in Example 8. However, the amount of mRNA was analyzed as follows. The nucleic acid-lipid particle dispersion was diluted and dissolved in 90% methanol, and the amount of mRNA in the nucleic acid-lipid particles was measured with an ultraviolet-visible spectrophotometer (manufactured by PerkinElmer, LAMBDA TM 465). The mRNA concentration was calculated by the following formula. {[Absorbance at 260 nm] - [Absorbance at 350 nm]} x 40 x dilution factor (μg / mL) The results are shown in Table 5.

[0117] [Reference Example 1] DNA Vaccine Model With reference to J. Yan et al. / Vaccine 27 (2009) 431-440, a plasmid expressing the fusion protein of HPV16 E6 and E7 was constructed. The translation region was the sequence described in GenBank Accession Number: Fj229356.

[0118] (Table 1) From the results of JPEG2025098089000017.jpg84170 and above, it was revealed that in these nucleic acid-lipid particles, more than 90% of the mRNA was encapsulated within the lipid particles and they had an average particle diameter of about 100 nm to about 130 nm.

[0119] (Table 2) From the results of JPEG2025098089000018.jpg51170 and above, it was revealed that in these nucleic acid-lipid particles, more than 75% of the mRNA was encapsulated within the lipid particles and they had an average particle diameter of about 70 nm to about 170 nm.

[0120] (Table 3) From the results of JPEG2025098089000019.jpg41170 and above, it was revealed that in these nucleic acid-lipid particles, more than 95% of the mRNA was encapsulated within the lipid particles and they had an average particle diameter of about 80 nm to about 110 nm.

[0121] (Table 4) From the results above 36170, it was revealed that in these nucleic acid-lipid particles, 95% or more of the mRNA was encapsulated within the lipid particles, and they had an average particle diameter of about 90 nm to about 130 nm.

[0122] (Table 5) From the results above 79167, it was revealed that in these nucleic acid-lipid particles, 95% or more of the mRNA was encapsulated within the lipid particles, and they had an average particle diameter of about 70 nm to about 120 nm.

[0123] [Test Example 1] Expression level of HPV genotype 16E7 vaccine antigen from cultured cells transfected with mRNA-encapsulating nucleic acid lipid particles (Figure 1) Per well of a 96-well plate, 2×10 4 cells of the human fetal kidney cell line HEK293T were seeded and cultured overnight at 37 °C under conditions of 5% CO2 concentration. Then, the mRNA-encapsulated nucleic acid-lipid particles of Examples 14 to 17 were added to HEK293T cells so that the final mRNA concentration was 0.3 - 10 μg / mL, and the cells were cultured at 37 °C under conditions of 5% CO2 concentration for 48 hours. After culturing, the 96-well plate was left standing at 4 °C for 1 hour, and 300 μL of PBS (-) (PBST) containing 0.05% Tween 20 was added per well and washed three times. Then, for the E7-expressing protein of HPV genotype 16 immobilized on the well of the plate (16E7), an anti-16E7 antibody labeled with horse radish peroxidase (HRP) was added and reacted at room temperature for 2 hours. After washing the well three times with PBST, an HRP substrate was added for detection. The 16E7 protein expression level was calculated by subtracting the absorbance at 540 nm, which was the background, from the absorbance at 450 nm of each well.

[0124] [Test Example 2] Induction level of cytotoxic T lymphocytes (CTLs) specific for the HPV genotype 16E7 vaccine antigen (Figures 2, 3, 5, 9) C57BL / 6J mice were purchased from CLEA Japan. All animal experiments were performed in accordance with the institutional guidelines of the National Institute of Biomedical Innovation, Health and Nutrition. All procedures involving animals were conducted under anesthesia, either by inhalation of isoflurane or by subcutaneous administration of ketalar / seractal.

[0125] Seven-week-old C57BL / 6 mice were inoculated intramuscularly in the thigh muscle with 4 - 10 μg of mRNA-encapsulated nucleic acid lipid particles per mouse twice at 10-day intervals. In addition, plasmid DNA was administered intramuscularly in the thigh at 40 μg per mouse by electroporation. The administration conditions for electroporation were 30 V, 50 ms ON / 100 ms OFF, 3 cycles. Electroporation was also performed twice at 10-day intervals. One week after the final immunization, peripheral blood was collected in the presence of heparin, or the spleen was collected 1 or 2 weeks after the final immunization, and peripheral blood mononuclear cells (PBMCs) and spleen cells were prepared and used as evaluation samples. The induction level of cytotoxic T lymphocytes (CTLs) specific for the HPV genotype 16E7 vaccine antigen in PBMCs and spleen cells was measured by flow cytometry (FACS) after immunostaining with antibodies against T cell surface markers and tetramer complexes of MHC class I molecules and 16E7 epitopes.

[0126] [Test Example 3] Measurement of antibody titer against the HPV genotype 16E7 vaccine antigen (Figure 4) Seven-week-old C57BL / 6 mice were inoculated intramuscularly in the thigh muscle with 10 μg of mRNA-encapsulated nucleic acid lipid particles per mouse twice at 10-day intervals. One week after the final immunization, peripheral blood was collected and plasma was prepared in the presence of heparin for use as evaluation samples. Total IgG binding to the HPV genotype 16E7 vaccine antigen in the plasma was measured using the ELISA method. For the solid-phase treatment of the ELISA method, 16E7 recombinant protein was immobilized overnight at 4°C on a 96-well plate at a concentration of 0.5 μg / mL. At the same time, a dilution series of mouse IgG protein with known concentrations was also prepared and immobilized on the same plate for use in the standard curve. Subsequently, after washing three times with PBST, blocking treatment was performed with PBST containing 1% BSA (1% BSA / PBST) for 1 hour. Plasma samples were serially diluted with 1% BSA / PBST and added to the 16E7-immobilized wells, and reacted at room temperature for 2 hours. Wells for the mouse IgG standard curve were added with 1% BSA / PBST and reacted in the same manner. After washing three times with PBST, HRP-labeled anti-mouse IgG antibody was added to each well and reacted at room temperature for 1 hour. After washing three times with PBST, HRP substrate was added to develop color, and 1N sulfuric acid solution was added as a color stop solution. The concentration of mouse IgG bound to 16E7 was calculated using the standard curve after subtracting the absorbance at 540 nm, which was used as the background, from the absorbance at 450 nm of each well.

[0127] [Test Example 4] Transplantation experiment of TC-1 cancer cells using C57BL / 6 mice and cancer regression effect of mRNA-encapsulating nucleic acid lipid particles (Figures 4 - 6) The TC-1 cancer cell line expressing E6 and E7 of HPV genotype 16 was administered subcutaneously into the mouse flank of C57BL / 6 mice, and the tumor size was measured over time. The mouse flank, which was the cancer transplantation site, was shaved before transplantation of the TC-1 cancer cells, and 1 × 10 5 cells per mouse were transplanted. The mRNA-encapsulated nucleic acid lipid particles of Example 20 were administered intramuscularly at 10 μg of mRNA per mouse 8 days after transplantation of the TC-1 cells. Administration of antibodies for removing CD4-positive cells and CD8-positive cells was started 2 days before administration of the mRNA-encapsulated nucleic acid lipid particles.

[0128] [Test Example 5] Depletion of CD4-positive and CD8-positive cells by administration of anti-CD4 antibody and anti-CD8 antibody (Figures 4 - 6) Into the abdominal cavity of 7-week-old C57BL / 6 mice, 100 μg of anti-CD4 antibody (GK1.5) and anti-CD8 antibody (53-6.72) per mouse were administered continuously for 3 days starting 2 days before the administration of the mRNA-encapsulated nucleic acid lipid particles. On the 3rd day after the antibody administration, the mRNA-encapsulated nucleic acid lipid particles of Example 20 were administered at a dose of 10 μg of mRNA per mouse into the thigh muscle.

[0129] [Test Example 6] Measurement of antibody titer against the OVA vaccine antigen (Figure 7) 7-week-old C57BL / 6 mice were inoculated intradermally at the base of the tail with 15 μg of mRNA-encapsulated nucleic acid lipid particles per mouse in terms of mRNA amount twice at 2-week intervals. One week after the final immunization, peripheral blood was collected and serum was prepared as an evaluation sample. Total IgG binding to the OVA vaccine antigen in plasma was measured using the ELISA method. In the solid-phase treatment of the ELISA method, recombinant OVA protein was immobilized on a 96-well plate at a concentration of 1 μg / mL at 4°C overnight. At the same time, a dilution series of mouse IgG protein with known concentrations was also prepared and immobilized on the same plate for the standard curve. Then, after washing 3 times with PBST, blocking treatment was performed with PBST containing 1% BSA (1% BSA / PBST) for 1 hour. Serum samples were serially diluted with 1% BSA / PBST and added to the wells immobilized with OVA, and reacted at room temperature for 2 hours. The wells for the mouse IgG standard curve were added with 1% BSA / PBST and reacted in the same manner. After washing 3 times with PBST, HRP-labeled anti-mouse IgG antibody was added to each well and reacted at room temperature for 1 hour. After washing 3 times with PBST, HRP substrate was added to develop color, and 1N sulfuric acid solution was added as a color stop solution. The concentration of mouse IgG bound to 16E7 was calculated using the standard curve after subtracting the absorbance at 540 nm as the background from the absorbance at 450 nm of each well.

[0130] [Test Example 7] OVA vaccine antigen-specific T cell cytokine production capacity (Figure 8) At the base of the tail of 7-week-old C57BL / 6 mice, nucleic acid-lipid particles encapsulating 15 μg of mRNA per mouse were inoculated twice at 2-week intervals. One week after the final immunization, the spleen was harvested and spleen cells were prepared. The spleen cells seeded in a 96-well culture plate were stimulated with an MHC class I-restricted epitope peptide of the OVA antigen and the OVA vaccine antigen protein, respectively, and cultured for 24 hours. The level of OVA antigen-specific cytokine production was evaluated using the cytokine ELISA method with the culture supernatant as a sample. In this study, IFN-γ, a representative cytokine produced by CTL and Th1, was measured.

[0131] [Test Example 8] HPV genotype 18E6E7 vaccine antigen-specific T cell cytokine production capacity (Figure 10) C57BL / 6J mice were purchased from CLEA Japan. All animal procedures were performed under inhalation anesthesia with isoflurane. Into the gastrocnemius muscle of 6-week-old C57BL / 6 mice, nucleic acid-lipid particles encapsulating 5 μg of mRNA per mouse were administered twice at 2-week intervals. One week after the final administration, the spleen was collected and spleen cells were prepared. The spleen cells were treated with HPV18E6 pool peptides (manufactured by JPT, catalog # PM-HPV18-E6), and the amount of IFN-γ in the culture supernatant after 48 hours of culture was measured by the cytokine ELISA method.

[0132] [Test Example 9] Induction level of cytotoxic T lymphocytes (CTLs) specific for the HPV genotype 16E6E7 vaccine antigen (Figure 11) C57BL / 6J mice were purchased from CLEA Japan. All animal procedures were performed under inhalation anesthesia with isoflurane. Into the gastrocnemius muscle of 6-week-old C57BL / 6 mice, nucleic acid-lipid particles encapsulating 5 μg of mRNA per mouse were administered twice at 2-week intervals. One week after the final administration, the spleen was collected and spleen cells were prepared. The induction level of cytotoxic T lymphocytes (CTL) specific for E7 of HPV genotype 16 (HPV16E7) in spleen cells was measured by flow cytometry after immunostaining with an antibody against the T cell surface marker and a complex of the MHC class I molecule-restricted HPV16E7 epitope and the MHC class I molecule.

[0133] [Test Example 10] HPV genotype 16E6E7 vaccine antigen-specific T cell cytokine production capacity (Figure 12) In the gastrocnemius muscle of 6-week-old C57BL / 6 mice, mRNA-encapsulated nucleic acid lipid particles at 5 μg per mouse in terms of mRNA were administered twice at 2-week intervals. One week after the final administration, the spleen was collected and spleen cells were prepared. Spleen cells were seeded in a 96-well culture plate and cultured for 24 hours under the treatment of an MHC class I-restricted epitope peptide of HPV16E7, and then the amount of IFN-γ in the culture supernatant was measured by cytokine ELISA.

[0134] [Results of Test Examples 1 to 10] E7 expression level of HPV genotype 16 by the mRNA-encapsulating nucleic acid lipid particles of Examples 14 - 17 (Figure 1) Using HEK293T cells, the antigen expression level from mRNA-encapsulated nucleic acid lipid particles in cultured cells was examined. The results are shown in Figure 1. In Examples 15 and 16, a tendency of higher E7 (16E7) protein expression level of HPV serotype 16 was observed compared with Examples 14 and 17. On the other hand, when compared with the wells untreated with mRNA-encapsulated nucleic acid lipid particles, since all of them induced 16E7 protein expression, the protein expression-inducing ability of mRNA-encapsulated nucleic acid lipid particles in cultured cells became clear.

[0135] Comparison of CTL induction levels between the DNA vaccine model and mRNA-encapsulating nucleic acid lipid particles (Figure 2) The DNA vaccine model of Reference Example 1 was constructed, and a comparative study was conducted on the CTL-inducing ability of the mRNA-encapsulated nucleic acid lipid particles of Examples 9, 11, and 13 in a mouse model. The results are shown in Figure 2. As a result of evaluating the level of induction of 16E7-specific CTL in peripheral blood 1 week after the final immunization, all three types of mRNA-encapsulated nucleic acid lipid particles showed a higher CTL-inducing ability compared with the DNA vaccine model (pDNA) (left figure). Also, as a result of evaluating the level of induction of 16E7-specific CTL in spleen cells 2 weeks after the final immunization, the mRNA-encapsulated nucleic acid lipid particles of Examples 9, 11, and 13 showed a CTL-inducing level equal to or higher than that of pDNA (right figure).

[0136] CTL induction ability in mice of Examples 14 to 17 of mRNA-encapsulating nucleic acid lipid particles (Figure 3) The induction levels of 16E7-specific CTLs by four types of mRNA-encapsulated nucleic acid lipid particles in C57BL / 6 mice were examined. The results are shown in Figure 3. The induction levels of 16E7-specific CTLs were observed in all four types of mRNA-encapsulated nucleic acid lipid particles, although there was variation among individuals.

[0137] Importance of CD4-positive and CD8-positive cells in antibody induction ability of mRNA-encapsulating nucleic acid lipid particles (Figure 4) After transplantation of TC-1 cancer cells, the antibody induction levels of mRNA-encapsulated nucleic acid lipid particles in mice from which CD4-positive cells and CD8-positive cells had been removed were examined. The results are shown in Figure 4. When comparing the control group (control group (No-depletion)) to which no cell-depleting antibody had been administered with the group administered the mRNA-encapsulated nucleic acid lipid particles of Example 20 (Example 20 (No-depletion)), 16E7-specific antibody levels were observed in the Example 20 (No-depletion) group. Also, the group administered the mRNA-encapsulated nucleic acid lipid particles from which CD8-positive cells had been removed (Example 20 (CD8-depletion)) also showed antibody induction levels equivalent to those of the Example 20 (No-depletion) group. However, in the group administered the mRNA-encapsulated nucleic acid lipid particles from which CD4-positive cells had been removed (Example 20 (CD4-depletion)), it became clear that the 16E7 antibody induction level had decreased compared to the Example 20 (No-depletion) group and the Example 20 (CD8-depletion) group. From the above results, it was suggested that CD4-positive cells might be important for the induction of 16E7 antigen-specific antibodies by administration of mRNA-encapsulated nucleic acid lipid particles.

[0138] Importance of CD4-positive and CD8-positive cells in CTL induction ability of mRNA-encapsulating nucleic acid lipid particles (Figure 5) After transplanting TC-1 cancer cells, the level of induction of 16E7-specific CTLs by mRNA-encapsulated nucleic acid lipid particles in mice from which CD4-positive cells and CD8-positive cells had been removed was examined. The results are shown in Fig. 5. When comparing the control group not administered with the cell-removing antibody (control group (No-depletion)) with the group administered with the mRNA-encapsulated nucleic acid lipid particles of Example 20 (Example 20 (No-depletion)), 16E7-specific CTL levels were observed in the Example 20 (No-depletion) group. On the other hand, the mRNA-encapsulated nucleic acid lipid particle-administered group from which CD4-positive cells had been removed (Example 20 (CD4-depletion)) showed a lower CTL induction level compared with the Example 20 (No-depletion) group. Furthermore, in the mRNA-encapsulated nucleic acid lipid particle-administered group from which CD8-positive cells had been removed (Example 20 (CD8-depletion)), it became clear that the 16E7-specific CTL induction level had decreased dramatically compared with the Example 20 (No-depletion) group. From the above results, it was suggested that CD8-positive cells might be essential for the induction of 16E7 antigen-specific CTLs by administration of mRNA-encapsulated nucleic acid lipid particles.

[0139] Importance of CD4-positive and CD8-positive cells in cancer growth inhibitory effect of mRNA-encapsulating nucleic acid lipid particles (Figure 6) After transplanting TC-1 cancer cells, the cancer regression effect of mRNA-encapsulated nucleic acid lipid particles in mice from which CD4-positive cells and CD8-positive cells had been removed was examined. The results are shown in Fig. 6. When comparing the control group without administration of cell depletion antibody (control group (No-depletion)) and the group administered with the mRNA-encapsulated nucleic acid lipid particles of Example 20 (Example 20 (No-depletion)), a growth inhibitory effect on TC-1 cancer tumors was observed in the Example 20 (No-depletion) group. Also, the group administered with mRNA-encapsulated nucleic acid lipid particles from which CD4-positive cells had been removed (Example 20 (CD4-depletion)) showed a TC-1 cancer growth inhibitory effect, similar to the Example 20 (No-depletion) group. However, in the group administered with mRNA-encapsulated nucleic acid lipid particles from which CD8-positive cells had been removed (Example 20 (CD8-depletion)), TC-1 cancer growth was not suppressed compared to the Example 20 (No-depletion) group, and the tumor size was equivalent to that of the control group (No-depletion). From the above results, it was suggested that CD8-positive cells might be essential for the cancer growth inhibitory effect by administration of mRNA-encapsulated nucleic acid lipid particles.

[0140] OVA-specific antibody induction levels of mRNA-encapsulating nucleic acid lipid particles of Examples 21 to 27 with different lipid compositions (Figure 7) C57BL / 6 mice were administered with the mRNA-encapsulated nucleic acid lipid particles of Examples 21 to 27 intramuscularly, and the level of induction of OVA-specific antibodies in the blood one week after the final immunization was examined. The results are shown in Fig. 7. The level of induction of OVA-specific antibodies was low with the mRNA-encapsulated nucleic acid lipid particles of Example 22, and equivalent in the other groups administered with mRNA-encapsulated nucleic acid lipid particles.

[0141] OVA-specific IFN-γ induction levels of mRNA-encapsulating nucleic acid lipid particles of Examples 21 to 27 with different lipid compositions (Figure 8) C57BL / 6 mice were administered with the mRNA-encapsulated nucleic acid lipid particles of Examples 21 to 27 intramuscularly, and one week after the final immunization, the level of induction of OVA-specific T cell cytokines from spleen cells was examined. The results are shown in Fig. 8. In the group immunized with the mRNA-encapsulated nucleic acid lipid particles of Example 26, the level of induction of IFN-γ in response to stimulation with the MHC class I-restricted epitope peptide of OVA and OVA protein was the lowest, and the OVA antigen-specific induction levels of Examples 21, 22, 25, and 27 were equivalent.

[0142] CTL induction levels in mice of mRNA-encapsulating nucleic acid lipid particles of Examples 28 to 32 with different phospholipid species and their contents (Figure 9) The induction levels of 16E7-specific CTLs of four types of mRNA-encapsulated nucleic acid lipid particles in C57BL / 6 mice were examined. The results are shown in Fig. 9. The induction levels of 16E7-specific CTLs were equivalent to those of Example 28 and Example 29 in which the phospholipid species was DOPC, Example 30 and Example 31 in which it was DOPE, and Example 32 in which it was DSPC.

[0143] HPV18E6-specific cytokine production induction levels of mRNA-encapsulating nucleic acid lipid particles of Examples 37 - 40 with different mRNA modifications (Figure 10) The mRNA-encapsulated nucleic acid lipid particles of Examples 37 to 40 were administered intramuscularly to C57BL / 6 mice, and one week after the final immunization, the amount of HPV18E6-specific T cell cytokines from spleen cells was examined. The results are shown in Fig. 10. In each group from Examples 37 to 40, induction of IFN-γ production in response to HPV18E6 pool peptide treatment was observed as compared with the NC group (Fig. 10).

[0144] CTL induction ability of mRNA-encapsulating nucleic acid lipid particles of Examples 41 - 52 with different lipid composition ratios (Figure 11) The mRNA-encapsulated nucleic acid lipid particles of Examples 41 to 52 were administered intramuscularly to C57BL / 6 mice, and one week after the final immunization, the induction level of 16E7-specific CTLs in spleen cells was evaluated. The results are shown in Fig. 11. The induction level of 16E7-specific CTLs was higher in all the mRNA-encapsulated nucleic acid lipid particles evaluated as compared with the NC group.

[0145] mRNA-encapsulating nucleic acid lipid particles with different lipid composition ratios, HPV16 E7-specific IFN-γ induction levels of Examples 41 to 52 (Figure 12) The mRNA-encapsulated nucleic acid lipid particles of Examples 41 to 52 were administered intramuscularly to C57BL / 6 mice, and one week after the final immunization, the induction level of HPV16E7-specific T cell cytokines from spleen cells was examined. The results are shown in Fig. 12. IFN-γ production was enhanced by treatment with the MHC class I-restricted epitope peptide of HPV16E7 in all the mRNA-encapsulated nucleic acid lipid particle administration groups as compared with the NC group. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Industrial Applicability

[0146] The present invention can be used for the prevention and / or treatment of infection by human papillomavirus.

Sequence Listing Free-Text

[0147] Template DNA for in vitro transcription (IVT) of <SEQ ID NO: 1> HPV16 E6-E7 fusion2. GCTAGC (NheI site): Base numbers 1-6 T7 promoter sequence: Base numbers 8-27 5'-UTR sequence of human β-globin: Base numbers 39-88 KOZAK sequence: Base numbers 89-94 Translation region of IgE leader sequence: Base numbers 95-148 Translation region of HPV16 type E6: Base numbers 149-598 Furin cleavage site: Base numbers 599-619 Translation region of HPV16 type E7: Base numbers 620-913 3'-UTR sequence of human β-globin: Base numbers 914-1045 poylA: Base numbers 1046-1147 ACTAGT (SpeI site): Base numbers 1152-1157 <Array number 2> HPV16 E6-E7 fusion2 mRNA -001. Template DNA for in vitro transcription (IVT) of HPV16 E6-E7 fusion10 with Array number 3. GCTAGC (NheI site): Base numbers 1 - 6 T7 promoter sequence: Base numbers 8 - 27 5'-UTR sequence of human β-globin: Base numbers 39 - 88 KOZAK sequence: Base numbers 89 - 94 Translation region of IgE leader sequence: Base numbers 95 - 148 Translation region of HPV16 type E6: Base numbers 149 - 598 Furin cleavage site: Base numbers 599 - 619 Translation region of HPV16 type E7: Base numbers 620 - 913 3’-UTR sequence of human β-globin: Base numbers 914 - 1045 poylA: Base numbers 1046 - 1147 ACTAGT (SpeI site): Base numbers 1152 - 1157 <Array number 4> HPV16 E6-E7 fusion10 mRNA -001 to 006. Template DNA of HPV16 E6-E7 fusion10 opt2 with Array Number 5. GCTAGC (NheI site): Base numbers 1 - 6 T7 promoter sequence: Base numbers 8 - 27 5'-UTR sequence of human β-globin: Base numbers 39 - 88 KOZAK sequence: Base numbers 89 - 94 Translation region of IgE leader sequence: Base numbers 95 - 148 Translation region of HPV16 E6: Base numbers 149 - 598 Furin cleavage site: Base numbers 599 - 619 Translation region of HPV16 E7: Base numbers 620 - 913 3’-UTR sequence of human β-globin: Base numbers 914 - 1045 poylA: Base numbers 1046 - 1147 ACTAGT (SpeI site): Base numbers 1152 - 1157 <Array No. 6> HPV16 E6-E7 fusion10 opt2 mRNA -001. Translation region of <Sequence number 7> Sequence number 7 OVA (Ovalbumin) Amino acid sequence of E6 antigen of HPV type 16, SEQ ID NO: 8. FQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKGLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRGMSCCRSSRTRRETQL Amino acid sequence of E7 antigen of HPV type 16, SEQ ID NO: 9. HGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVGPICSQKP <SEQ ID NO: 10> pMA-HPV18_fusion1_opt1 GCTAGCGTAATACGACTCACTATA A NheI sequence: base numbers 1 - 6 T7 promoter: base numbers 7 - 24 A : transcription start point: base number 25 5‘-UTR: base numbers 39 - 88 Kozak sequence: base numbers 89 - 94 IgE leader sequence: base numbers 95 - 148 HPV18 E6 sequence: base numbers 149 - 613 Furin recognition sequence: base numbers 614 - 634 HPV18 E7 sequence: base numbers 635 - 949 3‘-UTR: base numbers 950 - 1081 polyA sequence: base numbers 1082 - 1183 SpeI sequence: base numbers 1188 - 1193 <SEQ ID NO:11> HPV18 E6 - E7 fusion1 opt1 mRNA - 001 and 002 <Array number 12> pMA-HPV18_fusion1_opt2 GCTAGCGTAATACGACTCACTATA G NheI sequence: base numbers 1 - 6 T7 promoter: base numbers 7 - 24 G : transcription start point: base number 25 5‘-UTR: base numbers 39 - 88 Kozak sequence: base numbers 89 - 94 IgE leader sequence: base numbers 95 - 148 HPV18 E6 sequence: base numbers 149 - 613 Furin recognition sequence: base numbers 614 - 634 HPV18 E7 sequence: base numbers 635 - 949 3‘-UTR: base numbers 950 - 1081 polyA sequence: base numbers 1082 - 1183 SpeI sequence: base numbers 1188 - 1193 <SEQ ID NO:13> HPV18 E6 - E7 fusion1 opt2 mRNA - 001 and 002 <SEQ ID NO:14> Amino acid sequence of E6 antigen of HPV type 18. FEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKGIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQGHSCCNRARQERLQRRRETQV <SEQ ID NO:15> Amino acid sequence of E7 antigen of HPV type 18. HGPKATLQDIVLHLEPQNEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVGPWCASQQ <SEQ ID NO:16> Amino acid sequence of protease cleavage sequence RGRKRRS <SEQ ID NO:17> Amino acid sequence of E6 and E7 antigen fusion protein of HPV type 16 MDWTWILFLVAAATRVHSFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKGLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRGMSCCRSSRTRRETQLRGRKRRSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVGPICSQKP <SEQ ID NO:18> Amino acid sequence of E6 and E7 antigen fusion protein of HPV type 18 MDWTWILFLVAAATRVHSFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKGIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQGHSCCNRARQERLQRRRETQVRGRKRRSHGPKATLQDIVLHLEPQNEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVGPWCASQQ

Claims

1. A lipid particle encapsulating a nucleic acid capable of expressing the E6 and E7 antigens of human papillomavirus, wherein the lipid comprises a cationic lipid represented by general formula (Ia) or a pharma- ceutically acceptable salt thereof. 【Chemistry 1】 During the ceremony, R １ and R ２ is independently １ -C ３ represents an alkyl group; L １ is C ２ -C ４ C which may have one or more alkanoyloxy groups １７ -C １９ represents an alkenyl group; L ２ is C ２ -C ４ C which may have one or more alkanoyloxy groups １０ -C １９ Alkyl group, or C ２ -C ４ C which may have one or more alkanoyloxy groups １０ -C １９ represents an alkenyl group; p is 3 or 4.

2. R in general formula (Ia) １ and R ２ The particle according to claim 1 , wherein both are methyl groups.

3. 3. The particle according to claim 1 or 2, wherein p in general formula (Ia) is 3.

4. L in formula (Ia) １ C which may have one or more acetoxy groups １７ -C １９ The particle according to any one of claims 1 to 3, which is an alkenyl group.

5. L in formula (Ia) ２ C which may have one or more acetoxy groups １０ -C １２ C which may have one or more alkyl groups or acetoxy groups １０ -C １９ The particle according to any one of claims 1 to 4, which is an alkenyl group.

6. L in formula (Ia) ２ C which may have one or more acetoxy groups １０ -C １２ C which may have one or more alkyl groups or acetoxy groups １７ -C １９ The particle according to any one of claims 1 to 4, which is an alkenyl group.

7. L in formula (Ia) １ The particle according to any one of claims 1 to 6, wherein is an (R)-11-acetyloxy-cis-8-heptadecenyl group, a cis-8-heptadecenyl group, or a (8Z,11Z)-heptadecadienyl group.

8. L in formula (Ia) ２ The particle according to any one of claims 1 to 7, wherein is a decyl group, a cis-7-decenyl group, a dodecyl group, or an (R)-11-acetyloxy-cis-8-heptadecenyl group.

9. The cationic lipid has the following structural formula: 【Chemistry 2】 The particle according to claim 1 , wherein the particle is represented by the formula:

10. The cationic lipid has the following structural formula: 【Chemistry 3】 The particle according to claim 1 , wherein the particle is represented by the formula:

11. The cationic lipid has the following structural formula: 【Chemistry 4】 The particle according to claim 1 , wherein the particle is represented by the formula:

12. 11. The particle of claim 9 or 10, wherein the lipid further comprises an amphipathic lipid, a sterol, and a PEG lipid.

13. 12. The particle of claim 11, wherein the lipid further comprises an amphipathic lipid, a sterol, and a PEG lipid.

14. 13. The particle according to claim 12, wherein the amphiphilic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine.

15. 14. The particle according to claim 13, wherein the amphiphilic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine.

16. 15. The particle according to claim 12 or 14, wherein the sterol is cholesterol.

17. 16. The particle according to claim 13 or 15, wherein the sterol is cholesterol.

18. 17. The particle according to any one of claims 12, 14 and 16, wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine.

19. 18. The particle according to any one of claims 13, 15 and 17, wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine.

20. The particle according to any one of claims 12 to 19, wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 22.5% or less of amphipathic lipid, 15 to 55% of sterols, 40 to 65% of cationic lipid, and 1 to 5% of PEG lipid, and the ratio of the total lipid weight to the nucleic acid weight is 15 to 30.

21. 21. The particle according to claim 20, wherein the amphiphilic lipid is 5 to 22.5%.

22. 22. The particle according to claim 21, wherein the amphiphilic lipid is 10 to 22.5%.

23. A particle according to any one of claims 12, 14, 16 and 18, wherein the lipid composition of amphipathic lipid, sterols, cationic lipid and PEG lipid is, in molar amounts, 5 to 15% amphipathic lipid, 35 to 50% sterols, 40 to 55% cationic lipid and 1 to 3% PEG lipid, and the ratio of the total lipid weight to the nucleic acid weight is 15 to 30.

24. 24. The particle according to claim 23, comprising 10 to 15% amphiphilic lipid, 35 to 45% sterols, 40 to 50% cationic lipid, and 1 to 2% PEG lipid.

25. The particle according to any one of claims 13, 15, 17 and 19, wherein the lipid composition of amphipathic lipid, sterols, cationic lipid and PEG lipid is, in molar amounts, 15 to 22.5% amphipathic lipid, 15 to 40% sterols, 40 to 60% cationic lipid and 1 to 3% PEG lipid, and the ratio of the total lipid weight to the nucleic acid weight is 15 to 30.

26. 26. The particle of claim 25, comprising 45-60% cationic lipid and 1-2% PEG lipid.

27. 27. The particle according to any one of claims 20 to 26, wherein the ratio of total lipid weight to nucleic acid weight is 15 to 25.

28. 28. The particle according to claim 27, wherein the ratio of total lipid weight to nucleic acid weight is 15 to 22.

5.

29. The particle according to any one of claims 1 to 28, wherein the human papillomavirus is HPV type 16.

30. The particle according to claim 29, wherein the human papillomavirus is HPV type 16 and the E6 antigen of HPV type 16 consists of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

8.

31. The particle according to claim 29 or 30, wherein the human papillomavirus is HPV type 16, and the E7 antigen of HPV type 16 consists of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

9.

32. The particle according to any one of claims 29 to 31, wherein the human papillomavirus is HPV16 type, and the nucleic acid capable of expressing the E6 antigen and E7 antigen of the human papillomavirus encodes a fusion protein of the E6 antigen and E7 antigen of HPV16 type, the fusion protein consisting of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

17.

33. The particle according to any one of claims 29 to 32, wherein the human papillomavirus is HPV16 type, and the nucleic acid capable of expressing HPV16 type E6 antigen and E7 antigen is an mRNA comprising a cap structure (Cap), a 5' untranslated region (5'-UTR), a leader sequence, an E6 translation region, a protease cleavage sequence (Furin Cleavage site), an E7 translation region, a 3' untranslated region (3'-UTR) and a poly A tail (poly A).

34. 34. The particle according to claim 33, wherein the nucleic acid sequence capable of expressing the E6 and E7 antigens of HPV type 16 consists of a nucleotide sequence having at least 90% identity with any of the sequences of SEQ ID NO: 2, 4 or 6.

35. The particle according to any one of claims 1 to 28, wherein the human papillomavirus is HPV type 18.

36. The particle described in claim 35, wherein the human papillomavirus is HPV type 18 and the E6 antigen of HPV type 18 consists of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

14.

37. The particle according to claim 35 or 36, wherein the human papillomavirus is HPV type 18, and the E7 antigen of HPV type 18 consists of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

15.

38. The particle according to any one of claims 35 to 37, wherein the human papillomavirus is HPV18 type, and the nucleic acid capable of expressing the E6 antigen and E7 antigen of the human papillomavirus encodes a fusion protein of the E6 antigen and E7 antigen of HPV18 type, the fusion protein consisting of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

18.

39. The particle according to any one of claims 35 to 38, wherein the human papillomavirus is HPV18 type, and the nucleic acid capable of expressing HPV18 type E6 antigen and E7 antigen is an mRNA comprising a cap structure (Cap), a 5' untranslated region (5'-UTR), a leader sequence, an E6 translation region, a protease cleavage sequence (Furin Cleavage site), an E7 translation region, a 3' untranslated region (3'-UTR) and a poly A tail (poly A).

40. 40. The particle according to claim 39, wherein the nucleic acid sequence capable of expressing the E6 and E7 antigens of HPV type 18 consists of a nucleotide sequence having at least 90% identity with any of the sequences of SEQ ID NO: 11 or 13.

41. The particle according to any one of claims 1 to 40, wherein the nucleic acid comprises at least one modified nucleotide.

42. 42. The particle of claim 41, wherein the modified nucleotide comprises at least one pyrimidine nucleotide substituted at the 5-position and / or a pseudouridine nucleotide optionally substituted at the 1-position.

43. 42. The particle according to claim 41, wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine.

44. 42. The particle according to claim 41, wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methyluridine, and 1-methylpseudouridine.

45. The particles according to any one of claims 1 to 44, having an average particle size of 30 nm to 300 nm.

46. Use of particles according to any of claims 1 to 45 for the manufacture of a composition for preventing and / or treating infections with human papillomavirus.

47. 47. Use of the particles according to claim 46, wherein the infection is an infection with human papillomavirus of type HPV16 or type HPV18.

48. A composition comprising the particles according to any one of claims 1 to 45.

49. 49. The composition according to claim 48, for expressing human papillomavirus E6 and E7 antigens in vivo or in vitro.

50. 50. The composition according to claim 48 or 49, for use as a medicine.

51. The composition of claim 50 for inducing an immune response against human papillomavirus.

52. 52. The composition according to claim 50 or 51 for preventing and / or treating human papillomavirus infection.

53. A method for expressing human papillomavirus E6 and E7 antigens in vitro, comprising introducing the composition of claim 48 or 49 into a cell.

Images