HPV infectious disease vaccine

Abstract

Provided are lipid particles in which is encapsulated a nucleic acid, whereby: a vaccine for preventing and / or treating an infection by human papillomavirus type-6 and / or type-11 can be provided; and an E6 antibody and an E7 antibody to the human papillomavirus can be expressed. The lipid particles contain: a lipid that is a cationic lipid represented by general formula (Ia); or a pharmaceutically acceptable salt thereof. [In the formula, R1, R2, p, L1 and L2 are defined as described in the specification.]

Description

HPV infection vaccine

[0001] The present invention relates to a nucleic acid-lipid particle vaccine encapsulating mRNA encoding antigens of HPV (human papillomavirus) types 6 and / or 11.

[0002] Human papillomavirus (HPV) is a non-enveloped virus with a circular double-stranded DNA genome, and there are approximately 200 genotypes (Non-Patent Document 1). Among these, some genotypes cause infected cells to become cancerous. Genotypes 16 and 18, which have been shown to be correlated with the development of cancer, particularly cervical cancer, are classified as high-risk types (Non-Patent Document 2). On the other hand, the majority of genotypes, such as genotypes 6 and 11, cause epithelial hyperplasia but are benign and rarely cause malignant tumors, and are therefore classified as low-risk types (Non-Patent Document 3).

[0003] The HPV genome encodes eight viral proteins, which are classified into early genes (E1, E2, E4, E5, E6, E7) and late genes (L1 and L2) according to the timing of their expression in the viral life cycle. Early genes control viral replication and oncogenic transformation of infected cells, while L1 and L2 are structural proteins that form the outer shell (capsid) of the virus particle (Non-Patent Document 4).

[0004] The high-risk HPV viral proteins E6 and E7 cause abnormalities in cell proliferation in infected cells. This is because E6 and E7 inhibit the function of p53, which is responsible for inducing apoptotic cell death, and pRb, a member of the Rb family of proteins that regulates the cell cycle (Non-Patent Documents 5 and 6). It has also been suggested that E6 inhibits p53 and E7 inhibits p130, a member of the Rb family of proteins that regulates the cell cycle primarily on the epithelial side, in the case of low-risk HPV (Non-Patent Document 3). Furthermore, regions important for the oncogenic activity of E6 and E7 have been identified, and when using E6 and E7 as vaccine antigens, it is possible to enhance safety by inserting inactivating mutations (Non-Patent Documents 7-9).

[0005] HPV genotypes 6 and 11 infect basal cells through wounds in the mucosal epithelium of the respiratory tract and genital tract, but are usually eliminated by the host immune system. However, in immunocompromised individuals, the viral proteins E6 and E7 induce proliferation of mucosal epithelial cells, resulting in the formation of papillomas. This can lead to recurrent respiratory papillomatosis and genital condyloma. In rare cases, these viruses can transform into malignant cells (Non-Patent Document 3).

[0006] HPV infection is initiated by the adsorption of the L1 protein, which constitutes the capsid, to heparan sulfate proteoglycans present on the surface of the host cell membrane (Non-Patent Document 10). Because neutralizing antibodies that protect against HPV infection target the L1 protein, currently available preventive vaccines contain virus-like particle (VLP) antigens of the L1 protein as their active ingredient. Existing preventive vaccines also contain L1 VLP antigens of genotypes 6 and 11, which reduces the frequency of surgical procedures in the treatment of recurrent respiratory papillomatosis (Non-Patent Document 11).

[0007] Host defense immunity against HPV infection involves the induction of neutralizing antibodies, cytotoxic T cells (CTLs), and helper T cells. The nonstructural proteins E6 and E7 are particularly targeted antigens for CTL induction and have been attracting attention as therapeutic vaccine antigens for cervical cancer and cervical dysplasia caused by HPV infection (Non-Patent Document 12). Similarly, they have also been attracting attention as therapeutic vaccine antigens for the treatment of recurrent respiratory papillomatosis (Non-Patent Document 13).

[0008] Patent Document 1 describes the gene sequences of E6 and E7 fusion antigens for 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 E6 and E7 translational region sequences. Furthermore, mutations are introduced into the p53-binding region of E6 and the pRb-binding region of E7 to inactivate the oncogenic activity of E6 and E7. This gene sequence was introduced into a mammalian expression plasmid and used as a DNA gene vaccine against HPV, and its efficacy was evaluated in a mouse model. Immunization was performed by intramuscular administration into the thigh of mice using electroporation.

[0009] Virology 2013;445:2e10.J Natl Cancer Inst 1995;87:796-802.Virus Res. 2017, 231: 119-127.J Clin Virol 2005;32(Suppl.1):S7e15.Cell 1990;63:1129e36.Cancer Res 1996;56:4620e4.J Virol, 1989, p.2650-2656J Virol, 1992, p.1329-1335J Virol, 1994, p.5698-5705Proc Natl Acad Sci US A 2009; 106: 20458e63.J Infect Dis. 2019, 219(7): 1016-1025. Nat Rev Cancer. 2006, 6(10): 753-763. Hum Vaccin Immunother. 2012, 8(4): 470-478.

[0010] JP 2016-512553

[0011] An object of the present invention is to provide a vaccine for preventing and / or treating infection with human papillomavirus types 6 and / or 11.

[0012] The present inventors discovered that when mice were administered lipid particles encapsulating mRNA encoding the E6-E7 antigens of human papillomavirus types 6 and / or 11, an immune response specific to the antigens was observed, leading to the completion of the present invention.

[0013] The gist of the present invention is as follows: (1) Lipid particles encapsulating nucleic acids capable of expressing 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.

[0014]

[0015] In the formula, R 1 and R 2 each independently represents a C1-C3 alkyl group; L 1 represents a C17-C19 alkenyl group which may have one or more C2-C4 alkanoyloxy groups; L 2 represents a C10-C19 alkyl group which may have one or more C2-C4 alkanoyloxy groups, or a C10-C19 alkenyl group which may have one or more C2-C4 alkanoyloxy groups; and p is 3 or 4. (2) R in general formula (Ia) 1 and R 2 (3) The particle according to (1) or (2), wherein p in general formula (Ia) is 3. (4) The particle according to (1) or (2), wherein L in general formula (Ia) is 1. 1 (5) The particle according to any one of (1) to (3), wherein L in general formula (Ia) is a C17-C19 alkenyl group optionally having one or more acetoxy groups. 2 (6) The particle according to any one of (1) to (4), wherein L in general formula (Ia) is a C10-C12 alkyl group optionally having one or more acetoxy groups, or a C10-C19 alkenyl group optionally having one or more acetoxy groups. 2 ​(7) The particle according to any one of (1) to (4), wherein L in general formula (Ia) is a C10-C12 alkyl group optionally having one or more acetoxy groups, or a C17-C19 alkenyl group optionally having one or more acetoxy groups. 1 (8) The particles according to any one of (1) to (6), wherein L in general formula (Ia) is an (R)-11-acetyloxy-cis-8-heptadecenyl group, a cis-8-heptadecenyl group, or an (8Z,11Z)-heptadecadienyl group. 2 (9) The particles according to any one of (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.

[0016] (10) The particle according to (1), wherein the cationic lipid is represented by the following structural formula:

[0017] (11) The particle according to (1), wherein the cationic lipid is represented by the following structural formula:

[0018]

[0019] ​(1) The particle according to (1), wherein the lipid further comprises an amphiphilic lipid, a sterol, and a PEG-lipid. (12) The particle according to (9) or (10), wherein the lipid further comprises an amphiphilic lipid, a sterol, and a PEG-lipid. (13) The particle according to (11), wherein the lipid further comprises an amphiphilic lipid, a sterol, and a PEG-lipid. (14) The particle according to (12), wherein the amphiphilic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine. (15) The particle according to (13), wherein the amphiphilic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine. (16) The particle according to (12) or (14), wherein the sterol is cholesterol. (17) The particle according to (13) or (15), wherein the sterol is cholesterol. (18) The particle according to any one of (12), (14) and (16), wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxypoly(ethylene glycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine. (19) The particle according to any one of (13), (15) and (17), wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxypoly(ethylene glycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine.

[0020] (20) The particle according to any one of (12) to (19), wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 5 to 25% amphipathic lipid, 10 to 55% sterols, 40 to 65% cationic lipid, and 1 to 5% PEG lipid. (21) The particle according to (20), wherein the amphipathic lipid is 10 to 25%. (22) The particle according to any one of (12), (14), (16), or (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. (23) The particle according to (22), which contains 10-15% amphipathic lipid, 35-45% sterol, 40-50% cationic lipid, and 1-2.5% PEG lipid. (24) The particle according to (23), which contains 1-2% PEG lipid. (25) The particle according to any one of (13), (15), (17), and (19), which has a lipid composition of amphipathic lipid, sterol, cationic lipid, and PEG lipid, in molar amounts of 10-25% amphipathic lipid, 10-50% sterol, 40-65% cationic lipid, and 1-3% PEG lipid. (26) The particle according to (25), which contains 10-45% sterol, 42.5-65% cationic lipid, and 1-2.5% PEG lipid. (27) The particle according to (26), which contains 1-2% PEG lipid. (28) The particle according to any one of (20) to (27), wherein the ratio of total lipid weight to nucleic acid weight is 15 to 30. (29) The particle according to (28), wherein the ratio of total lipid weight to nucleic acid weight is 15 to 25. (30) The particle according to (29), wherein the ratio of total lipid weight to nucleic acid weight is 17.5 to 22.5. (31) The particle according to any one of (1) to (30), wherein the human papillomavirus is HPV6. (32) The particle according to (31), wherein the human papillomavirus is HPV6 and the E6 antigen of HPV6 consists of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 12. (33) The particle according to (31) or (32), wherein the human papillomavirus is HPV6 and the E7 antigen of HPV6 consists of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 13.(34) The particle according to any one of (31) to (33), wherein the human papillomavirus is HPV6, and the nucleic acid capable of expressing the E6 and E7 antigens of the human papillomavirus encodes a fusion protein of the HPV6 E6 and E7 antigens, the fusion protein consisting of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 17. (35) The particle according to any one of (31) to (34), wherein the human papillomavirus is HPV6, and the nucleic acid capable of expressing the E6 and E7 antigens of the HPV6 is mRNA comprising a cap structure (Cap), a 5' untranslated region (5'-UTR), a leader sequence, a translated region of E6, a furin cleavage site (Furin Cleavage Site), a translated region of E7, a 3' untranslated region (3'-UTR), and a polyA tail (polyA). (36) The particle according to (35), wherein the sequence of the nucleic acid capable of expressing HPV6 E6 and E7 antigens consists of a nucleotide sequence having at least 90% identity with the sequence of SEQ ID NO: 5. (37) The particle according to any of (31) to (34), wherein the human papillomavirus is HPV6, and the nucleic acid capable of expressing HPV6 E6 and E7 antigens is mRNA comprising a cap structure (Cap), a 5'-untranslated region (5'-UTR), a leader sequence, a translated region of E6, a protease cleavage sequence (Furin Cleavage Site), a translated region of E7, and a 3'-untranslated region (3'-UTR). (38) The particle according to (37), wherein the composition consisting of the cap structure (Cap), 5' untranslated region (5'-UTR), leader sequence, E6 translation region, protease cleavage sequence (Furin Cleavage Site), E7 translation region and 3' untranslated region (3'-UTR) consists of a nucleotide sequence having at least 90% identity with the sequence from base 1 to base 1018 of SEQ ID NO: 5. (39) The particle according to any of (1) to (30), wherein the human papillomavirus is HPV type 11.

[0021] (40) The particle according to (39), wherein the human papillomavirus is HPV type 11 and the E6 antigen of HPV type 11 consists of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 14. (41) The particle according to (39) or (40), wherein the human papillomavirus is HPV type 11 and the E7 antigen of HPV type 11 consists of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 15. (42) The particle according to any of (39) to (41), wherein the human papillomavirus is HPV type 11 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 HPV type 11 consisting of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 18. (43) The particle according to any one of (39) to (42), wherein the human papillomavirus is HPV11, and the nucleic acid capable of expressing the HPV11 E6 and E7 antigens is mRNA comprising a cap structure (Cap), a 5'-untranslated region (5'-UTR), a leader sequence, the E6 translation region, a furin cleavage site, the E7 translation region, a 3'-untranslated region (3'-UTR), and a polyA tail (polyA). (44) The particle according to (43), wherein the sequence of the nucleic acid capable of expressing the HPV11 E6 and E7 antigens consists of a nucleotide sequence having at least 90% identity with the sequence of SEQ ID NO: 8. (45) A particle described in any of (39) to (42), wherein the human papillomavirus is HPV11 type, and the nucleic acid capable of expressing the E6 antigen and E7 antigen of HPV11 type is mRNA comprising a cap structure (Cap), a 5' untranslated region (5'-UTR), a leader sequence, the E6 translation region, a protease cleavage sequence (Furin Cleavage Site), the E7 translation region, and a 3' untranslated region (3'-UTR).(46) The particle according to (45), wherein the composition consisting of the cap structure (Cap), 5'-untranslated region (5'-UTR), leader sequence, E6 translated region, protease cleavage site (Furin Cleavage Site), E7 translated region and 3'-untranslated region (3'-UTR) consists of a nucleotide sequence having at least 90% identity to the sequence from bases 1 to 1018 of SEQ ID NO: 8. (47) The particle according to any of (1) to (30), wherein the human papillomavirus is HPV type 6 or 11. (48) The particle according to (47), wherein the human papillomavirus is HPV type 6 or 11, 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 HPV type 6 and the E6 antigen and E7 antigen of HPV type 11, the fusion protein consisting of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 19. (49) Particles described in (47) or (48), wherein the human papillomavirus is HPV type 6 or 11, and the nucleic acid capable of expressing the E6 and E7 antigens of HPV type 6 and the E6 and E7 antigens of HPV type 11 is mRNA comprising a cap structure (Cap), a 5' untranslated region (5'-UTR), a leader sequence, the translation region of E6 of HPV type 6, the translation region of E7 of HPV type 6, a protease cleavage sequence (Furin Cleavage Site), the translation region of E6 of HPV type 11, the translation region of E7 of HPV type 11, a 3' untranslated region (3'-UTR), and a polyA tail (polyA).

[0022] (50) The particle according to (49), wherein the sequence of the nucleic acid capable of expressing the HPV6 E6 and E7 antigens and the HPV11 E6 and E7 antigens consists of a nucleotide sequence having at least 90% identity with the sequence of SEQ ID NO: 11. (51) The particle according to (47) or (48), wherein the human papillomavirus is HPV6 and 11, and the nucleic acid capable of expressing the HPV6 E6 and E7 antigens and the HPV11 E6 and E7 antigens is mRNA comprising a cap structure (Cap), a 5'-untranslated region (5'-UTR), a leader sequence, the HPV6 E6 translation region, the HPV6 E7 translation region, a protease cleavage site (Furin Cleavage Site), the HPV11 E6 translation region, the HPV11 E7 translation region, and a 3'-untranslated region (3'-UTR). (52) The particle according to (51), wherein the composition consisting of the cap structure (Cap), 5' untranslated region (5'-UTR), leader sequence, HPV6 E6 translation region, HPV6 E7 translation region, protease cleavage sequence (Furin Cleavage Site), HPV11 E6 translation region, HPV11 E7 translation region, and 3' untranslated region (3'-UTR) consists of a nucleotide sequence having at least 90% identity with the sequence from base 1 to base 1798 of SEQ ID NO: 11. (53) The particle according to any one of (1) to (52), wherein the nucleic acid contains at least one modified nucleotide. (54) The particle according to (53), wherein the modified nucleotide contains at least one pyrimidine nucleotide substituted at position 5 and / or pseudouridine optionally substituted at position 1. (55) The particle according to (53), wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine. (56) The particle according to (53), wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methyluridine, and 1-methylpseudouridine. (57) The particle according to any one of (1) to (56), wherein the average particle diameter is 30 to 300 nm.(58) Use of the particles according to any one of (1) to (57) for producing a composition for preventing and / or treating infection with human papillomavirus. (59) Use of the particles according to any one of (1) to (57) for producing a composition for preventing and / or treating a disease caused by infection with human papillomavirus.

[0023] (60) Use of the particles according to (59), wherein the disease caused by human papillomavirus infection is recurrent respiratory papillomatosis or condyloma acuminata. (61) Use of the particles according to any of (58) to (60), wherein the infection is infection with human papillomavirus type 6 or type 11. (62) A composition comprising the particles according to any of (1) to (57). (63) The composition according to (62), for expressing E6 antigen and E7 antigen of human papillomavirus in vivo or in vitro. (64) The composition according to (62) or (63), for use as a medicine. (65) The composition according to (64), for inducing an immune response against human papillomavirus. (66) The composition according to (64) or (65), for preventing and / or treating human papillomavirus infection. (67) The composition according to (64) or (65), for preventing and / or treating diseases caused by human papillomavirus infection. (68) The composition according to (67), wherein the disease caused by human papillomavirus infection is recurrent respiratory papillomatosis or condyloma acuminata. (69) The composition according to any one of (66) to (68), wherein the infection is infection with human papillomavirus type HPV6 or HPV11.

[0024] (70) A method for expressing human papillomavirus E6 antigen and E7 antigen in vitro, comprising introducing the composition according to (62) or (63) into a cell. (71) A method for expressing human papillomavirus E6 antigen and E7 antigen in vivo, comprising administering the composition according to any one of (62) to (66) to a mammal. (72) A method for inducing an immune response against human papillomavirus, comprising administering the composition according to (64) or (65) to a mammal. (73) A method for preventing and / or treating human papillomavirus infection, comprising administering the composition according to any one of (64) to (69) to a mammal.

[0025] The present invention makes it possible to prevent and / or treat infections caused by human papillomavirus types 6 and / or 11. The present invention also makes it possible to prevent and / or treat diseases caused by infections with human papillomavirus types 6 and / or 11 (such as recurrent respiratory papillomatosis and condyloma acuminata). Furthermore, the particles of the present invention have excellent properties in terms of metabolic stability, in vitro activity, in vivo activity, rapidity of onset of efficacy, duration of efficacy, physical stability, drug interactions, safety, etc., and are useful as pharmaceuticals for treating or preventing the above-mentioned diseases.

[0026] HPV6E6- and HPV11E6-specific IFN-γ production levels in C57BL / 6 mice following mRNA-encapsulated nucleic acid lipid particles. Examples 4, 7, and 10: mRNA-encapsulated nucleic acid lipid particle administration group; NC: negative control group administered with buffer. No peptides: negative control group not treated with peptide; HPV6E6 peptides: HPV6E6 pool peptide treatment group; HPV11E6 peptides: HPV11E6 pool peptide treatment group. Bar graphs show the average values ​​for each group, and error bars show the standard deviation. HPV6E6- and HPV11E6-specific IFN-γ production levels in C57BL / 6 mice following mRNA-encapsulated nucleic acid lipid particles. Examples 5, 8, and 11: mRNA-encapsulated nucleic acid lipid particle administration group; NC: negative control group administered with buffer. 11E6; No peptides; negative control group not treated with peptides; HPV6E6 peptides; HPV6E6 pool peptide treatment group; HPV11E6 peptides; HPV11E6 pool peptide treatment group. Bar graphs show the mean values ​​for each group, and error bars show the standard deviation. HPV6E6 and HPV11E6-specific IFN-γ production levels for mRNA-encapsulated nucleic acid-lipid particles in C57BL / 6 mice. Examples 6, 9, and 12; mRNA-encapsulated nucleic acid-lipid particle administration group; NC; negative control group administered with buffer. No peptides; negative control group not treated with peptides; HPV6E6 peptides; HPV6E6 pool peptide treatment group; HPV11E6 peptides; HPV11E6 pool peptide treatment group. Bar graphs show the mean values ​​for each group, and error bars show the standard deviation. Figure 11E6 shows the nucleotide sequence (SEQ ID NO: 1) of template plasmid DNA for IVT of HPV6 E6-E7 fusion. Figure 1 shows the nucleotide sequence of a sense primer (SEQ ID NO: 2) and an antisense primer (SEQ ID NO: 3). Figure 2 shows the nucleotide sequence of an HPV6 E6-E7 fusion template DNA (SEQ ID NO: 4). Figure 3 shows the nucleotide sequence of an HPV6 E6-E7 fusion mRNA (SEQ ID NO: 5). Figure 4 shows the nucleotide sequence of a template plasmid DNA for IVT of HPV11 E6-E7 fusion (SEQ ID NO: 6). Figure 5 shows the nucleotide sequence of an HPV11 E6-E7 fusion template DNA (SEQ ID NO: 7).Figure 1 shows the nucleotide sequence of HPV11 E6-E7 fusion mRNA (SEQ ID NO: 8). Figure 2 shows the nucleotide sequence of HPV6 E6-E7 HPV11 E6-E7 fusion template plasmid DNA (SEQ ID NO: 9). Figure 3 shows the nucleotide sequence of HPV6 E6-E7 HPV11 E6-E7 fusion template DNA (SEQ ID NO: 10). FIG. 1 shows the nucleotide sequence of HPV6 E6-E7 HPV11 E6-E7 fusion mRNA (SEQ ID NO: 11). FIG. 2 shows the amino acid sequence of HPV6 E6 antigen (SEQ ID NO: 12). FIG. 3 shows the amino acid sequence of HPV6 E7 antigen (SEQ ID NO: 13). FIG. 4 shows the amino acid sequence of HPV11 E6 antigen (SEQ ID NO: 14). FIG. 5 shows the amino acid sequence of HPV11 E7 antigen (SEQ ID NO: 15). FIG. 6 shows the amino acid sequence of a protease cleavage sequence (SEQ ID NO: 16). FIG. 7 shows the amino acid sequence of HPV6 E6 antigen and E7 antigen fusion protein (SEQ ID NO: 17). FIG. 8 shows the amino acid sequence of HPV11 E6 antigen and E7 antigen fusion protein (SEQ ID NO: 18). FIG. 9 shows the amino acid sequences of HPV6 E6 antigen and E7 antigen fusion protein and HPV11 E6 antigen and E7 antigen fusion protein (SEQ ID NO: 19). FIG. 11 shows the IgE leader sequence (SEQ ID NO: 20).

[0027] The present invention will be described in detail below.

[0028] The following terms are used in this specification:

[0029] "Lipid particle": As used herein, the term "lipid particle" refers to a particle containing amphipathic lipids, sterols, cationic lipids, and PEG-lipids as constituent lipids.

[0030] "Capable of being expressed": As used herein, "capable of being expressed" means that a protein of interest can be produced in a cell in vitro or in vivo.

[0031] "C1-C3 alkyl group" refers to a straight or branched chain alkyl group having 1 to 3 carbon atoms. Examples include a methyl group, an ethyl group, a propyl group, and an isopropyl group.

[0032] "C2-C4 alkanoyl group" refers to an alkanoyl group having 2 to 4 carbon atoms. Examples include an acetyl group, a propionyl group, a butyryl group, and an isobutyryl group.

[0033] "C2-C4 alkanoyloxy group": A group in which the above C2-C4 alkanoyl group is bonded to an oxygen atom. Examples include an acetyloxy group, a propionyloxy group, a butyryloxy group, and an isobutyryloxy group.

[0034] "C17-C19 alkenyl group" refers to a straight-chain or branched-chain alkenyl group having 17 to 19 carbon atoms. In this specification, the C17-C19 alkenyl group includes any of a C17-C19 alkadienyl group, a C17-C19 alktrienyl group, and a C17-C19 alktetraenyl group. Examples include a heptadecenyl group, an octadecenyl group, a nonadecenyl group, a heptadecadienyl group, an octadecadienyl group, a nonadecadienyl group, a heptadecatrienyl group, an octadecatrienyl group, and a nonadecatrienyl group.

[0035] "C17-C19 alkenyl group optionally having one or more C2-C4 alkanoyloxy groups" refers to a group in which a hydrogen atom at any position of the above C17-C19 alkenyl group is substituted with the above C2-C4 alkanoyloxy group. Examples include an 11-acetyloxy-8-heptadecenyl group and an 11-propionyloxy-8-heptadecenyl group.

[0036] "C10-C19 alkyl group" refers to a straight-chain or branched-chain alkyl group having 10 to 19 carbon atoms. Examples include decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, and nonadecyl groups.

[0037] "A C10-C19 alkyl group which may have one or more C2-C4 alkanoyloxy groups" refers to a group in which a hydrogen atom at any position of the above C10-C19 alkyl group is substituted with the above C2-C4 alkanoyloxy group.

[0038] "C10-C19 alkenyl group" refers to a straight-chain or branched-chain alkenyl group having 10 to 19 carbon atoms. In this specification, the C10-C19 alkenyl group includes a C10-C19 alkadienyl group, a C10-C19 alkatrienyl group, and a C10-C19 alkatetraenyl group. Examples of such alkyl groups include decenyl, uncenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, decadienyl, undecadienyl, dodecadienyl, tridecadienyl, tetradecadienyl, pentadecadienyl, hexadecadienyl, heptadecadienyl, octadecadienyl, nonadecadienyl, decatrienyl, undecatrienyl, dodecatrienyl, tridecatrienyl, tetradecatrienyl, pentadecatrienyl, hexadecatrienyl, heptadecatrienyl, octadecatrienyl, and nonadecatrienyl groups.

[0039] "C10-C19 alkenyl group optionally having one or more C2-C4 alkanoyloxy groups" refers to a group in which a hydrogen atom at any position of the above C10-C19 alkenyl group is substituted with the above C2-C4 alkanoyloxy group. Examples include an 11-acetyloxy-8-heptadecenyl group and an 11-propionyloxy-8-heptadecenyl group.

[0040] "C17-C19 alkenyl group optionally having one or more acetyloxy groups" refers to a group in which a hydrogen atom at any position of the above C17-C19 alkenyl group is substituted with an acetyloxy group. Examples include an 11-acetyloxy-8-heptadecenyl group and an 11-propionyloxy-8-heptadecenyl group.

[0041] "C10-C12 alkyl group optionally having one or more acetyloxy groups" refers to a group in which a hydrogen atom at any position of the above C10-C12 alkyl group is substituted with an acetyloxy group.

[0042] "C10-C19 alkenyl group optionally having one or more acetyloxy groups" refers to a group in which a hydrogen atom at any position of the above C10-C19 alkenyl group is substituted with an acetyloxy group. Examples include an 11-acetyloxy-8-heptadecenyl group and an 11-propionyloxy-8-heptadecenyl group.

[0043] "Treatment": As used herein, treatment refers to recovery, remission, alleviation, and / or delay of worsening of clinical symptoms of an infectious disease caused by a virus, bacteria, or the like, or a disease caused by such an infection (e.g., precancerous lesion, cancer, etc.) in a patient who has developed such a disease.

[0044] "Prevention": As used herein, prevention means reducing the incidence of diseases caused by infectious diseases such as viruses or bacteria. Prevention includes reducing the risk of progression of diseases caused by infectious diseases such as viruses or bacteria, or reducing the severity of such diseases. The particles of the present invention are effective in preventing and / or treating the above-mentioned diseases by inducing a protective immune response.

[0045] "Identity": As used herein, identity refers to the relationship between the sequences of two or more nucleotide or amino acid sequences, as determined by comparing the sequences, as known in the art. In the art, "identity" also refers to the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between a sequence of two or more nucleotide sequences or two or more amino acid sequences. Identity can be assessed by calculating the percent of identical matches between the smaller of two or more sequences and a gap alignment (if any) addressed by a particular mathematical model or computer program (i.e., "algorithm"). Specifically, identity can be assessed using software such as ClustalW2 provided by the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI), but is not limited to any software commonly used by those skilled in the art.

[0046] The present invention provides lipid particles encapsulating a nucleic acid capable of expressing the E6 and E7 antigens of human papillomavirus type 6 and / or type 11, wherein the lipid comprises a cationic lipid represented by general formula (Ia) or a pharmaceutically acceptable salt thereof:

[0047]

[0048] In the formula, R 1 and R 2 each independently represents a C1-C3 alkyl group; L 1 represents a C17-C19 alkenyl group which may have one or more C2-C4 alkanoyloxy groups; L 2 represents a C10-C19 alkyl group which may have one or more C2-C4 alkanoyloxy groups, or a C10-C19 alkenyl group which may have one or more C2-C4 alkanoyloxy groups; and p is 3 or 4.

[0049] R in general formula (Ia) 1 and R 2 are independently a C1-C3 alkyl group, and preferably both are methyl groups.

[0050] In the general formula (Ia), p is 3 or 4, preferably 3.

[0051] L in general formula (Ia) 1 represents a C17-C19 alkenyl group which may have one or more C2-C4 alkanoyloxy groups, and is preferably a C17-C19 alkenyl group which may have one or more acetoxy groups. 1 Specific examples of the alkyl group include an (R)-11-acetyloxy-cis-8-heptadecenyl group, a cis-8-heptadecenyl group, and a (8Z,11Z)-heptadecadienyl group.

[0052] L in general formula (Ia) 2 ​represents a C10-C19 alkyl group which may have one or more C2-C4 alkanoyloxy groups, or a C10-C19 alkenyl group which may have one or more C2-C4 alkanoyloxy groups, and is preferably a C10-C12 alkyl group which may have one or more acetoxy groups, or a C10-C19 alkenyl group which may have one or more acetoxy groups. Alternatively, L in general formula (Ia) 2 is also preferably a C10-C12 alkyl group which may have one or more acetoxy groups, or a C17-C19 alkenyl group which may have one or more acetoxy groups. 2 Specific examples of the alkyl group include a decyl group, a cis-7-decenyl group, a dodecyl group, and an (R)-11-acetyloxy-cis-8-heptadecenyl group.

[0053] Specific examples of cationic lipids that are components constituting the particles of the present invention include those having the following structural formula:

[0054]

[0055]

[0056]

[0057] Examples include (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyl diacetate, 3-dimethylaminopropyl(9Z,12Z)-octacosa-19,22-dien-11-yl carbonate, and (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacosa-9-en-7-yl acetate, each of which is represented by the formula:

[0058] The cationic lipid represented by general formula (Ia) may be one type of compound or a combination of two or more types of compounds.

[0059] A method for producing cationic lipids of general formula (Ia) is described in WO 2015 / 005253.

[0060] The lipids of the present invention may further comprise amphipathic lipids, sterols and PEG lipids.

[0061] The amphipathic lipid is a lipid that has affinity for both polar and nonpolar solvents, and specific examples thereof include distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, and combinations thereof. The amphipathic lipid used in the particles of the present invention is preferably distearoylphosphatidylcholine and / or dioleoylphosphatidylethanolamine, and more preferably distearoylphosphatidylcholine.

[0062] The sterols are sterols having a hydroxy group, and specific examples thereof include cholesterol.

[0063] The PEG lipid is a PEG-modified lipid, specifically, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine, or a combination thereof, preferably 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol. The average molecular weight of the PEG lipid is not particularly limited, but is, for example, 1,000 to 5,000, preferably 1,500 to 3,000, and more preferably 1,800 to 2,200.

[0064] The lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is not particularly limited, but may be, for example, 5-25% amphipathic lipids, 10-55% sterols, 40-65% cationic lipids, and 1-5% PEG lipids in molar amounts. Preferably, the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 10-25% amphipathic lipids, 10-55% sterols, 40-65% cationic lipids, and 1-5% PEG lipids. More preferably, the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 10-22.5% amphipathic lipids, 15-55% sterols, 40-65% cationic lipids, and 1-5% PEG lipids. In addition, the proportion of PEG lipid in the lipid composition is preferably 1-3% by molar amount, more preferably 1-2%, even more preferably 1.2-2%, even more preferably 1.25-2%, even more preferably 1.3-2%, even more preferably 1.5-2%. In addition, the ratio of total lipid weight to nucleic acid weight in the lipid composition is not particularly limited, but may be 15-30, preferably 15-25, more preferably 15-22.5, even more preferably 17.5-22.5.

[0065] When 3-dimethylaminopropyl (9Z,12Z)-octacosa-19,22-dien-11-yl carbonate or (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyl diacetate is used as the cationic lipid, the lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is not particularly limited, but for example, in molar amounts, amphipathic lipid is 5 to 25%, sterols is 10 to 55%, cationic lipid is 40 to 65%, and PEG lipid is 1 to 5%, and amphipathic lipid is 5 to 15%, sterols is 20 to 55%, cationic lipid is 40 to 65%, and PEG lipid is 1 to 5%. It is more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 5 to 15% amphipathic lipids, 35 to 50% sterols, 40 to 55% cationic lipids, and 1 to 3% PEG lipids; it is even more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 10 to 15% amphipathic lipids, 35 to 45% sterols, 40 to 50% cationic lipids, and 1 to 2.5% PEG lipids; and it is even more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 10 to 15% amphipathic lipids, 35 to 45% sterols, 40 to 50% cationic lipids, and 1 to 2% PEG lipids. In the lipid composition, the PEG lipid content is preferably 1.2 to 2%, more preferably 1.25 to 2%, even more preferably 1.3 to 2%, and even more preferably 1.5 to 2%. The ratio of total lipid weight to nucleic acid weight in the lipid composition is not particularly limited, but may be 15 to 30, preferably 15 to 25, more preferably 15 to 22.5, and even more preferably 17.5 to 22.5.

[0066] When (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate is used as the cationic lipid, the lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is not particularly limited, but for example, the molar amounts are preferably 5 to 25% amphipathic lipid, 10 to 55% sterols, 40 to 65% cationic lipid, and 1 to 5% PEG lipid, or 10 to 25% amphipathic lipid, 10 to 50% sterols, 40 to 65% cationic lipid, and 1 to 3% amphipathic lipid, sterols, cationic lipid, and PEG lipid. It is more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 10 to 25% amphipathic lipids, 10 to 45% sterols, 42.5 to 65% cationic lipids, and 1 to 2.5% PEG lipids; it is even more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 15 to 22.5% amphipathic lipids, 15 to 40% sterols, 45 to 65% cationic lipids, and 1 to 2% PEG lipids; and it is even more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 17.5 to 22.5% amphipathic lipids, 15 to 40% sterols, 45 to 65% cationic lipids, and 1 to 2% PEG lipids. In the lipid composition, the PEG lipid content is preferably 1.2 to 2%, more preferably 1.25 to 2%, even more preferably 1.3 to 2%, and even more preferably 1.5 to 2%. The ratio of total lipid weight to nucleic acid weight in the lipid composition is not particularly limited, but may be 15 to 30, preferably 15 to 25, even more preferably 15 to 22.5, and even more preferably 17.5 to 22.5.

[0067] Specific lipid combinations in the present invention include distearoylphosphatidylcholine, dioleoylphosphatidylcholine, or dioleoylphosphatidylethanolamine as amphipathic lipids, cholesterol as sterols, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyl diacetate, 3-dimethylaminopropyl(9Z,12Z)-octacosa-19,22-dien-11-yl carbonate, or (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacosa-9-en-7-yl acetate as cationic lipids, and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol or N-[methoxy]-2-methyl-2-methylpropanol as PEG lipids. Poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine may be used in combination. Also preferred is a lipid combination using distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine as the amphipathic lipid, cholesterol as the sterol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate or (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate as the cationic lipid, and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol as the PEG lipid. A more preferred specific lipid combination in the present invention is distearoylphosphatidylcholine as the amphipathic lipid, cholesterol as the sterol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate or (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate as the cationic lipid, and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol as the PEG lipid.

[0068] In the present invention, the nucleic acid encapsulated in the lipid particles is capable of expressing the E6 and E7 antigens of human papillomavirus types 6 and / or 11. The E6 and E7 antigens of human papillomavirus types 6 and / or 11 expressed by the nucleic acid encapsulated in the lipid particles may be a fusion protein of the E6 and E7 antigens of human papillomavirus type 6, a fusion protein of the E6 and E7 antigens of human papillomavirus type 11, or a fusion protein of the E6 and E7 antigens of human papillomavirus type 6 with the E6 and E7 antigens of human papillomavirus type 11, and a protease cleavage sequence may be contained between adjacent antigens. Furthermore, the antigens may be fused to a signal peptide, and an IgE leader sequence may be used as a signal peptide for extracellular secretion of the antigen. The signal peptide is preferably fused to the N-terminus of the antigen. For example, the fusion protein of a signal peptide and an antigen has the amino acid sequence of SEQ ID NO: 17, and the amino acid sequence from positions 1 to 18 of the amino acid sequence of SEQ ID NO: 17 is the amino acid sequence of the IgE leader sequence.

[0069] The amino acid sequence of HPV6 E6 antigen is shown in SEQ ID NO: 12. The nucleic acid to be encapsulated in the lipid particles preferably encodes HPV6 E6 antigen consisting of an amino acid sequence having at least 95%, preferably 96%, and more preferably 97% identity to the amino acid sequence of SEQ ID NO: 12.

[0070] The amino acid sequence of HPV6 E7 antigen is shown in SEQ ID NO: 13. The nucleic acid to be encapsulated in the lipid particles preferably encodes HPV6 E7 antigen consisting of an amino acid sequence having at least 95%, preferably 96%, and more preferably 97% identity to the amino acid sequence of SEQ ID NO: 13.

[0071] The amino acid sequence of HPV type 11 E6 antigen is shown in SEQ ID NO: 14. The nucleic acid to be encapsulated in the lipid particles preferably encodes HPV type 11 E6 antigen consisting of an amino acid sequence having at least 95%, preferably 96%, and more preferably 97% identity to the amino acid sequence of SEQ ID NO: 14.

[0072] The amino acid sequence of HPV type 11 E7 antigen is shown in SEQ ID NO: 15. The nucleic acid to be encapsulated in the lipid particles preferably encodes HPV type 11 E7 antigen consisting of an amino acid sequence having at least 95%, preferably 96%, and more preferably 97% identity to the amino acid sequence of SEQ ID NO: 15.

[0073] 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 is cleaved by Furin protein, and examples include 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).

[0074] The amino acid sequence of the fusion protein of HPV6 E6 antigen and E7 antigen is shown in SEQ ID NO: 17. The nucleic acid to be encapsulated in the lipid particles preferably encodes a fusion protein of HPV6 E6 antigen and E7 antigen, the fusion protein consisting of an amino acid sequence having at least 95%, preferably 96%, and more preferably 97% identity to the amino acid sequence of SEQ ID NO: 17.

[0075] The amino acid sequence of the fusion protein of HPV type 11 E6 antigen and E7 antigen is shown in SEQ ID NO: 18. The nucleic acid to be encapsulated in the lipid particles preferably encodes a fusion protein of HPV type 11 E6 antigen and E7 antigen, the fusion protein consisting of an amino acid sequence having at least 95%, preferably 96%, and more preferably 97% identity to the amino acid sequence of SEQ ID NO: 18.

[0076] The amino acid sequence of the fusion protein of HPV6 E6 and E7 antigens with HPV11 E6 and E7 antigens is shown in SEQ ID NO: 19. The nucleic acid to be encapsulated in the lipid particles preferably encodes a fusion protein of HPV6 E6 and E7 antigens with HPV11 E6 and E7 antigens, the fusion protein consisting of an amino acid sequence having at least 95%, preferably 96%, and more preferably 97% identity to the amino acid sequence of SEQ ID NO: 19.

[0077] The amino acid sequence identity is a numerical representation of the percentage of amino acid identity relative to the full-length sequence, where amino acids that are completely identical to each other are considered to be the same amino acid. The sequence identity in the present invention is calculated using sequence analysis software GENETYX-SV / RC (manufactured by Genetyx Corporation), and this algorithm is commonly used in the technical field. The amino acids encoded by the nucleic acids encapsulated in the lipid particles of the present invention may contain amino acid mutations (substitutions), deletions, insertions, and / or additions, as long as they maintain a certain level of identity with SEQ ID NOs: 12-20.

[0078] The amino acids encoded by the nucleic acids to be encapsulated in the lipid particles of the present invention maintain the above-mentioned sequence identity, and may have several amino acids substituted, deleted, inserted and / or added at several positions (preferably 5 positions or less, more preferably 3, 2 or 1 position) in the amino acid sequence of SEQ ID NO: 12-20, with several amino acids per position (preferably 10 positions or less, more preferably 7 positions or less, even more preferably 5, 4, 3, 2 or 1 position).

[0079] The nucleic acid capable of expressing the E6 and E7 antigens of human papillomavirus type 6 and / or type 11 may be an mRNA comprising a cap structure (Cap), a 5'-untranslated region (5'-UTR), a leader sequence, the E6 translation region, a furin cleavage site (Furin Cleavage Site), the E7 translation region, a 3'-untranslated region (3'-UTR), and a poly(A) tail. The cap structure (Cap) is present at the 5' end of many eukaryotic mRNAs and contains a 7-methylguanosine moiety. Examples of cap structures include cap0, cap1, cap2, and cap structures obtained using ARCA (Anti-Reverse Cap Analog). The cap structure is as shown in the following structural formula:

[0080]

[0081] [In the formula, Base represents any unmodified or modified nucleic acid base, and RNA represents any polynucleotide.]

[0082] [In the formula, Base represents any unmodified or modified nucleic acid base, and RNA represents any polynucleotide.]

[0083] [In the formula, Base represents any unmodified or modified nucleic acid base, and RNA represents any polynucleotide.]

[0084]

[0085] [In the formula, Base represents any unmodified or modified nucleic acid base, and RNA represents any polynucleotide.]

[0086] The cap structure of the mRNA of the present invention is preferably cap0 or cap1, more preferably cap1.

[0087] The sequences of the 5' untranslated region and 3' untranslated region are not particularly limited, and the untranslated region of stable mRNA such as α-globin, β-globin, actin, or GAPDH can be used. The untranslated region used in the nucleic acid encapsulated in the lipid particles of the present invention is preferably the untranslated region of β-globin. For example, the 5' untranslated region of β-globin can be a sequence containing bases 15 to 64 in SEQ ID NO: 2, and the 3' untranslated region of β-globin can be a sequence containing bases 887 to 1018 in SEQ ID NO: 2.

[0088] Examples of the sequence of the 5'-untranslated region (5'-UTR) include the sequence of nucleotides 1 to 70 in SEQ ID NO: 5, the sequence of nucleotides 1 to 70 in SEQ ID NO: 8, and the sequence of nucleotides 1 to 70 in SEQ ID NO: 11. Examples of the sequence of the leader sequence include the sequence of nucleotides 71 to 124 in SEQ ID NO: 5, the sequence of nucleotides 71 to 124 in SEQ ID NO: 8, and the sequence of nucleotides 71 to 124 in SEQ ID NO: 11. The sequence of the E6 translation region is a sequence that can express all or part of the amino acid sequence of the E6 antigen and may include an initiation codon and / or a termination codon, such as the sequence of nucleotides 125 to 571 in SEQ ID NO: 5, the sequence of nucleotides 125 to 571 in SEQ ID NO: 8, and the sequences of nucleotides 125 to 571 and 905 to 1351 in SEQ ID NO: 11. Examples of the protease cleavage sequence (furin cleavage site) are the sequence of nucleotides 572 to 592 in SEQ ID NO: 5, the sequence of nucleotides 572 to 592 in SEQ ID NO: 8, and the sequences of nucleotides 572 to 592, 884 to 904, and 1352 to 1372 in SEQ ID NO: 11. The sequence of the E7 translation region is a sequence that can express all or part of the amino acid sequence of the E7 antigen and may include an initiation codon and / or a termination codon, such as the sequence of nucleotides 593 to 886 in SEQ ID NO: 5, the sequence of nucleotides 593 to 886 in SEQ ID NO: 8, and the sequences of nucleotides 593 to 883 and 1373 to 1666 in SEQ ID NO: 11. Examples of the sequence of the 3' untranslated region (3'-UTR) are the sequence of nucleotides 887 to 1018 in SEQ ID NO: 5, the sequence of nucleotides 887 to 1018 in SEQ ID NO: 8, and the sequence of nucleotides 1667 to 1798 in SEQ ID NO: 11. Examples of the sequence of the polyA tail (polyA) are the sequence of nucleotides 1019 to 1118 in SEQ ID NO: 5, the sequence of nucleotides 1019 to 1118 in SEQ ID NO: 8, and the sequence of nucleotides 1799 to 1898 in SEQ ID NO: 11.The cap structure (Cap), 5'-untranslated region (5'-UTR), leader sequence, E6 coding region, protease cleavage site (Furin Cleavage Site), E7 coding region, 3'-untranslated region (3'-UTR), and polyA tail (polyA) sequences may be modified. The nucleic acid sequence capable of expressing HPV6 E6 and E7 antigens preferably consists of a nucleotide sequence having at least 90%, preferably 95%, and more preferably 97% identity to the sequence of SEQ ID NO: 5. The nucleic acid sequence capable of expressing HPV11 E6 and E7 antigens preferably consists of a nucleotide sequence having at least 90%, preferably 95%, and more preferably 97% identity to the sequence of SEQ ID NO: 8. Furthermore, the nucleic acid sequence capable of expressing HPV6 and 11 E6 and E7 antigens preferably consists of a nucleotide sequence having at least 90%, preferably 95%, and more preferably 97% identity to the sequence of SEQ ID NO: 11.

[0089] The length of the poly A tail is not particularly limited, but is, for example, 10 to 250 bases long, preferably 15 to 120 bases long, more preferably 15 to 115 bases long, and particularly preferably 20 to 110 bases long.

[0090] The mRNA of the present invention may be an mRNA having a sequence comprising a cap structure (Cap), a 5' untranslated region (5'-UTR), a translated region of E6, a protease cleavage sequence (Furin Cleavage Site), a translated region of E7, and a 3' untranslated region (3'-UTR), wherein the portion consisting of the cap structure (Cap), the 5' untranslated region (5'-UTR), the translated region of E6, a protease cleavage sequence (Furin Cleavage Site), a translated region of E7, and a 3' untranslated region (3'-UTR) has at least 90%, preferably 95%, and more preferably 97% identity to any of the sequences set forth in positions 1 to 1018 of SEQ ID NO: 5, positions 1 to 1018 of SEQ ID NO: 8, or positions 1 to 1798 of SEQ ID NO: 11.

[0091] The nucleic acid to be encapsulated in the lipid particles may be in any form as long as it is capable of expressing the E6 and E7 antigens of human papillomavirus type 6 and / or type 11. Examples of such nucleic acids include single-stranded DNA, single-stranded RNA (e.g., mRNA), a single-stranded polynucleotide consisting of a mixture of DNA and RNA, double-stranded DNA, double-stranded RNA, a DNA-RNA hybrid polynucleotide, and a double-stranded polynucleotide consisting of two types of polynucleotides consisting of a mixture of DNA and RNA, with mRNA being preferred.

[0092] The nucleotides constituting the nucleic acid to be encapsulated in the lipid particles may be natural or modified nucleotides, but it is preferable that the nucleic acid contains at least one modified nucleotide.

[0093] The modified nucleotide may be one in which any of the base, sugar, and phosphodiester bond is modified, and the modification site may be one or more.

[0094] Examples of base modifications include 5-methylation, 5-fluoroation, and N4-methylation of cytosine, 5-methylation (thymine) and 5-fluoroation of uracil, N6-methylation of adenine, and N2-methylation of guanine.

[0095] An example of a sugar modification is 2'-O-methylation of D-ribofuranose.

[0096] An example of a modification of a phosphodiester bond is a phosphorothioate bond.

[0097] The modified nucleotide is preferably one in which the base moiety is modified, and examples thereof include a pyrimidine nucleotide substituted at the 5th position and a pseudouridine optionally substituted at the 1st position. Specific examples include 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine. The 1-alkylpseudouridine may be 1-(C1-C6 alkyl)pseudouridine, preferably 1-methylpseudouridine or 1-ethylpseudouridine. More preferred examples of the modified nucleotide include 5-methylcytidine, 5-methyluridine, and 1-methylpseudouridine. Particularly preferred examples of the modified nucleotide include a combination of 5-methylcytidine and 5-methyluridine, or a combination of 5-methylcytidine and 1-methylpseudouridine.

[0098] Nucleic acids capable of expressing the E6 and E7 antigens of human papillomaviruses (e.g., HPV6 and HPV11) of the present invention can be produced by in vitro transcription from DNA having the desired nucleotide sequence. The 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 (e.g., AmpliScribe T7 High Yield Transcription Kit (Epicentre) and mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies)). The DNA used to produce single-stranded RNA can be cloned DNA, such as a plasmid DNA or a DNA fragment. Plasmid DNA or DNA fragments may be commercially available or may be prepared 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 DG et al., Science, 2008, 319(5867), 1215-1220).

[0099] To obtain mRNA with improved stability and / or safety, some or all of the unmodified nucleotides in the mRNA can be replaced with modified nucleotides by substituting some or all of the unmodified nucleoside-5'-triphosphates with modified nucleoside-5'-triphosphates in an in vitro transcription reaction (Kormann, M., Nature Biotechnology, 2011, 29, 154-157.).

[0100] To obtain mRNA with improved stability and / or safety, a cap structure (the Cap0 structure described above) can be introduced at the 5' end of mRNA by using a capping enzyme after in vitro transcription. Furthermore, Cap0 can be converted to Cap1 by treating mRNA with Cap0 with 2'-O-methyltransferase. Commercially available capping enzymes and 2'-O-methyltransferases can be used (e.g., Vaccinia Capping System, M2080; mRNA Cap 2'-O-Methyltransferase, M0366, both manufactured by New England Biolabs). When using commercially available products, mRNA with a cap structure can be produced according to the protocol provided with the product.

[0101] A cap structure at the 5' end of mRNA can also be introduced by methods other than those using enzymes. For example, adding ARCA or CleanCap® to an in vitro transcription reaction can introduce a cap analog structure possessed by ARCA or a Cap1 structure derived from CleanCap® into mRNA. ARCA and CleanCap® are commercially available products (ARCA, N-7003; CleanCap Reagent AG, N-7113, both manufactured by TriLink BioTechnologies). When using commercially available products, mRNA with a cap structure can be produced according to the protocol provided with the product.

[0102] In the present invention, the nucleic acid to be encapsulated in lipid particles may be purified by methods such as desalting, HPLC (reverse phase, gel filtration, ion exchange, affinity), PAGE, ultrafiltration, etc. By removing impurities through the purification process, the production of inflammatory cytokines in a living body to which the nucleic acid is administered can be reduced.

[0103] The nucleic acid-encapsulating lipid particles of the present invention can be produced by methods such as the thin film method, reverse phase evaporation, ethanol injection method, ether injection method, dehydration-rehydration method, surfactant dialysis method, hydration method, and freeze-thaw method. 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, they can be produced using Precision Nanosystems' NanoAssemblr (registered trademark) according to the method described in the accompanying protocol.

[0104] The particles of the present invention may have an average particle size of 30 to 300 nm, preferably 30 to 200 nm, and more preferably 30 to 100 nm. The average particle size can be obtained by measuring the volume average particle size based on the principle of dynamic light scattering using an instrument such as a Zeta Potential / Particle Sizer NICOMP (registered trademark) 380ZLS (PARTICLE SIZING SYSTEMS).

[0105] The particles of the present invention can be used to produce a composition for preventing and / or treating diseases caused by human papillomavirus type 6 and / or type 11 infection (recurrent respiratory papillomatosis, condyloma acuminata, etc.).

[0106] The particles of the present invention can be used to express the E6 and E7 antigens of human papillomavirus types 6 and / or 11 in vivo or in vitro. Accordingly, the present invention provides a method for expressing the E6 and E7 antigens of human papillomavirus types 6 and / or 11 in vitro, comprising introducing a composition containing the particles into cells. The present invention also provides a method for expressing the E6 and E7 antigens of human papillomavirus types 6 and / or 11 in vivo, comprising administering a composition containing the particles to a mammal. By expressing the E6 and E7 antigens of human papillomavirus types 6 and / or 11 in vivo, an immune response against human papillomavirus types 6 and / or 11 can be induced. As a result, infection with human papillomavirus types 6 and / or 11 can be prevented and / or treated. Accordingly, the present invention provides a method for inducing an immune response against human papillomavirus types 6 and / or 11, comprising administering a composition containing the particles to a mammal. The present invention also provides a method for preventing and / or treating infection with human papillomavirus types 6 and / or 11, which comprises administering a composition containing the above-described particles to a mammal.

[0107] The particles of the present invention can be used as pharmaceuticals or experimental reagents. The particles of the present invention are typically added to a carrier such as water, buffer solution, or physiological saline, and the resulting formulation (composition) can be introduced into cells (in vitro) or administered to mammals (in vivo). When administered to mammals, the carrier should be a pharmaceutically acceptable carrier (e.g., physiological saline). The particles of the present invention can also be formulated into creams, pastes, ointments, gels, lotions, and other formulations using fats, fatty oils, lanolin, petrolatum, paraffin, wax, resins, plastics, glycols, higher alcohols, glycerin, water, emulsifiers, suspending agents, and other base materials.

[0108] The particles of the present invention can be administered orally or parenterally to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, pigs, monkeys, cats, dogs, horses, goats, sheep, and cows by methods such as intramuscular administration, intravenous administration, rectal administration, transdermal administration, transmucosal administration, subcutaneous administration, and intradermal administration.

[0109] When the particles of the present invention are administered to humans, for example, a single dose of about 0.001 to 1 mg, preferably 0.01 to 0.2 mg, of mRNA per adult may be administered once or several times by intramuscular injection, subcutaneous injection, intradermal injection, intravenous drip injection, or intravenous injection, but the dose and number of administrations may be varied as appropriate depending on the type of disease, symptoms, age, administration method, etc.

[0110] When used as an experimental reagent, the particles of the present invention can be introduced into cells in which the E6 and E7 antigens of human papillomavirus types 6 and / or 11 are to be expressed (e.g., HEK293 cells and their derivatives (HEK293T cells, FreeStyle 293 cells, and Expi293 cells), CHO cells, C2C12 mouse myoblasts, and immortalized mouse dendritic cells (MutuDC1940)), allowing the in vitro expression of the E6 and E7 antigens of human papillomavirus types 6 and / or 11. The expression of the E6 and E7 antigens of human papillomavirus types 6 and / or 11 can be analyzed by detecting the E6 and E7 antigen proteins of human papillomavirus types 6 and / or 11 in a sample by Western blotting or by detecting peptide fragments specific to the E6 and E7 antigens of human papillomavirus types 6 and / or 11 by mass spectrometry.

[0111] In the present invention, "treatment" means recovery, remission, alleviation and / or delay of worsening of clinical symptoms of an infectious disease caused by a virus, bacteria, etc., or a disease caused by such an infection (e.g., recurrent respiratory papillomatosis, genital warts, precancerous lesions, cancer, etc.) in a patient who has developed such a disease.

[0112] In the present invention, "prevention" means reducing the incidence of diseases caused by infectious diseases such as viruses or bacteria. Prevention includes reducing the risk of progression of diseases caused by infectious diseases such as viruses or bacteria, or reducing the severity of such diseases. The particles of the present invention are effective in preventing and / or treating the above-mentioned diseases by inducing a protective immune response.

[0113] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0114] Example 1 Preparation of HPV6 E6-E7 fusion mRNA-001 (1) Preparation of template DNA for in vitro transcription (IVT) of HPV6 E6-E7 fusion A plasmid was constructed to prepare template DNA to be used in in vitro transcription (IVT). A plasmid (pMA-HPV6) was prepared by introducing a DNA fragment (SEQ ID NO: 1) containing a sequence in which the following sequences were linked in order: GCTAGC (NheI site), T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, IgE leader sequence-HPV6 type E6-Furin Cleavage Site-HPV6 type E7 translation region, human β-globin 3'-UTR sequence, poly A tail, and ACTAGT (SpeI site). 8 ng of the plasmid was treated with the restriction enzyme SpeI and dissolved in Nuclease-free water (566.4 μL) using 10x Buffer for KOD-Plus-Ver. 2 (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 2 mM dNTP mix (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 25 mM MgSO4 (48 μL, Toyobo Co., Ltd. catalog # KOD-211), 50 μM sense primer (4.8 μL, SEQ ID NO: 2), 50 μM antisense primer (4.8 μL, SEQ ID NO: 3), KOD Plus polymerase (16 μL, Toyobo Co., Ltd. catalog # HPV6 E6-E7 fusion DNA was amplified by adding 100% ribosomal DNA (KOD-211) and incubating at 98°C for 1 minute, followed by 20 cycles of 98°C for 5 seconds, 55°C for 15 seconds, and 68°C for 2 minutes, followed by a further incubation at 68°C for 1 minute. After the reaction, the template DNA (SEQ ID NO: 4) was purified using the Wizard SV Gel and PCR Clean-Up System (Promega catalog # A9281).

[0115] (2) Preparation of HPV6 E6-E7 fusion mRNA-001 by in vitro transcription 479 μg / mL template DNA obtained in Example 1-(1) (57 μL), 100 mM CleanCap AG (55 μL, TriLink catalog # T-7113), 100 mM ATP (55 μL, Hongene catalog # R1331), 100 mM GTP (55 μL, Hongene catalog # R2331), 100 mM 5-methylcytidine-5'-triphosphate (55 μL, Hongene catalog # R3-029), 100 mM 5-methyluridine-5'-triphosphate (55 μL), nuclease-free water (438 μL, Qiagen catalog # 129114), T7 Transcription 5x buffer (220 μL, Promega catalog # P140X), enzyme mix, and T7 RNA polymerase (110 μL, Promega catalog # P137X) were mixed and incubated at 37°C for 4 hours. This was mixed with RQ1 RNase-Free DNase (27.5 μL, Promega catalog # M6101) and incubated at 37°C for 15 minutes. 8M LiCl solution (550 μL, Sigma-Aldrich catalog # L7026) was added and the mixture was left standing overnight at -20°C. After centrifugation (4°C, 4000 × g, 30 minutes), the supernatant was discarded. 70% ethanol was added, followed by centrifugation (4°C, 4000 × g, 10 minutes). The supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water and purified using the RNeasy Maxi kit (Qiagen catalog # 75162) according to the accompanying manual. The resulting eluate (3 mL, 4809 μg in UV equivalent), nuclease-free water (88 μL), rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001) buffer solution (450 μL), and enzyme (962 μL) were mixed and incubated at 37°C for 30 minutes.The target mRNA was obtained by purification using an RNeasy Maxi kit according to the accompanying manual (3 mL, 4.1 mg in UV equivalent). The obtained mRNA had the sequence set forth in SEQ ID NO: 5. Analysis using the LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) confirmed that it was the desired length.

[0116] Example 2 Preparation of HPV11 E6-E7 fusion mRNA-002 (1) Preparation of template DNA for IVT of HPV11 E6-E7 fusion A plasmid was constructed to prepare template DNA to be used as an IVT template. A plasmid (pMA-HPV11) was prepared by introducing a DNA fragment (SEQ ID NO: 6) containing a sequence in which the following sequences were linked in order: GCTAGC (NheI site), T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, IgE leader sequence-HPV11 type E6-Furin Cleavage Site-HPV11 type E7 translation region, human β-globin 3'-UTR sequence, poly A tail, and ACTAGT (SpeI site). Eight ng of the plasmid was treated with the restriction enzyme SpeI and dissolved in nuclease-free water (566.4 μL) using 10x Buffer for KOD-Plus-Ver. 2 (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 2 mM dNTP mix (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 25 mM MgSO4 (48 μL, Toyobo Co., Ltd. catalog # KOD-211), 50 μM sense primer (4.8 μL, SEQ ID NO: 2), 50 μM antisense primer (4.8 μL, SEQ ID NO: 3), KOD Plus polymerase (16 μL, Toyobo Co., Ltd. catalog # HPV6 E6-E7 DNA was amplified by adding 100% ribosomal DNA (KOD-211) and incubating at 98°C for 1 minute, followed by 20 cycles of 98°C for 5 seconds, 55°C for 15 seconds, and 68°C for 2 minutes, followed by a further incubation at 68°C for 1 minute. After the reaction, the template DNA (SEQ ID NO: 7) was purified using the Wizard SV Gel and PCR Clean-Up System (Promega catalog # A9281).

[0117] (2) Preparation of HPV11 E6-E7 fusion mRNA-002 by in vitro transcription 467 μg / mL template DNA obtained in Example 1-(1) (59 μL), 100 mM CleanCap AG (55 μL, TriLink catalog # T-7113), 100 mM ATP (55 μL, Hongene catalog # R1331), 100 mM GTP (55 μL, Hongene catalog # R2331), 100 mM 5-methylcytidine-5'-triphosphate (55 μL, Hongene catalog # R3-029), 100 mM 5-methyluridine-5'-triphosphate (55 μL), nuclease-free water (436 μL, Qiagen catalog # 129114), T7 Transcription 5x buffer (220 μL, Promega catalog # P140X), enzyme mix, and T7 RNA polymerase (110 μL, Promega catalog # P137X) were mixed and incubated at 37°C for 4 hours. This was mixed with RQ1 RNase-Free DNase (27.5 μL, Promega catalog # M6101) and incubated at 37°C for 15 minutes. 8M LiCl solution (550 μL, Sigma-Aldrich catalog # L7026) was added and the mixture was left standing overnight at -20°C. After centrifugation (4°C, 4000 × g, 30 minutes), the supernatant was discarded. 70% ethanol was added, followed by centrifugation (4°C, 4000 × g, 10 minutes). The supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water and purified using the RNeasy Maxi kit (Qiagen catalog # 75162) according to the accompanying manual. The resulting eluate (3 mL, 4769 μg in UV equivalent), nuclease-free water (96 μL), rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001) buffer solution (450 μL), and enzyme (954 μL) were mixed and incubated at 37°C for 30 minutes.The target mRNA was obtained by purification using an RNeasy Maxi kit according to the accompanying manual (3 mL, 3.9 mg in UV equivalent). The obtained mRNA had the sequence set forth in SEQ ID NO: 8. Analysis using the LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) confirmed that it was the desired length.

[0118] Example 3 Preparation of HPV6 E6-E7 HPV11 E6-E7 fusion mRNA-003 (1) Preparation of template DNA for IVT of HPV6 E6-E7 HPV11 E6-E7 fusion A plasmid was constructed to prepare template DNA to be used as an IVT template. A plasmid (pMA-HPV6_HPV11) was prepared by introducing a DNA fragment (SEQ ID NO: 9) containing a sequence in which GCTAGC (NheI site), T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, IgE leader sequence-HPV6 type E6-Furin Cleavage Site-HPV6 type E7-Furin Cleavage Site-HPV11 type E6-Furin Cleavage Site-HPV11 type E7 translation region, β-globin 3'-UTR sequence, poly A tail, and ACTAGT (SpeI site) were linked in this order. 8 ng of the plasmid treated with the restriction enzyme SpeI was dissolved in nuclease-free water (566.4 μL) and 10× Buffer for KOD-Plus-Ver. 2 (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 2 mM dNTP mix (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 25 mM MgSO4 (48 μL, Toyobo Co., Ltd. catalog # KOD-211), 50 μM sense primer (4.8 μL, SEQ ID NO: 2), 50 μM antisense primer (4.8 μL, SEQ ID NO: 3), KOD Plus polymerase (16 μL, Toyobo Co., Ltd. catalog # HPV6 E6-E7 DNA was amplified by adding 100% ribosomal DNA (KOD-211) and incubating at 98°C for 1 minute, followed by 20 cycles of 98°C for 5 seconds, 55°C for 15 seconds, and 68°C for 2 minutes, followed by a further incubation at 68°C for 1 minute. After the reaction, the template DNA (SEQ ID NO: 10) was purified using the Wizard SV Gel and PCR Clean-Up System (Promega catalog # A9281).

[0119] (2) Preparation of HPV6 E6-E7 HPV11 E6-E7 fusion mRNA-003 by in vitro transcription 413 μg / mL template DNA (67 μL) obtained in Example 1-(1), 100 mM CleanCap AG (55 μL, TriLink catalog # T-7113), 100 mM ATP (55 μL, Hongene catalog # R1331), 100 mM GTP (55 μL, Hongene catalog # R2331), 100 mM 5-methylcytidine-5′-triphosphate (55 μL, Hongene catalog # R2331), 100 mM ATP ... The mixture was mixed with 100 mM 5-methyluridine-5'-triphosphate (55 μL), nuclease-free water (428 μL, Qiagen catalog # 129114), T7 Transcription 5x buffer (220 μL, Promega catalog # P140X), enzyme mix, and T7 RNA polymerase (110 μL, Promega catalog # P137X), and incubated at 37°C for 4 hours. This was mixed with RQ1 RNase-Free DNase (27.5 μL, Promega catalog # M6101) and incubated at 37°C for 15 minutes. 8M LiCl solution (550 μL, Sigma-Aldrich catalog # L7026) was added and the mixture was left standing overnight at -20°C. After centrifugation (4°C, 4000 × g, 30 minutes), the supernatant was discarded. 70% ethanol was added, followed by centrifugation (4°C, 4000 × g, 10 minutes). The supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water and purified using the RNeasy Maxi kit (Qiagen catalog # 75162) according to the accompanying manual. The resulting eluate (3 mL, 4345 μg in UV equivalent), nuclease-free water (181 μL), rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001) buffer solution (450 μL), and enzyme (869 μL) were mixed and incubated at 37°C for 30 minutes.The target mRNA was obtained by purification using an RNeasy Maxi kit according to the accompanying manual (3 mL, 3.8 mg in UV equivalent). The obtained mRNA had the sequence set forth in SEQ ID NO: 11. Analysis using the LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) confirmed that it was the desired length.

[0120] Examples 4 to 12 Preparation of mRNA-Encapsulated Nucleic Acid Lipid Particles Using the mRNA Described in Examples 1 to 3 (1) Preparation of mRNA-Encapsulated Nucleic Acid Lipid Particles Distearoylphosphatidylcholine (1,2-Distearoyl-sn-glycero-3-phosphocholine: hereinafter referred to as DSPC, NOF CORPORATION), cholesterol (Cholesterol: hereinafter referred to as Chol, Sigma-Aldrich, Inc.), (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate (the compound described in Example 28 of WO 2015 / 005253) (hereinafter referred to as LP), and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol having a molecular weight of approximately 2000 were used. Glycol (1,2-Dimyristoyl-sn-Glycero-3-Methoxypolyethylene Glycol, hereinafter referred to as PEG-DMG, NOF CORPORATION) was dissolved in ethanol at the molar ratio shown in Table 1 to give a total lipid concentration of 5 mM.

[0121] Meanwhile, the mRNA obtained in Examples 1 to 3 was diluted with citrate buffer (20 mM citrate buffer, pH 4.0) and prepared. The above lipid solution and mRNA solution were mixed in a microchannel using a NanoAssemblr BenchTop (Precision Nanosystems Inc.) so that the total lipid weight ratio to mRNA was the value listed in Table 1 and the volume ratio was 1:3, to obtain a crude dispersion of nucleic acid-lipid particles. The nucleic acid-lipid particle dispersion was dialyzed (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) for 12 to 18 hours against approximately 25 to 50 times the amount of buffer, thereby removing ethanol and obtaining a dispersion of purified mRNA-encapsulated nucleic acid-lipid particles. LP was synthesized according to the method described in Example 28 of WO2015 / 005253.

[0122] (2) Evaluation of the Characteristics of mRNA-Encapsulated Nucleic Acid-Lipid Particles The characteristics of the dispersion containing the prepared nucleic acid-lipid particles were evaluated. The methods for evaluating each characteristic are described below.

[0123] (2-1) mRNA Encapsulation Rate The mRNA encapsulation rate was measured using the Quant-iT RiboGreen RNA Assay kit (Invitrogen) according to the package insert. Specifically, mRNA in a dispersion of nucleic acid-lipid particles was quantified in the presence and absence of 0.015% Triton X-100 surfactant, and the encapsulation rate was calculated using 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 (%)

[0124] (2-2) Ratio of mRNA to Lipid The amount of mRNA in a nucleic acid-lipid particle dispersion was measured by one of the following methods: The nucleic acid-lipid particle dispersion was diluted with 1.0% Triton X-100 and measured by reverse phase chromatography (System: Agilent 1260 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).

[0125] 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 using an ultraviolet-visible spectrophotometer (Perkin-Elmer, LAMBDA TM The mRNA concentration was calculated using the following formula: {[absorbance at 260 nm] - [absorbance at 350 nm]} x 40 x dilution factor (μg / mL).

[0126] The amount of each lipid in the nucleic acid-lipid particle dispersion was measured by reverse phase chromatography (System: DIONEX UltiMate 3000, Column: XSelect CSH C18 (130 Å, 3.5 μm, 3.0 mm × 150 mm) (Waters catalog # 186005263), Buffer A: 0.2% formic acid, Buffer B: 0.2% formic acid, methanol, (B%): 75-100% (0-6 min), 100% (6-15 min), Flow Rate: 0.45 mL / min, Temperature: 50°C, Detection: Corona CAD (Charged Aerosol Detector)). The ratio of total lipid to mRNA was calculated using the following formula: [total lipid concentration] / [mRNA concentration] (wt / wt).

[0127] (2-3) Average Particle Diameter The particle diameter of the nucleic acid-lipid particles was measured using a Zeta Potential / Particle Sizer NICOMP™ 380ZLS (PARTICLE SIZING SYSTEMS). The average particle diameter in the table represents the volume average particle diameter, and ± indicates deviation. The results of the characteristic evaluation are shown in Table 2.

[0128]

[0129]

[0130] From the above results, it was revealed that these nucleic acid-lipid particles had 95% or more of the mRNA encapsulated within the lipid particles, and had an average particle size of about 110 nm to about 140 nm.

[0131] [Test Example 1] Cytokine Production of HPV Genotype 6E6E7 Vaccine Antigen-Specific T Cells (Figure 1) C57BL / 6J mice were purchased from CLEA Japan. All animal procedures were performed under isoflurane inhalation anesthesia. Six-week-old C57BL / 6 mice were administered 5 μg of mRNA-encapsulated nucleic acid lipid particles per mouse into the gastrocnemius muscle twice, at two-week intervals. One week after the final administration, spleens were harvested and splenocytes were prepared. Spleen cells were treated with HPV6E6 pool peptide (JPT, catalog # PM-HPV06-E6) or HPV11E6 pool peptide (JPT, catalog # PM-HPV11-E6). After 48 hours of incubation, the culture supernatant was diluted 2-fold or 30-fold, and IFN-γ levels were measured by cytokine ELISA.

[0132] [Test Example 2] Cytokine Production Ability of HPV Genotype 11E6E7 Vaccine Antigen-Specific T Cells (Figure 2) Six-week-old C57BL / 6 mice were injected twice into the gastrocnemius muscle with 5 μg of mRNA-encapsulated nucleic acid lipid particles per mouse, at two-week intervals. One week after the final injection, the spleen was harvested and splenocytes were prepared. Spleen cells were treated with HPV6E6 pool peptide (JPT, catalog # PM-HPV06-E6) or HPV11E6 pool peptide (JPT, catalog # PM-HPV11-E6). After 48 hours of incubation, the culture supernatant was diluted 2-fold or 30-fold, and IFN-γ levels were measured by cytokine ELISA. When the IFN-γ levels using the 30-fold diluted culture supernatant exceeded the upper limit of detection of the calibration curve, the upper limit was multiplied by the 30-fold dilution factor to calculate the data.

[0133] [Test Example 3] Cytokine Production of HPV Genotype 6E6E7-Genotype 11E6E7 Vaccine Antigen-Specific T Cells (Figure 3) Six-week-old C57BL / 6 mice were administered 5 μg of mRNA-encapsulated nucleic acid lipid particles per mouse into the gastrocnemius muscle twice, at two-week intervals. One week after the final administration, spleens were harvested and splenocytes were prepared. Spleen cells were treated with HPV6E6 pool peptide (JPT, catalog # PM-HPV06-E6) or HPV11E6 pool peptide (JPT, catalog # PM-HPV11-E6). After 48 hours of incubation, the culture supernatant was diluted 2-fold or 30-fold, and IFN-γ levels were measured by cytokine ELISA. When the IFN-γ levels using the 30-fold diluted culture supernatant exceeded the upper limit of detection of the calibration curve, the upper limit was multiplied by the 30-fold dilution factor to calculate the data.

[0134] [Results of Test Examples 1 to 3]

[0135] (Results of Test Example 1) HPV6E6- and HPV11E6-specific IFN-γ induction levels in mRNA-encapsulated nucleic acid lipid particles of Examples 4, 7, and 10 with different lipid composition ratios C57BL / 6 mice were intramuscularly administered with the mRNA-encapsulated nucleic acid lipid particles of Examples 4, 7, and 10, and one week after the final immunization, the HPV6E6- and HPV11E6-specific T cell cytokine induction levels from spleen cells were examined. The results are shown in Figure 1. Compared to the NC group, IFN-γ production was enhanced by HPV6E6 pool peptide treatment in all mRNA-encapsulated nucleic acid lipid particle administration groups. IFN-γ production was also induced by HPV11E6 pool peptide treatment.

[0136] (Results of Test Example 2) HPV6E6- and HPV11E6-specific IFN-γ induction levels in mRNA-encapsulated nucleic acid lipid particles of Examples 5, 8, and 11 with different lipid composition ratios C57BL / 6 mice were intramuscularly administered with the mRNA-encapsulated nucleic acid lipid particles of Examples 5, 8, and 11, and the HPV6E6- and HPV11E6-specific T cell cytokine induction levels from spleen cells were examined one week after the final immunization. The results are shown in Figure 2. Compared to the NC group, IFN-γ production was enhanced by HPV11E6 pool peptide treatment in all mRNA-encapsulated nucleic acid lipid particle administration groups. IFN-γ production was also induced by HPV6E6 pool peptide treatment.

[0137] (Results of Test Example 3) HPV6E6- and HPV11E6-specific IFN-γ induction levels in mRNA-encapsulated nucleic acid lipid particles of Examples 6, 9, and 12 with different lipid composition ratios C57BL / 6 mice were intramuscularly administered the mRNA-encapsulated nucleic acid lipid particles of Examples 6, 9, and 12, and one week after the final immunization, the HPV6E6- and HPV11E6-specific T cell cytokine induction levels from spleen cells were examined. The results are shown in Figure 3. Compared to the NC group, IFN-γ production was induced by treatment with HPV6E6 pool peptide and HPV11E6 pool peptide in both mRNA-encapsulated nucleic acid lipid particle administration groups, indicating that the HPV genotype 6E6E7-genotype 11E6E7 fusion vaccine induced a cellular immune response equivalent to the HPV6E6-specific cellular immune response obtained when the HPV genotype 6E6E7 vaccine was administered alone, and the HPV11E6-specific cellular immune response obtained when the HPV genotype 11E6E7 vaccine was administered alone.

[0138] The present invention can be used to prevent and / or treat infection with human papillomavirus types 6 and / or 11.

[0139] SEQ ID NO: 1: Template plasmid DNA for IVT of HPV6 E6-E7 fusion SEQ ID NO: 2: Sense primer SEQ ID NO: 3: Antisense primer SEQ ID NO: 4: HPV6 E6-E7 fusion template DNA SEQ ID NO: 5: HPV6 E6-E7 fusion mRNA SEQ ID NO: 6: Template plasmid DNA for IVT of HPV11 E6-E7 fusion SEQ ID NO: 7: HPV11 E6-E7 fusion template DNA SEQ ID NO: 8: HPV11 E6-E7 fusion mRNA SEQ ID NO: 9: HPV6 E6-E7 HPV11 E6-E7 fusion template plasmid DNA SEQ ID NO: 10: HPV6 E6-E7 HPV11 E6-E7 fusion template DNA SEQ ID NO: 11: HPV6 E6-E7 HPV11 E6-E7 fusion mRNA SEQ ID NO: 12: Amino acid sequence of HPV6 E6 antigen SEQ ID NO: 13: Amino acid sequence of HPV6 E7 antigen SEQ ID NO: 14: Amino acid sequence of HPV11 E6 antigen SEQ ID NO: 15: Amino acid sequence of HPV11 E7 antigen SEQ ID NO: 16: Amino acid sequence of protease cleavage sequence SEQ ID NO: 17: Amino acid sequence of HPV6 E6 antigen and E7 antigen fusion protein SEQ ID NO: 18: Amino acid sequence of HPV11 E6 antigen and E7 antigen fusion protein SEQ ID NO: 19: Amino acid sequences of HPV6 E6 antigen and E7 antigen and HPV11 E6 antigen and E7 antigen fusion protein SEQ ID NO: 20: IgE leader sequence

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 each independently represents a C1-C3 alkyl group; L 1 represents a C17-C19 alkenyl group which may have one or more C2-C4 alkanoyloxy groups; L 2 represents a C10-C19 alkyl group which may have one or more C2-C4 alkanoyloxy groups, or a C10-C19 alkenyl group which may have one or more C2-C4 alkanoyloxy groups; p is 3 or 4.

2. R in general formula (Ia) 1 and R 2 and are both 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 3. The particle according to claim 1 or 2, wherein is a C17-C19 alkenyl group optionally having one or more acetoxy groups.

5. L in formula (Ia) 2 3. The particle according to claim 1, wherein is a C10-C12 alkyl group optionally having one or more acetoxy groups, or a C10-C19 alkenyl group optionally having one or more acetoxy groups.

6. L in formula (Ia) 2 3. The particle according to claim 1 or 2, wherein is a C10-C12 alkyl group optionally having one or more acetoxy groups, or a C17-C19 alkenyl group optionally having one or more acetoxy groups.

7. L in formula (Ia) 1 3. The particle according to claim 1 or 2, 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 claim 1 or 2, 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】 or 【Chemistry 3】 The particle according to claim 1, represented by the formula:

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

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

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

13. 12. The particle according to claim 11, wherein the amphiphilic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine.

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. 12. The particle according to claim 11, wherein the sterol is cholesterol.

16. 13. The particle according to claim 12, wherein the sterol is cholesterol.

17. 12. A particle according to any of claims 11, wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxypoly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine.

18. 13. A particle according to any of claims 12, wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxypoly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine.

19. The particle according to claim 11, wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 5 to 25% amphipathic lipid, 10 to 55% sterols, 40 to 65% cationic lipid, and 1 to 5% PEG lipid.

20. A particle as described in claim 13, having a lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids, in molar amounts, of 5 to 25% amphipathic lipids, 10 to 55% sterols, 40 to 65% cationic lipids, and 1 to 5% PEG lipids.

21. 21. The particle according to claim 19 or 20, wherein the amphiphilic lipid is 10 to 25%.

22. The particle according to claim 11, 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.

23. 23. The particle according to claim 22, comprising 10-15% amphiphilic lipid, 35-45% sterols, 40-50% cationic lipid, and 1-2.5% PEG lipid.

24. 24. The particle of claim 23, wherein the PEG lipid is 1-2%.

25. 13. The particle according to claim 12, wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 10 to 25% amphipathic lipid, 10 to 50% sterols, 40 to 65% cationic lipid, and 1 to 3% PEG lipid.

26. 26. The particle according to claim 25, comprising 10 to 45% sterols, 42.5 to 65% cationic lipid, and 1 to 2.5% PEG lipid.

27. 27. The particle of claim 26, wherein the PEG lipid is 1-2%.

28. 26. A particle according to any one of claims 19, 20, 22 and 25, wherein the ratio of total lipid weight to nucleic acid weight is 15 to 30.

29. 29. The particle according to claim 28, wherein the ratio of total lipid weight to nucleic acid weight is 15 to 25.

30. 30. The particle according to claim 29, wherein the ratio of total lipid weight to nucleic acid weight is 17.5 to 22.

5.

31. 3. The particle according to claim 1 or 2, wherein the human papillomavirus is HPV6.

32. The particle described in claim 31, wherein the human papillomavirus is HPV6 and the E6 antigen of HPV6 consists of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

12.

33. The particle described in claim 31, wherein the human papillomavirus is HPV6 and the E7 antigen of HPV6 consists of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

13.

34. The particle described in claim 31, wherein the human papillomavirus is HPV6 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 HPV6 type, the fusion protein consisting of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

17.

35. The particle described in claim 31, wherein the human papillomavirus is HPV6 type, and the nucleic acid capable of expressing HPV6 type E6 antigen and E7 antigen is an mRNA including 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 polyA tail (polyA).

36. 36. The particle according to claim 35, wherein the nucleic acid sequence capable of expressing HPV type 6 E6 and E7 antigens consists of a nucleotide sequence having at least 90% identity with the sequence of SEQ ID NO:

5.

37. The particle described in claim 31, wherein the human papillomavirus is HPV6 type, and the nucleic acid capable of expressing HPV6 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, and a 3' untranslated region (3'-UTR).

38. The particle described in claim 37, wherein the composition consisting of the cap structure (Cap), 5' untranslated region (5'-UTR), leader sequence, E6 translation region, protease cleavage sequence (Furin Cleavage Site), E7 translation region and 3' untranslated region (3'-UTR) is composed of a nucleotide sequence having at least 90% identity to the sequence from position 1 to position 1018 of SEQ ID NO:

5.

39. 3. The particle according to claim 1 or 2, wherein the human papillomavirus is HPV type 11.

40. The particle described in claim 39, wherein the human papillomavirus is HPV type 11 and the E6 antigen of HPV type 11 consists of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

14.

41. The particle described in claim 39, wherein the human papillomavirus is HPV type 11 and the E7 antigen of HPV type 11 consists of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

15.

42. The particle described in claim 39, wherein the human papillomavirus is HPV type 11, 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 HPV type 11, the fusion protein consisting of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

18.

43. The particle described in claim 39, wherein the human papillomavirus is HPV type 11, and the nucleic acid capable of expressing HPV type 11 E6 and E7 antigens is an mRNA including 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 polyA tail (polyA).

44. The particle described in claim 43, wherein the nucleic acid sequence capable of expressing HPV type 11 E6 and E7 antigens consists of a nucleotide sequence having at least 90% identity with the sequence of SEQ ID NO:

8.

45. The particle described in claim 39, wherein the human papillomavirus is HPV type 11, and the nucleic acid capable of expressing HPV type 11 E6 and E7 antigens 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 and a 3' untranslated region (3'-UTR).

46. The particle described in claim 45, wherein the composition consisting of the cap structure (Cap), 5' untranslated region (5'-UTR), leader sequence, E6 translation region, protease cleavage sequence (Furin Cleavage Site), E7 translation region and 3' untranslated region (3'-UTR) consists of a nucleotide sequence having at least 90% identity to the sequence from position 1 to position 1018 of SEQ ID NO:

8.

47. 3. The particle according to claim 1 or 2, wherein the human papillomavirus is HPV type 6 or 11.

48. The particle described in claim 47, wherein the human papillomavirus is HPV type 6 or 11, 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 HPV type 6 and the E6 antigen and E7 antigen of HPV type 11, the fusion protein consisting of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

19.

49. The particle described in claim 47, wherein the human papillomavirus is HPV types 6 and 11, and the nucleic acid capable of expressing the E6 and E7 antigens of HPV6 and the E6 and E7 antigens of HPV11 is an mRNA including a cap structure (Cap), a 5' untranslated region (5'-UTR), a leader sequence, the translation region of E6 of HPV6, the translation region of E7 of HPV6, a protease cleavage sequence (Furin Cleavage Site), the translation region of E6 of HPV11, the translation region of E7 of HPV11, a 3' untranslated region (3'-UTR) and a polyA tail (polyA).

50. The particle described in claim 49, wherein the nucleic acid sequence capable of expressing HPV type 6 E6 antigens and E7 antigens and HPV type 11 E6 antigens and E7 antigens consists of a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO:

11.

51. The particle described in claim 47, wherein the human papillomavirus is HPV types 6 and 11, and the nucleic acid capable of expressing the E6 and E7 antigens of HPV6 and the E6 and E7 antigens of HPV11 is an mRNA comprising a cap structure (Cap), a 5' untranslated region (5'-UTR), a leader sequence, the translation region of E6 of HPV6, the translation region of E7 of HPV6, a protease cleavage sequence (Furin Cleavage Site), the translation region of E6 of HPV11, the translation region of E7 of HPV11, and the 3' untranslated region (3'-UTR).

52. The particle described in claim 51, wherein the composition consisting of the cap structure (Cap), 5' untranslated region (5'-UTR), leader sequence, translation region of E6 of HPV6 type, translation region of E7 of HPV6 type, protease cleavage sequence (Furin Cleavage Site), translation region of E6 of HPV11 type, translation region of E7 of HPV11 type and 3' untranslated region (3'-UTR) is composed of a nucleotide sequence having at least 90% identity to the sequence from the 1st to the 1798th positions of SEQ ID NO:

11.

53. 3. The particle of claim 1 or 2, wherein the nucleic acid comprises at least one modified nucleotide.

54. 54. The particle of claim 53, 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.

55. 54. The particle of claim 53, wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine.

56. 54. The particle of claim 53, wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methyluridine, and 1-methylpseudouridine.

57. 3. The particles according to claim 1 or 2, having an average particle size of 30 to 300 nm.

58. 3. Use of particles according to claim 1 or 2 for the manufacture of a composition for the prevention and / or treatment of infection with human papillomavirus or diseases due to infection with human papillomavirus.

59. Use of the particles according to claim 58, wherein the disease caused by infection with human papillomavirus is recurrent respiratory papillomatosis or genital warts.

60. 59. The use of particles according to claim 58, wherein the infection is an infection with human papillomavirus type HPV6 or type HPV11.

61. A composition comprising the particles according to claim 1 or 2.

62. The composition of claim 61 for expressing human papillomavirus E6 and E7 antigens in vivo or in vitro.

63. 62. The composition of claim 61 for use as a medicine.

64. The composition of claim 63 for inducing an immune response against human papillomavirus.

65. 64. The composition according to claim 63 for preventing and / or treating human papillomavirus infection or a disease caused by human papillomavirus infection.

66. The composition according to claim 65, wherein the disease caused by human papillomavirus infection is recurrent respiratory papillomatosis or genital warts.

67. The composition of claim 65, wherein the infection is infection with human papillomavirus type HPV6 or HPV11.

68. A method for expressing human papillomavirus E6 and E7 antigens in vitro, comprising introducing the composition of claim 61 into a cell.

69. A method for expressing human papillomavirus E6 and E7 antigens in vivo, comprising administering the composition of claim 61 to a non-human mammal.

70. 64. A method of inducing an immune response against human papillomavirus comprising administering to a non-human mammal the composition of claim 63.

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