RNA strand with secondary structure having function of carrying genome into murine norovirus particles, artificial murine norovirus particles containing the RNA strand, and method for producing the same

Abstract

To provide safer means of carrying RNA to a target in the body.SOLUTION: We have found that the above problems can be solved by using, as means for carrying foreign RNA to a target in the body, artificial murine norovirus (MNV) obtained by introducing foreign RNA into MNV by using an RNA strand having a connection end with foreign RNA at both or one end, the artificial MNV being a portion having a secondary structure of RNA having packaging ability in MNV included in genomic RNA (+)4901-5200nt of MNV, or a portion having the secondary structure introduced with a mutation to the extent that it retains the packaging ability in MNV.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to an RNA strand with a secondary structure such as a stem-loop, which can be used to produce artificial murine norovirus particles containing foreign RNA molecules. [Background technology]

[0002] In recent years, various RNA-based medical technologies, such as vaccine technology and gene therapy technology, have been rapidly advancing. These medical technologies require a means to deliver the desired RNA molecules intact to the target site in the body, and currently, lipid nanoparticles (LNPs) fulfill this role. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2023-166489 [Patent Document 2] Japanese Patent Application Publication No. 2023-144134 DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0004] As mentioned above, LNPs are widely used as a means of delivering RNA, but their high inflammatory potential is a problem. In particular, polyethylene glycol (PEG) present on the surface of LNPs is believed to be one of the causes of anaphylaxis.

[0005] An object of the present invention is to provide a highly safe means for delivering RNA to a target in the body, which can overcome the current situation. [Means for solving the problem]

[0006] To solve the above problems, the present inventors focused on the replication mechanism of noroviruses. Because it is currently difficult to grow and culture human noroviruses using established cell lines, they investigated the function of the stem-loop structure in the genome of murine norovirus (hereinafter also referred to as MNV).

[0007] Norovirus is known to have a life cycle as shown in Figure 1. When norovirus 11 infects a host cell 10, its gene (positive RNA strand) is expressed as polyprotein 12 within the host cell. Non-structural proteins 13 expressed therein act to synthesize a negative RNA strand using the positive RNA strand as a template, followed by synthesis of a positive strand using the negative strand as a template (positive strand 14). This results in the production of a double-stranded RNA genome, nascent genomic RNA 15, within the infected host cell 10. The nascent genomic RNA 15 is composed, from the 5' end, of a 5' untranslation site (UTR), ORF1, ORF2, ORF3, a 3' UTR, and poly(A). Meanwhile, subgenomic RNA 16, composed of ORF2 and ORF3, is synthesized in large quantities within the infected host cell 10 in parallel with the production of the nascent genomic RNA 15. Using the accessory genomic RNA 16 as a template, norovirus structural proteins (VP1, VP2) 17 are expressed within the host-infected cells 10, and the nascent genomic RNA 15 and structural proteins 17 assemble (assembly 18) to produce "nascent norovirus particles 19" within the host-infected cells 10, which contain the nascent genomic RNA within virus particles made of the structural proteins, and these particles are then released outside the host-infected cells 10.

[0008] One of the distinctive features of the norovirus life cycle is that nascent norovirus particles contain the nascent genomic RNA but do not contain the accessory genomic RNA, which is synthesized in large quantities. The starting point for the invention of this invention was to address the question of how the nascent genomic RNA and accessory genomic RNA are distinguished and only the nascent genomic RNA is packaged.

[0009] The 5'-terminal nucleotide sequences of the MNV nascent genomic RNA and accessory genomic RNA are almost identical [nascent genomic RNA GUGAAAUGAGGAUG (SEQ ID NO: 9), accessory genomic RNA GUGAAUGAGGAUG (SEQ ID NO: 10)]. Nevertheless, as mentioned above, the two are clearly distinguished during packaging. Therefore, the possibility that this 5'-terminal nucleotide sequence is the MNV packaging signal is ruled out. If it were a packaging signal, both the nascent genomic RNA and the accessory genomic RNA should be contained in nascent MNV particles. Furthermore, since no particles have been found in which the accessory genomic RNA is packaged, it is clear that a packaging signal is not present within the accessory genomic RNA sequence.

[0010] Therefore, the inventors hypothesized that there exists a "means that acts as a signal for packaging the nascent MNV genomic RNA into viral particles" in ORF1, which is present in the nascent MNV genomic RNA but not in the accessory genomic RNA, and began their investigations.

[0011] As a result, the present inventors found that an RNA strand with a secondary structure falling within the range of MNV genomic RNA (+)4928-5004 nt functions as a means for packaging the nascent MNV genomic RNA strand into viral particles, particularly due to its stem-loop structure. Utilizing this property, the present inventors discovered that by providing an RNA strand that satisfies certain conditions and falls within the range of MNV genomic RNA (+)4901-5200 nt and has a linking means for foreign RNA at either or both ends, artificial MNV particles in which foreign RNA is encapsulated within MNV viral particles can be obtained, thereby completing the present invention.

[0012] The contents of the present invention are as follows.

[0013] First, an RNA strand (hereinafter also referred to as the RNA strand of the present invention) is provided, which is a portion of the genomic RNA (+) 4901-5200 nt, preferably 4901-5090 nt, and more preferably 4928-5090 nt, of murine norovirus (MNV) RNA (+) that has a secondary structure of RNA with packaging ability in MNV, or a portion of the secondary structure into which a mutation has been introduced to the extent that packaging ability in MNV is maintained, and which has a linkage end to a foreign RNA at both or one end of the portion; Second, the present invention provides an RNA strand of the present invention, wherein a foreign RNA is linked to one or both of its termini; Third, artificial murine norovirus particles (hereinafter also referred to as artificial MNV particles of the present invention) are provided, which contain within the MNV particles an RNA strand having a secondary structure of an RNA contained in MNV genomic RNA (+) 4901-5200 nt, preferably 4901-5090 nt, more preferably 4928-5090 nt, or an RNA strand having such a secondary structure into which a mutation has been introduced to the extent that packaging ability in MNV is maintained, and which has ends at either or both ends to which a foreign RNA is linked, and at either or both ends to which the foreign RNA is linked; Fourth, a method for producing artificial MNV particles (hereinafter also referred to as the first production method of the present invention) is provided, which comprises coexisting an RNA strand having a secondary structure of an RNA contained in MNV genomic RNA (+) 4901-5200 nt, preferably 4901-5090 nt, and more preferably 4928-5090 nt, or an RNA strand having such secondary structure mutated to the extent that it retains packaging ability in MNV, and which has a linking end to a foreign RNA at both or one of its ends, and an RNA strand encoding an MNV structural protein, in an MNV receptor-expressing cell, and producing artificial MNV particles in the cell containing an RNA strand linked to the foreign RNA within MNV particles formed by structural proteins expressed in the cell from the RNA strand encoding the MNV structural protein; Fifth, we provide a method for producing artificial MNV particles (hereinafter also referred to as the second production method of the present invention), which involves associating an RNA strand having a secondary structure of RNA contained in MNV genomic RNA (+) 4901-5200 nt, preferably 4901-5090 nt, and more preferably 4928-5090 nt, or an RNA strand having such secondary structure with a mutation introduced to the extent that it retains packaging ability in MNV, and which has a linking end to foreign RNA at both or one of its ends, and to which foreign RNA is linked at both or one of the linking ends, with MNV structural proteins in an aqueous solvent, and generating artificial MNV particles containing the RNA strand linked to the foreign RNA in the aqueous solvent (hereinafter also referred to as the second production method of the present invention).

[0014] An "RNA strand with secondary structure" is not simply a nucleic acid (RNA) sequence that encodes amino acids, but rather a planar or three-dimensional structure formed in a biological environment by the interaction of nucleic acid molecules in that sequence as a substance. In the present invention, "portions of MNV genomic RNA (+) 4901-5200 nt, 4901-5090 nt, and 4928-5090 nt that have the secondary structure of RNA with packaging ability in MNV" include the RNA region shown in SEQ ID NO: 7. Furthermore, "portions with the above secondary structure into which mutations have been introduced to the extent that packaging ability in MNV is maintained" include the RNA regions shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 8. These will be described below.

[0015] The "linking end to the foreign RNA" can be, for example, a restriction enzyme recognition site or the like as a linking sequence, but any sequence may be added. Alternatively, the foreign RNA can be directly linked without modifying the linking end.

[0016] The production of the MNV particles of the present invention by packaging in a form that maintains the secondary structure of the RNA strand is efficient when carried out in MNV receptor-expressing cells, as specified in the first production method of the present invention. The MNV receptor-expressing cells can be an animal cell line into which a known MNV receptor gene such as CD300lf has been introduced, and one example is the "Huh7.5.1CD300lfC12-2 cells" used in "(3) Infection Test 2" of Verification Test 2, which will be described later.

[0017] Furthermore, as defined in the second production method of the present invention, packaging of the RNA strand in a form that maintains its secondary structure for producing the MNV particles of the present invention can be performed in an aqueous solvent (in vitro). In this case, examples of MNV structural proteins that are associated with the "RNA strand of the present invention to which a foreign RNA has been added" in the aqueous solvent include a set of murine norovirus structural proteins such as recombinant VP1, VP2, and VPg, or disassembled virus-like particles (VLPs). [Effects of the Invention]

[0018] The present invention provides an RNA strand for packaging foreign RNA, which can be used to produce artificial murine norovirus (MNV) particles containing the foreign RNA by packaging the foreign RNA into the MNV particles, the artificial MNV particles, and a method for producing the artificial MNV particles. [Brief explanation of the drawings]

[0019] [Figure 1] 1 is a diagram showing the life cycle of norovirus. [Figure 2] 1 is a diagram showing the predicted secondary structure of the target region (wild type) of the MNV genomic RNA (+) strand to accomplish the present invention. [Figure 3] FIG. 1 is an alignment diagram showing the specific details of synonymous substitution mutations used in verifying the present invention. [Figure 4] 1 is a diagram showing the secondary structure of the target region predicted from the synonymous substitution mutations used to verify the present invention. [Figure 5] 1 is a diagram showing an outline of the method for introducing MNV genomic RNA with synonymous substitution mutations into a cultured cell line and the verification test method after the introduction. [Figure 6] This figure shows the results of an immunofluorescence staining test in which the presence or absence of viral proteins was examined in each cultured cell line after the genomic RNA of MNV with the synonymous substitution mutations shown in Figure 5 was introduced into the cultured cell line. [Figure 7] This figure shows the results of examining whether or not cytopathic effect (CPE) occurs in MNV receptor-expressing cells when the culture supernatant of a cultured cell line into which MNV genomic RNA with the synonymous substitution mutation shown in Figure 5 had been introduced is contacted with the receptor-expressing cells. DETAILED DESCRIPTION OF THE INVENTION

[0020] The embodiments of the present invention are disclosed primarily as validation tests for the RNA strands of the present invention.

[0021] [Verification test 1] Prediction of the structure of the MNV genome RNA (+) strand (1) Wild-type structure We used RNAfold (http: / / ma.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi) (hereafter referred to as the "prediction software"), a software for predicting RNA secondary structure, to predict the secondary structure near the target site in ORF1 of MNV. The nucleotide sequence of the (+) strand of MNV (wild-type) genomic RNA has been published (AB435515). We predicted the secondary structure of ORF1 of MNV and identified a region roughly ranging from 4901 to 5200 nt where several stem-loop structures were observed as the target region (MNV-S7). We predicted that the stem-loop structures observed within 4901 to 5090 nt, and more specifically, within 4928 to 5090 nt, are involved in packaging of the MNV genomic RNA, and verified this prediction.

[0022] The predicted shape of the secondary structure (secondary structure formed as a centroid form) of the above-mentioned target region (MNV-S7) 20 (wild type: WT) by prediction software is shown in Figure 2. Target region 20 is roughly composed of three stem-loop structures (stem-loops 21, 22, and 23), and the inventors predicted that stem-loop 21 is deeply involved in packaging of MNV genomic RNA. Stem-loop 11 has three loop structures 211, 212, and 213, which form its basic structure.

[0023] (2) Structure of synonymous substitution mutants When genomic RNA is expressed as a protein based on its base sequence, it is translated using a "triplet code," i.e., three bases correspond to one amino acid. In the triplet code, even if the third base is different, the corresponding amino acid is the same. In this case, even if the translated amino acid is the same, the physical and chemical properties of the bases themselves are different. Therefore, the third base of the triplet significantly affects the secondary structure of the RNA strand, resulting in different "functions of the RNA strand itself." The present inventors predicted that wild-type stem-loop 11 plays a major role in packaging of MNV genomic RNA. Therefore, they introduced synonymous mutations into the third base of the wild-type triplet in this region without affecting the translated amino acid, and examined the effects of such synonymous mutations on MNV genomic RNA packaging. Seven mutation patterns, M1-M7 (M7 is a synonymous mutation in stem-loop 22), were designed. Figure 3 shows an alignment of the base sequences (4801-5260 nt) showing the specific mutations to be introduced. From top to bottom in Figure 3, they are M1, M2, M3, M4, M5, M6, WT (wild type), and M7. The ranges of "4901-5200 nt" and "4901-5090 nt" defined in the present invention are also shown in Figure 3.

[0024] Each mutation is specifically shown below in the format of "wild-type base sequence position mutant base." M1:G4955C,A4958U M2:C4946G,A4958U,G4967C M3:G4955C,A4958U,G4967C M4:C4946G,G4955C,A4958U,G4967C M5:U4985A,C4988G,G4991U M6:C4946A,G4967C,C4979U,U4985A,C4988G,G4991U M7:U5018C,U5021C,G5024U

[0025] Furthermore, the specific sequences of "4928-5004nt" of WT and M1-M6 are shown in the sequence listing.

[0026] Specifically, they are shown as follows: M1: SEQ ID NO: 1, M2: SEQ ID NO: 2, M3: SEQ ID NO: 3, M4: SEQ ID NO: 4, M5: SEQ ID NO: 5, M6: SEQ ID NO: 6, WT: SEQ ID NO: 7.

[0027] The specific sequence of "4928-5040nt" of M7 is shown in the sequence listing as SEQ ID NO:8.

[0028] Figure 4 shows the secondary structures of the target regions predicted from the synonymous substitution mutations (M1-M6, including WT) used in verifying the present invention. In Figure 4, WT is the same as that shown in Figure 2. The secondary structures of stem-loop 22 in target region 20 (M1) caused by M1 mutation, target region 20 (M2) caused by M2 mutation, target region 20 (M3) caused by M3 mutation, target region 20 (M4) caused by M4 mutation, target region 20 (M5) caused by M5 mutation, and target region 20 (M6) caused by M6 mutation were unchanged from those in wild-type (WT) 20, but the wild-type stem-loop 21 was predicted to have a unique secondary structure due to each synonymous substitution mutation. Specifically, stem-loop 21 is shown as M1 (M1), M2 (M2), M3 (M3), M4 (M4), M5 (M5), and M6 (M6). The M7 mutation is not expected to affect the secondary structure of stem-loop 21.

[0029] [Verification test 2] Introduction of synonymous substitution mutations (Figure 5) (1) Introduction of MNV genomic RNA into cultured cells Site-directed mutations were introduced into the DNA sequences complementary to the 5' untranslated region (UTR), ORF1, ORF2, ORF3, 3' UTR, and poly(A) of the MNV genomic RNA downstream of the T7 promoter in a pT7 plasmid (self-made). A delta virus-derived ribozyme was then inserted immediately below the poly(A) sequence at the 3' end of ORF3 of each recombinant plasmid. The plasmid was cleaved at a unique restriction enzyme site immediately following the ribozyme to generate linear double-stranded DNA. This was then treated with T7 RNA polymerase to synthesize single-stranded RNA, which was then capped at the 5' end to generate RNA constructs for wild-type and synonymous substitution mutants capable of infecting MNV. These RNA constructs were then electroporated into HEK293T cells and cultured for 72 hours.

[0030] (2) Infection test 1 We examined whether each RNA construct introduced into HEK293T cells was expressed as a viral protein (VP1:ORF2 expression product, VP2:ORF2 expression product) in the cells by immunofluorescence staining. The results are shown in Figure 6.

[0031] As shown in Figure 6, except for the negative control (NC: mock transfection of T7 plasmid), the WT and all mutated RNA constructs (M1-M6) were found to be expressed as VP1 and VP2 in HEK293T cells. Therefore, it was concluded that introducing secondary structure mutations into stem-loop 21 does not affect intracellular viral protein synthesis. Furthermore, although not shown, the M7 mutant RNA construct was expressed as VP1 and VP2, similar to the WT and M1-M6, and did not affect intracellular viral protein synthesis.

[0032] (3) Infection test 2 The 72-hour culture supernatant of a cultured cell line transfected with the genomic RNA of MNV carrying the synonymous substitution mutations described in (1) above was contacted with MNV receptor-expressing cells (Huh7.5.1CD300lfC12-2 cells) to examine whether or not cytopathic effect (CPE) occurred in the receptor-expressing cells. Observation of CPE indicates that the transfected gene was packaged as a viral particle within the infected cell, generating nascent MNV, which destroyed the infected cell upon extracellular release. The results are shown in Figure 7.

[0033] As shown in Figure 7, WT showed the strongest CPE, and M1, M2, M3, and M5 showed weaker CPE. In contrast, M4 and M6, like NC, showed no CPE.

[0034] These results demonstrated that the packaging efficiency was reduced or even completely eliminated except for the intact stem-loop 21 (WT). Therefore, stem-loop 21 is an essential structure for packaging the MNV genomic RNA. Although not shown, M7 showed CPE comparable to that of WT.

[0035] [Establishment of the present invention] As described above, it has been revealed that the secondary structure of stem-loop 21 in the wild-type MNV genomic RNA is an essential structure for packaging the genomic RNA into virus particles. It has also been revealed that even when a mutation affecting the secondary structure of stem-loop 21 is introduced, the packaging ability may or may not be affected. Furthermore, it has also been revealed that the secondary structure of stem-loop 22 does not affect the packaging ability of MNV genomic RNA.

[0036] In other words, the "RNA strand with a secondary structure consisting of wild-type MNV genomic RNA (+) 4928-5004 nt (SEQ ID NO: 7)" corresponding to the above stem-loop 21 is an essential part that controls the packaging ability of the genomic RNA in MNV, and is included in "the part having the secondary structure of RNA with packaging ability in MNV in the range of MNV genomic RNA (+) 4901-5200 nt and 4901-5090 nt defined in the present invention."

[0037] Furthermore, as is clear from the above test results, even if there is a mutation that affects the secondary structure of the stem-loop 21, in the case of the RNA regions represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 5, these RNA regions are also included in the ``portions having the secondary structure of RNA with packaging ability in MNV in the above ranges of 4901-5200 nt and 4901-5090 nt.''

[0038] Furthermore, the RNA region represented by a mutation (sequence number 8) that affects the secondary structure of stem-loop 22 but does not affect the secondary structure of stem-loop 22 is also included in the "portion having a secondary structure of RNA with packaging ability in MNV in the above range of 4901-5200 nt and range of 4901-5090 nt."

[0039] Based on these findings, it was conceived that an RNA strand having a secondary structure of RNA with packaging ability in MNV, which is contained in the range of MNV genomic RNA (+) 4901-5200 nt, preferably 4901-5090 nt, or a portion having the above secondary structure into which mutations have been introduced to the extent that packaging ability in MNV is maintained, and which has a linking end to a foreign RNA at both ends or one end, can be packaged into MNV viral particles with a foreign RNA strand linked to the linking end, thereby producing artificial MNV viral particles containing a desired foreign RNA, and the present invention was created based on this conceivable result.

[0040] The above artificial MNV particles have lost their pathogenicity and can be used in the medical field, such as in vaccines and gene therapy, as a carrier of foreign RNA, replacing the currently used LNP. [Explanation of symbols]

[0041] 10: Host cells or host-infected cells 11: Norovirus 12: Polyprotein 13: Nonstructural proteins 14: Synthesis of norovirus genomic RNA 15: Genomic RNA of emerging MNV 16: Norovirus minor genomic RNA 17: Structural protein 18: Assembly 19: Nascent norovirus particles 20: Target region of MNV genomic RNA 21, 22, 23: stem-loop structure 211, 212, 213: Loop structure 20(M1), 20(M2), 20(M3), 20(M4), 20(M5), 20(M6): Target region of MNV genomic RNA into which synonymous substitution mutations were introduced 21(M1), 21(M2), 21(M3), 21(M4), 21(M5), 21(M6): Target stem-loop structures of MNV genomic RNAs with synonymous substitution mutations

Claims

1. An RNA strand having a secondary structure of RNA with packaging ability in murine norovirus contained in the genomic RNA (+) 4901-5200 nt of murine norovirus, or a portion having the above secondary structure into which mutations have been introduced to the extent that packaging ability in murine norovirus is maintained, and having a linkage end to a foreign RNA at both or one end.

2. The RNA strand according to claim 1, wherein the RNA strand is a portion having a secondary structure of an RNA that has packaging ability in murine norovirus and is contained in the genomic RNA (+) 4901-5200 nt of murine norovirus, or a portion having the above secondary structure into which a mutation has been introduced to the extent that packaging ability in murine norovirus is maintained, and a foreign RNA is linked to one or both of the linking ends of the portion.

3. In the RNA strand, the portion having the secondary structure of RNA with packaging ability in murine norovirus contained in genomic RNA (+) 4901-5200 nt of murine norovirus includes the RNA region represented by SEQ ID NO: 7; The portion having the secondary structure into which mutations have been introduced to the extent that packaging ability in murine norovirus is maintained includes an RNA region represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 8; The RNA strand of claim 1 or 2.

4. Artificial murine norovirus particles containing an RNA strand having a secondary structure of RNA contained in the genomic RNA (+) 4901-5200 nt of murine norovirus, or an RNA strand having the secondary structure into which a mutation has been introduced to the extent that packaging ability in murine norovirus is maintained, the RNA strand having a linking end to a foreign RNA at both or one of both ends, and the foreign RNA linked to one or both of the linking ends.

5. In the artificial murine norovirus particle, the portion having the secondary structure of an RNA with packaging ability in murine norovirus contained in the genomic RNA (+) 4901-5200 nt of the murine norovirus includes an RNA region represented by SEQ ID NO: 7; The portion having the secondary structure into which mutations have been introduced to the extent that packaging ability in murine norovirus is maintained includes an RNA region represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 8; The artificial murine norovirus particle according to claim 4.

6. A method for producing artificial murine norovirus particles, comprising: allowing an RNA strand having a secondary structure of RNA contained in murine norovirus genomic RNA (+) 4901-5200 nt, or an RNA strand having such secondary structure into which a mutation has been introduced to the extent that packaging ability in murine norovirus is maintained, and which has a linking end to foreign RNA at both or one of both ends, and an RNA strand encoding a structural protein of murine norovirus, to coexist in a murine norovirus receptor-expressing cell; and producing artificial murine norovirus particles in the cell, the murine norovirus particles being made of structural proteins expressed in the cell from the RNA strand encoding the murine norovirus structural protein, and containing the RNA strand linked to the foreign RNA.

7. A method for producing artificial murine norovirus particles, comprising: associating an RNA strand having a secondary structure of RNA contained in murine norovirus genomic RNA (+) 4901-5200 nt, or an RNA strand having such secondary structure into which a mutation has been introduced to the extent that packaging ability in murine norovirus is maintained, with murine norovirus structural proteins in an aqueous solvent; and producing artificial murine norovirus particles containing the RNA strand linked to the foreign RNA in the aqueous solvent.

8. In the production method, the portion having the secondary structure of an RNA having packaging ability in murine norovirus contained in genomic RNA (+) 4901-5200 nt of murine norovirus comprises an RNA region represented by SEQ ID NO: 7; The portion having the secondary structure into which mutations have been introduced to the extent that packaging ability in murine norovirus is maintained includes an RNA region represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 8; A method for producing the artificial murine norovirus particles according to claim 6 or 7.

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