Nucleic acid for use in treatment of severe fever with thrombocytopenia syndrome virus, and conjugate of targeting molecule and said nucleic acid

Abstract

The present disclosure relates to a nucleic acid for use in a knockdown technique against severe fever with thrombocytopenia syndrome virus (SFTSV), and a conjugate of the nucleic acid and an antibody. The nucleic acid is capable of knocking down the RNA genome of SFTSV in cells. The nucleic acid can exhibit an antiviral effect against SFTSV.

Description

Nucleic acid for use in treating severe fever with thrombocytopenia syndrome virus, and conjugates of said nucleic acid with targeting molecules

[0001] The present disclosure relates to nucleic acids for use in the treatment of severe fever with thrombocytopenia syndrome virus, and conjugates of said nucleic acids with targeting molecules.

[0002] Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus that causes severe fever with thrombocytopenia syndrome (SFTS). This virus is a tripartite, negative-sense, single-stranded RNA virus belonging to the Banyangvirus genus in the Phenuiviridae family. Its particle diameter is approximately 100 nm, and it expresses glycoproteins on its surface, which are involved in infection. Other viruses in the Phenuiviridae family include Rift Valley fever virus (RVFV) and Heartland virus (HRTV). The three segments of this virus—the S segment, the M segment, and the L segment—each encode a different gene. The S segment encodes nucleoproteins and nonstructural proteins, the M segment encodes glycoproteins, and the L segment encodes RNA-dependent RNA polymerase. In recent years, cases of human infection through bites or direct contact with SFTSV-infected companion animals have been reported. Cases of human-to-human transmission via blood or bodily fluids have also been reported. The incubation period for SFTS is said to be 6 to 14 days. Symptoms of SFTS include fever, headache, general fatigue, gastrointestinal symptoms such as diarrhea and vomiting, and impaired consciousness. Blood tests in SFTS patients have confirmed thrombocytopenia and leukopenia. SFTS patients may experience multiple organ failure, with a mortality rate of approximately 10 to 30%, making SFTS known as an infectious disease with a poor prognosis. In the final stage of fatal SFTSV infection, SFTSV targets B cells that differentiate into plasmablasts and macrophages in secondary lymphoid organs, and the majority of SFTSV-infected cells are B-lineage lymphocytes (Non-Patent Document 1).

[0003] Suzuki et al., J Clin Invest. (2020) 3;130(2):799-812

[0004] The present disclosure provides nucleic acids for use in the treatment of severe fever with thrombocytopenia syndrome virus, and conjugates of the nucleic acids with targeting molecules.

[0005] According to the present disclosure, in an in vitro expression system of SFTSV genomic RNA segments, any knockdown nucleic acid designed to target any segment effectively knocked down the targeted segment. However, in SFTSV infection experiments, knockdown nucleic acids designed to target various genomic regions were successfully confirmed to have antiviral effects. It was also revealed that there are particularly preferred target regions for exerting antiviral effects. The present disclosure includes inventions based on such findings.

[0006] According to the present disclosure, for example, the following inventions are provided: (1) A knockdown nucleic acid that targets the S segment of the minus-strand single-stranded RNA genome of SFTSV or a plus strand having a sequence complementary to said region. {The nucleic acid exerts an antiviral effect against SFTSV (particularly intracellular SFTSV after infecting a cell).} (2) The nucleic acid according to (1) above, which targets the 91-838 region of the S segment of the RNA genome or a plus strand having a sequence complementary to said region. (3) The nucleic acid according to (1) or (2) above, which targets the 150-300 region of the S segment of the RNA genome or a plus strand having a sequence complementary to said region; the 450-650 region of the S segment of the RNA genome or a plus strand having a sequence complementary to said region; or the 780-830 region of the S segment of the RNA genome or a plus strand having a sequence complementary to said region. (4) The nucleic acid according to any one of (1) to (3) above, which is a small interfering RNA (siRNA). (5) The nucleic acid according to any one of (1) to (4) above, which is capable of reducing the SFTSV genome level to 50% or less (preferably 40% or less) 72 hours after SFTSV infection of Vero9013 cells in an in vitro culture system of Vero9013 cells, compared to a negative control. (6) The nucleic acid according to any one of (1) to (4) above, which is capable of reducing the SFTSV genome level to 30% or less (preferably 20% or less) 72 hours after SFTSV infection of Vero9013 cells in an in vitro culture system of Vero9013 cells, compared to a negative control. (7) The nucleic acid according to any one of (1) to (4) above, which is capable of reducing the SFTSV genome level to 10% or less 72 hours after SFTSV infection of Vero9013 cells in an in vitro culture system of Vero9013 cells, compared to a negative control. (15) A conjugate of a targeting means (particularly a targeting molecule, i.e., a target-binding molecule, for example, an antibody or an antigen-binding fragment thereof) and a knockdown-type nucleic acid, wherein the targeting means and the knockdown-type nucleic acid are linked via a linker, and the knockdown-type nucleic acid targets the RNA genome of SFTSV. {The conjugate exerts an antiviral effect against intracellular SFTSV after infecting a cell.}(16) The conjugate according to (15) above, wherein the knockdown-type nucleic acid targets the S segment of the RNA genome. (17) The conjugate according to (15) or (16) above, wherein the knockdown-type nucleic acid targets a region of 91 to 838 of the S segment of the RNA genome. (18) The conjugate according to any of (15) to (17) above, wherein the knockdown-type nucleic acid targets: a region of 200 to 240 of the S segment of the RNA genome or a plus strand having a sequence complementary to said region; a region of 400 to 750 of the S segment of the RNA genome or a plus strand having a sequence complementary to said region; or a region of 780 to 820 of the S segment of the RNA genome or a plus strand having a sequence complementary to said region. (19) The conjugate according to any of (15) to (18) above, wherein the knockdown-type nucleic acid is siRNA.

[0007] (21) The nucleic acid according to any one of the above, wherein the nucleic acid comprises a modified nucleic acid. (22) The conjugate according to any one of the above, wherein the nucleic acid comprises a modified nucleic acid. (23) A conjugate according to any one of the above, wherein the sugar moiety is modified with 2'-O-methyl (2'-OMe), 2'-methoxyethyl (2'-MOE), 2'-fluoro (2'-F), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), or T-O-dimethylaminoethyloxye. methylacetamide (2'-O-DMAEOE) modification or 2'-O-N-methylacetamide (2'-O-NMA) modification, replacement of the sugar moiety with a morpholino, replacement of the phosphodiester bond forming the internucleotide bond with a phosphorothioate bond, replacement of the phosphodiester bond with a phosphorodithioate bond, replacement of the phosphodiester bond with a phosphoramidate bond, linkage of the oxygen atom at the 2' position to the carbon atom at the 4' position, for example, LNA, ENA, BNA COC , BNA NC(24) The nucleic acid according to (21) above, which has a modification or modified base selected from the group consisting of 2'-OMe modification, 2'-MOE modification, 2'-F modification of the sugar moiety, replacement of the sugar moiety with a morpholino, replacement of the internucleotide phosphodiester bond with a phosphorothioate bond, nucleic acids having a modification linking the oxygen atom at the 2' position and the carbon atom at the 4' position (e.g., LNA, ENA, BNA, cMOE, cEt, AmNA, GluNA, scpBNA), and peptide nucleic acid (PNA). (25) The nucleic acid according to (21) or (23) above, which exhibits higher knockdown efficiency for target messenger RNA (mRNA) compared to a nucleic acid having the same sequence but without modified bases. (26) The conjugate according to (22) or (24) above, which exhibits higher knockdown efficiency for target mRNA compared to a conjugate having the same sequence in which the nucleic acid does not contain modified bases. (49) A composition comprising any of the conjugates above. (50) A pharmaceutical composition comprising any of the conjugates above.

[0008] The results of evaluating the susceptibility of each cultured cell to viral infection are shown below. The cells were seeded at 5 × 10 adherent cells (Huh7.5.1-8 cells, Vero cell line). 4 cells / well, 2 x 10 suspension cells (K562 cells) 5Cells were seeded at 1000 cells / well in a 48-well plate and cultured at 37°C for 24 hours. Viral infection was performed with the SFTSV-SPL010 strain at moi = 1 (adherent cells) or 0.25 (suspension cells) for 2 hours at 37°C. 24 hours after infection, total RNA was collected from the cells and culture supernatant, and SFTSV genomic RNA was quantified by reverse transcription-quantitative PCR. The vertical axis of panel (a) represents the number of SFTSV genomic RNA copies per mL of culture supernatant. The vertical axis of panel (b) represents the number of SFTSV genomic RNA copies per μg of total RNA. Each cell line was run in duplicate. The in silico selection process for siRNA candidate sequences is shown. (a) shows the virus strains used in the study. (b) shows the specific in silico selection process for siRNA candidate sequences. Using the SFTSV-SPL010 genomic RNA sequence (S segment: AB817999, M segment: AB817991, L segment: AB817983) as a template, 50 siRNAs with conserved sequences among SFTSV strains at siRNA target loci with no homology to humans were selected as seed regions. Using the psiCHECK2 system, the knockdown effect of genomic RNA by siRNAs targeting the S, M, and L segments is shown. siRNAs were added at 5 pmol / well. Ibid. Ibid. Concentration-dependent knockdown activity at 0.05, 0.5, and 5 pmol / well is shown for siRNAs that demonstrated knockdown activity at 5 pmol / well using the psiCHECK2 plasmid assay. A similar experiment was performed using siRNA against mouse myostatin (siMSTN; J. Controlled Release 237, 1-13, 2016), whose effectiveness has been confirmed in animals, as a positive control, confirming equivalent knockdown activity. The antiviral effects of candidate siRNAs in the SFTSV infection system are shown. (a) shows the effect of siRNA addition on the SFTSV genome copy number in K562 cells and Vero9013 cells. (b) shows the results of evaluating the antiviral activity of each candidate siRNA. The antiviral effects of additionally designed siRNAs against SFTSV in the SFTSV infection system are shown.This figure shows the antiviral effect of additionally designed siRNAs against SFTSV in an SFTSV infection system. This figure shows the antiviral effect of additionally designed siRNAs against SFTSV in an SFTSV infection system. This figure shows that siRNA (25 nM) targeting a region including 91-838 of the S segment exerts an siRNA antiviral effect against SFTSV. This figure shows the concentration-dependent knockdown effect of siSFTSV-Sr#2 and #3. This figure shows the inhibitory effect of siSFTSV-Sr#2 and #3 (25 nM) on the copy number of SFTSV genomic RNA. This figure shows the chemical modification patterns (patterns A to F) of siRNAs used in the examples. This figure shows the knockdown activity of siRNA (#2) with various chemical modification patterns. This figure shows the knockdown activity of siRNA (#3) with various chemical modification patterns. This figure shows the antiviral effect of siRNA (25 nM) with various chemical modification patterns. This figure shows the knockdown activity of unmodified siRNA (siSFTSV#2 and #3). Figure 1 shows the effect of unmodified siRNA (siSFTSV#2 and #3) (25 nM) on suppressing the copy number of SFTSV genomic RNA (HB29 strain and YG1 strain) genomic RNA. Figure 2 shows the effect of unmodified siRNA (siSFTSV#2 and #3) (25 nM) on suppressing the copy number of SFTSV genomic RNA (HB29 strain and YG1 strain) genomic RNA. Figure 3 shows the effect of RI7-siSFTSV conjugate (siSFTSV#2) on suppressing the copy number of SFTSV genomic RNA against infection with SFTSV (SPL010 strain, 0.02 FFU). Figure 4 shows the time course of survival rate after administration of the SFTSV-SPL010 strain at the indicated FFU to interferon α, β receptor and interferon γ receptor double-deficient mice (AG129 mice). Figure 5 shows the change in body weight after administration of the SFTSV-SPL010 strain at the indicated FFU to interferon α, β receptor and interferon γ receptor double-deficient mice (AG129 mice). 1 shows the time course of survival rate after administration of the SFTSV-SPL010 strain at the indicated FFU to interferon α, β receptor and interferon γ receptor double-deficient mice (AG129 mice). 2 shows the change in body weight after administration of the SFTSV-SPL010 strain at the indicated FFU to interferon α, β receptor and interferon γ receptor double-deficient mice (AG129 mice). Modes for carrying out the invention

[0009] As used herein, "subject" means an animal (e.g., a mammal (e.g., a primate (e.g., a human))).

[0010] As used herein, the term "antibody" refers to immunoglobulin, including polyclonal and monoclonal antibodies. Monoclonal antibodies are preferred. The origin of the antibody is not particularly limited, but examples include non-human animal antibodies (e.g., antibodies from non-human mammals) and human antibodies. The antibody may also be a chimeric antibody or a humanized antibody. The antibody may also be a bispecific antibody. Humanized or human antibodies are preferred. Antibodies include subclasses of IgG, IgA, IgE, IgM, and IgD. Human IgG includes subclasses of IgG1, IgG2, IgG3, and IgG4. Chimeric, humanized, or human monoclonal antibodies are preferred. When used as a pharmaceutical, chimeric monoclonal antibodies are preferred, and more preferably, humanized or human monoclonal antibodies are used, each of which is preferably isolated. Chimeric and humanized monoclonal antibodies can be produced from antibodies of non-human animals using known methods. Antibodies have a structure in which two heavy chains and two light chains are associated. The heavy chain consists of a heavy chain variable region (VH), a heavy chain constant region (CH1, CH2, and CH3), and a hinge region located between the heavy chain variable region and the heavy chain constant region. The light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The heavy chain variable region and the light chain variable region each have three complementarity-determining regions (CDRs), which characterize the antigen specificity of the antibody. The CDRs are referred to from the N-terminus of the heavy chain and light chain, respectively, as heavy chain CDR1 (also referred to as HCDR1 or CDR-H1), heavy chain CDR2, and heavy chain CDR3, and light chain CDR1 (also referred to as LCDR1 or CDR-L1), light chain CDR2, and light chain CDR3. Antibodies include full-length antibodies.

[0011] As used herein, the term "antigen-binding fragment" refers to a portion of an antibody that retains its ability to bind to an antigen. The antigen-binding fragment may comprise the heavy chain variable region, the light chain variable region, or both of the antibody of the present invention. The antigen-binding fragment may be chimerized or humanized. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv (single-chain Fv), diabody, sc(Fv)2 (single-chain (Fv)2), and half-molecule Ig. Antigen-binding fragments of these antibodies may be used in the present invention. Such antibody fragments can be obtained, for example, by treating the antibody with an enzyme, but are not limited to this. For example, Fab can be obtained by digesting the antibody with papain. Alternatively, F(ab')2 can be obtained by digesting the antibody with pepsin, and Fab' can be obtained by further reduction. These fragments can be expressed and purified as recombinant proteins in prokaryotic or eukaryotic expression systems.

[0012] As used herein, "treating" means therapy or prevention.

[0013] As used herein, "nucleic acid" includes natural nucleic acids such as natural DNA and natural RNA, antisense oligonucleotides (ASOs), editing oligonucleotides and guide oligonucleotides used to introduce mutations into genomic DNA and mRNA, modified nucleic acids (including artificial nucleic acids) such as modified DNA and modified RNA, and combinations thereof.

[0014] As used herein, a "modified nucleic acid" refers to a nucleic acid having one or more modifications or substitutions at the base moiety, at the sugar moiety (e.g., the ribose or deoxyribose moiety), or at the internucleotide bond (i.e., the bond between nucleic acid monomer units). Modified nucleic acids are widely used for purposes such as improving nucleic acid stability, improving affinity for target nucleic acids, reducing immunostimulatory activity, and improving the duration of action. Various modified nucleic acids have been developed, and those skilled in the art can use them as appropriate. In one non-limiting example, a knockdown-type nucleic acid consists solely of modified nucleic acids. Examples of modified nucleic acids include nucleic acids modified with fluorescent dyes, biotinylated nucleic acids, and nucleic acids into which cholesteryl groups have been introduced. To enhance stability, RNA may be modified with 2'-OMe, 2'-F, or 2'-MOE at the sugar moiety relative to the base, or may be a nucleic acid in which the sugar moiety is substituted with morpholino (PMO; see, e.g., U.S. Pat. No. 9,469,664B). US Pat. No. 9,469,664B discloses modified nucleic acids in which the phosphorus in the intersubunit (internucleotide bond) is replaced with a tertiary amine. In US Pat. No. 9,469,664B, the phosphorus in the intersubunit (internucleotide bond) is modified with dimethylamine, and may have the structure -P(=O)(-N(CH3)2)-O-. The phosphodiester bond in the internucleotide bond may also be replaced with a phosphorothioate bond. The phosphodiester bond in the internucleotide bond may also be replaced with a phosphorodiamidate bond. The phosphodiester bond in the internucleotide bond may also be replaced with a boranophosphate bond. The phosphodiester bond in the internucleotide bond may also be replaced with a peptide bond (e.g., peptide nucleic acid). Modified nucleic acids with modified sugars include nucleic acids in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged. Examples of such artificial nucleic acids include locked nucleic acid (LNA), which is a cross-linked DNA in which the oxygen atom at the 2' position and the carbon atom at the 4' position are cross-linked via methylene; ENA, in which the oxygen atom at the 2' position and the carbon atom at the 4' position are cross-linked via ethylene; and BNA, in which the oxygen atom at the 2' position and the carbon atom at the 4' position are cross-linked via -CHOCH-. COC, a BNA in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -NR-CH2- (where R is a methyl or hydrogen atom). NC Examples of such modified nucleic acids include bridged nucleic acids (BNAs) such as cMOE, in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -CH(OCH)-, cEt, in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -CH(CH)-, AmNA, in which the carbon atoms at the 2' position and the carbon atom at the 4' position are bridged via an amide, GluNA, in which the carbon atoms at the 2' position and the carbon atom at the 4' position are bridged via a guanidide, scpBNA, in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via a methylene to form a cyclopropane at the 6' position, and peptide nucleic acids (PNAs), in which the backbone of a polymer is formed by amide-linking N-(2-aminoethyl)glycine instead of deoxyribose or ribose. Modified nucleic acids can confer one or more, two or more, or all of the following: improved nucleic acid stability, improved affinity for the target nucleic acid, reduced immunostimulatory activity, and improved duration of action.

[0015] As used herein, a "knockdown nucleic acid" refers to a nucleic acid that can target DNA or RNA based on its base sequence to reduce the production of the RNA or the protein encoded by the RNA, thereby inhibiting the function of the RNA. Knockdown can be achieved by various methods, such as regulating RNA degradation or splicing, or rewriting the genetic information of the DNA or RNA, thereby suppressing the normal expression (i.e., knockdown) of the protein encoded by the DNA or RNA. Examples of knockdown nucleic acids include artificial RNAs for gene silencing, such as siRNA and short hairpin RNA (shRNA), microRNA (miRNA), guide RNA, genome editing oligonucleotides, RNA editing oligonucleotides, antisense oligonucleotides (ASO, PMO), and gapmers. Guide RNA can be used together with a fusion protein of a dCas9 protein lacking endonuclease activity and a transcription repression domain. This inhibits transcription of the target gene in a guide RNA sequence-dependent manner. Furthermore, when used with an RNA-guided nuclease such as Cas9, which has nuclease activity, the target genome sequence is modified, resulting in 100% knockdown of the mRNA of the originally expressed sequence. This is specifically called knockout. When used alone, genome editing oligos can alter the gene sequence through homologous recombination and reduce the mRNA of the original sequence. RNA editing oligos bind complementarily to the target mRNA, but intentionally introducing mismatches can serve as substrates for endogenous base editing enzymes. One such enzyme is adenosine deaminase (ADAR), which converts adenine at the mismatch site to inosine, thereby reducing the originally expressed mRNA. Gapmers are nucleic acids that contain modified nucleic acids at both ends (wing regions) of a single-stranded DNA (gap region). The modified wing regions can, for example, improve in vivo stability and / or improve RNA binding ability. This presents the gap region as a substrate for RNase H, resulting in RNA degradation.The knockdown nucleic acid may have mismatches or gaps with the target sequence as long as knockdown is induced, but preferably has a strand with a sequence complementary to the target RNA. In a preferred embodiment, the knockdown nucleic acid can induce RNA degradation. Viruses of the Banyanvirus genus (particularly SFTSV) are minus-strand single-stranded RNAs, and within cells, RNA-dependent RNA polymerase synthesizes plus-strand RNA using the minus-strand single-stranded RNA as a template, which functions as mRNA. Since suppression of mRNA naturally leads to inhibition of functional protein production from the RNA genome, it has an inhibitory effect on protein expression from Banyanvirus viruses (particularly SFTSV). Therefore, targeting of genomic RNA can be achieved not only by targeting the minus strand of genomic RNA, but also by targeting the plus strand having a sequence complementary to the genomic RNA. Those skilled in the art can design knockdown nucleic acids as appropriate. Various algorithms exist for determining the target region of RNA. In particular, in order to minimize off-target effects on human genes, the target region can be set in a region that is not homologous to the sequence found in humans. A nucleic acid having a sequence that is completely complementary to the target RNA is preferably used as the knockdown nucleic acid, but the sequence of the knockdown nucleic acid does not necessarily have to be completely complementary to the target RNA, and can be appropriately designed by those skilled in the art. For example, the knockdown nucleic acid can be appropriately selected from sequences that have 85% or more, 90% or more, or 95% or more identity to the complementary sequence of the target RNA.

[0016] As used herein, the term "conjugate" refers to a bond in which two or more substances are covalently linked. In a conjugate, the two or more substances may be directly linked or linked via a linker. In the present invention, one of the two or more substances is a targeting molecule (preferably an antibody or an antigen-binding fragment thereof), and the other is a drug (particularly a knockdown nucleic acid). In addition, in the present invention, the linker may be a cleavable linker or a non-cleavable linker.

[0017] As used herein, a "targeting means" refers to a means for targeting a biomolecule expressed in a living organism, has affinity and selectivity for the biomolecule, and is suitable for delivering a target molecule linked to the targeting means to the biomolecule in the living organism. The targeting means may be a means for binding to the surface of an SFTSV-infected cell or a molecule expressed on the surface. Since the targeting means is specifically composed of a molecule, the "targeting means" will hereinafter be referred to as a "targeting molecule." As used herein, a "targeting molecule" refers to a molecule for targeting a biomolecule expressed in a living organism, has affinity and selectivity for the biomolecule, and is suitable for delivering a target molecule linked to the targeting molecule to the biomolecule in the living organism. Examples of targeting molecules include, but are not limited to, aptamers (e.g., DNA aptamers and RNA aptamers), cyclic peptides, antibodies, and antigen-binding fragments thereof. The targeting molecule preferably has a sulfhydryl group for reaction with a linker drug moiety. The targeting molecule targets an SFTSV-infected cell or an antigen expressed on the cell. As long as the targeting molecule targets SFTSV-infected cells, it is acceptable for it to also target non-infected cells.

[0018] As used herein, a "targeting molecule-drug conjugate" refers to a conjugate between a targeting molecule and a drug. Linking to a targeting molecule confers affinity for an antigen to the drug, which can increase the efficiency of drug delivery to a target site in the body. Also, as used herein, a "conjugate of a targeting molecule and a knockdown-type nucleic acid" refers to a compound in which a targeting molecule and a knockdown-type nucleic acid are linked via a linker, and refers to a targeting molecule-drug conjugate in which the drug is a knockdown-type nucleic acid. As used herein, an "antibody-drug conjugate" refers to an antibody-drug conjugate (ADC). However, antibody-drug conjugates also include antibody antigen-binding fragment-drug conjugates in which an antibody antigen-binding fragment and a drug are linked via a linker. Linking to an antibody confers affinity for an antigen to the drug, which can increase the efficiency of drug delivery to a target site in the body. As used herein, the term "antibody-drug conjugate" is used to include conjugates of an antibody antigen-binding fragment and a drug. As used herein, a "conjugate of an antibody and a knockdown-type nucleic acid" refers to a compound in which an antibody and a knockdown-type nucleic acid are linked via a linker, and refers to an antibody-drug conjugate in which the drug is a knockdown-type nucleic acid. Antibodies and antigen-binding fragments thereof are examples of targeting molecules.

[0019] As used herein, the term "human monoclonal antibody" refers to any antibody in which the variable and constant domain sequences are human sequences. The term encompasses antibodies that have sequences derived from human genes but that have been altered to, for example, reduce potential immunogenicity, increase affinity, or remove cysteines that may cause undesired folding. The term also encompasses such antibodies that have been recombinantly produced in non-human cells, which may provide glycosylation not typical of human cells. These antibodies can be prepared in a variety of ways, as described below.

[0020] As used herein, the term "chimeric monoclonal antibody" refers to a monoclonal antibody that combines antibody regions from two or more different species. Examples of chimeric monoclonal antibodies include monoclonal antibodies in which the variable regions of a mouse antibody are linked to the constant regions of a human antibody. As used herein, the term "human chimeric monoclonal antibody" includes the VH and VL domains of an antibody from a non-human mammalian species and the CH and CL domains of a human antibody. Non-human mammals may include rodents as well as camels and llamas. One or more CDRs of a chimeric antibody may be derived from a human antibody. In one example, a CDR from a human antibody may be combined with a CDR from an antibody from a non-human mammal, such as a mouse or rat. In another example, all CDRs may be derived from a human antibody. In another example, CDRs from multiple human antibodies may be combined in a chimeric antibody. For example, a chimeric antibody may comprise the light chain CDR1 of a first human antibody, the light chain CDR2 of a second human antibody, and the light chain CDR3 of a third human antibody, and the heavy chain CDRs may be derived from one or more other antibodies.

[0021] As used herein, the term "humanized monoclonal antibody" refers to a monoclonal antibody derived from a non-human mammal in which amino acid residues characteristic of the antibody sequence of the non-human mammalian species are replaced with residues found at the corresponding positions in human antibodies. This "humanization" process is believed to reduce the immunogenicity of the resulting antibody in humans. Antibodies derived from non-human mammals can be humanized using techniques well known in the art, such as those described in Winter et al., Immunol. Today, 14:43-46 (1993). Humanized monoclonal antibodies can be engineered, for example, by using recombinant DNA techniques to replace the CH1, CH2, CH3, hinge, and / or framework domains of an antibody derived from a non-human mammal with corresponding human sequences (e.g., the techniques described in WO 92 / 02190 and U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085). As used herein, the term "humanized monoclonal antibody" includes within its meaning chimeric human monoclonal antibodies and CDR-grafted antibodies. The CDR-grafted antibodies of the present invention are obtained by replacing the CDRs of the VH and VL of a human antibody with the CDRs of the VH and VL, respectively, of an antibody from a non-human animal.

[0022] As used herein, the term "severe fever with thrombocytopenia syndrome virus" refers to a virus of the genus Bundavirus in the family Fenuviridae of the order Bunyaviridae, which has been identified as a pathogen of viral severe fever with thrombocytopenia syndrome (SFTS) (e.g., NCBI classification ID: 1003835). The particle diameter is approximately 100 nm, and along with the envelope, glycoproteins involved in infection are expressed on the surface. Other viruses belonging to the family Fenuviridae are known as Rift Valley fever virus (RVFV, e.g., NCBI classification ID: 11588) and Heartland virus (HRTV, e.g., NCBI classification ID: 1216928). Severe fever with thrombocytopenia syndrome virus is also referred to as SFTSV. SFTSV has a negative-sense single-stranded RNA genome (approximately 11.3 kb). The RNA genome of SFTSV is divided into three segments, specifically the L segment (approximately 6.3 kb), the M segment (approximately 3.3 kb), and the S segment (approximately 1.7 kb). The L segment (e.g., GenBank: AB817983.1) contains RNA encoding an RNA-dependent RNA polymerase (RdRP). The M segment (e.g., GenBank: AB817991.1) contains RNA encoding glycoprotein N (Gn) and the precursor (GP) of glycoprotein (Gc). The S segment contains RNA encoding two proteins, the nucleoprotein (N) and the nonstructural protein (NS). The RNA encoding the two proteins is typically separated by a 54-base intergenic region on the S segment. N is a protein approximately 246 amino acids long. N may be, for example, N having the amino acid sequence registered as GenBank: BAN58201.1 or a sequence corresponding to the amino acid sequence, for example, encoded by positions 44 to 781 of the S segment (e.g., the sequence registered as GenBank: AB817999.1). NS is a protein approximately 293 amino acids in length.The NS may be, for example, an NS having the amino acid sequence registered as GenBank: BAN58202.1 or a sequence corresponding to the amino acid sequence, for example, encoded by positions 836 to 1717 of the S segment (e.g., the sequence registered as GenBank: AB817999.1). SFTSVs include those belonging to a clade selected from the group consisting of J1, J2, J3, C1, C2, C3, C4, and C5. Various specific strains of SFTSV are known, including, for example, the HB29 strain, the YG1 strain, the SPL003A strain, the SPL004A strain, the SPL005A strain, the SPL010A strain, the SPL030A strain, and the SPL035A strain (see The Journal of Infectious Diseases, Volume 212, Issue 6, Pages 889-898, 2015).

[0023] As used herein, the term "transferrin receptor" refers to a transmembrane glycoprotein that is expressed on the cell surface and specifically binds to transferrin, a blood iron transport protein. Specifically, the term "transferrin receptor" includes any of the following, or a polypeptide having an amino acid sequence derived therefrom: (1) a polypeptide corresponding to human transferrin receptor 1 (TfR1; CD71; UniProt Accession No. P02786); (2) a polypeptide corresponding to human transferrin receptor 2 (TfR2; UniProt Accession No. Q9UP52); and (3) a variant that shares at least 80% sequence identity with either of the amino acid sequences (1) or (2) and retains transferrin binding ability. Both TfR1 and TfR2 are known to bind to transferrin via their extracellular domains and are involved in the endocytic mechanism by which transferrin is internalized into cells as a receptor-ligand complex. Furthermore, because TfR1 is highly expressed in proliferating cells and cancer cells, it has been widely studied and used as a tumor marker and a target for antibody drugs. The transferrin receptor preferred as a target herein is CD71. The TfR1 antibody must bind to TfR1 expressed on cells. The extracellular domain of TfR1 may be the region from C89 to the C-terminus.

[0024] Knockdown Nucleic Acids of the Present Disclosure Knockdown nucleic acids of the present disclosure target the RNA genome of severe fever with thrombocytopenia syndrome virus (SFTSV). In some embodiments, knockdown nucleic acids of the present disclosure target the S segment of the RNA genome. In some embodiments, knockdown nucleic acids of the present disclosure target the NS-encoding region of the S segment. In some embodiments, knockdown nucleic acids of the present disclosure target the N-encoding region of the S segment. In some embodiments, knockdown nucleic acids of the present disclosure target the M segment of the RNA genome. In some embodiments, knockdown nucleic acids of the present disclosure target the GP-encoding region of the M segment. GP is a precursor of glycoproteins Gn and Gc. In some embodiments, knockdown nucleic acids of the present disclosure target the L segment of the RNA genome. In some embodiments, knockdown nucleic acids of the present disclosure target the RdRP-encoding region of the L segment. Thus, it was expected that targeting regions encoding proteins important for SFTSV proliferation and formation would effectively exert antiviral effects against SFTSV. Targeting can be achieved, for example, by designing a nucleic acid to have a sequence complementary to the target RNA. Targeting can also be achieved by using a knockdown-type nucleic acid having a sequence that does not cause specific knockdown in host cells. The knockdown-type nucleic acid can be a single-stranded nucleic acid (e.g., ASO and gapmer, etc.) or a double-stranded nucleic acid (e.g., siRNA and shRNA, etc.) designed to have a sequence complementary to the target RNA.

[0025] In a preferred embodiment, the knockdown nucleic acid of the present disclosure targets the S segment of the RNA genome. Targeting the S segment is advantageous for obtaining an antiviral effect, and the knockdown nucleic acid of the present disclosure preferably targets the S segment of the RNA genome and has an antiviral effect against SFTSV. In a preferred embodiment, the knockdown nucleic acid of the present disclosure targets the region encoding N of the S segment. Alternatively, in a preferred embodiment, the knockdown nucleic acid of the present disclosure targets the region of positions 1-882 (i.e., the region encoding N), 1-870, 1-860, 1-850, 1-840, 1-838, 1-830, or 1-820 of the S segment, and more preferably targets the region of 50-882, 60-882, 70-882, 80-882, 91-882, 50-870, 60-870, 70-870, 80-870, 91-870, ... The targeting region is: -860, 60-860, 70-860, 80-860, 91-860, 50-850, 60-850, 70-850, 80-850, 91-850, 90-840, 50-840, 60-840, 70-840, 80-840, 91-838, 90-840, 50-838, 60-838, 70-838, 80-838, 44-781, or 91-838.

[0026] More preferably, the knockdown nucleic acid of the present disclosure targets positions 150-300, 450-650, or 780-830 of the S segment. In a preferred embodiment, the knockdown nucleic acid of the present disclosure reduces the amount of S segment RNA in SFTSV-infected cells by 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. In a preferred embodiment, the knockdown nucleic acid of the present disclosure reduces the amount of SFTSV genomic RNA in SFTSV-infected cells by 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. The reduction in the amount of S segment RNA can be measured, for example, by lipofecting siRNA into Vero9013 cells infected with the SFTSV-YG1 strain.

[0027] In some embodiments, knockdown-type nucleic acids can be designed so as not to significantly affect gene expression in a host, and the sequence of the knockdown-type nucleic acid can be designed so that a Blast search (with default parameters) based on the sequence does not yield a hit against human mRNA.

[0028] In some embodiments, the knockdown nucleic acid comprises a guide strand and a passenger strand, each of which may be the same or different in length, and the guide strand and the passenger strand have complementary sequences. In some embodiments, the knockdown nucleic acid comprises a guide strand and a passenger strand, each of which may be the same or different in length, and the guide strand and the passenger strand have complementary sequences. Here, the guide strand is a strand having a sequence complementary to a minus-strand RNA virus.

[0029] In one embodiment, the knockdown nucleic acid comprises a guide strand and a passenger strand, each of which may be the same or different in length. The guide strand and passenger strand are complementary and have one or more mismatches, but are capable of hybridizing to each other under physiological conditions. In this embodiment, the guide strand and passenger strand can target different sequences with one or more mismatches, which is useful, for example, in knocking down the RNA genome of an RNA virus, particularly an RNA virus (e.g., a minus strand) that utilizes an RNA-dependent RNA polymerase. SFTSV is one such virus, and the knockdown nucleic acid of the present disclosure can target different SFTSV variants using the guide strand and passenger strand. Examples of SFTSV variants include, but are not limited to, those belonging to a clade selected from the group consisting of J1, J2, J3, C1, C2, C3, C4, and C5. The knockdown nucleic acids of the present disclosure may target different variants belonging to the same clade using the guide strand and passenger strand, or may target variants belonging to different clades. Examples of SFTSV variants include, but are not limited to, HB29 strain, YG1 strain, SPL003A strain, SPL004A strain, SPL005A strain, SPL010A strain, SPL030A strain, SPL057A strain, SPL060A strain, SPL066A strain, SPL087A strain, SPL100A strain, SPL129A strain, 2011YPQ12 strain, 2011YSC60 strain, AHL / China / 2011 strain, JS2011-062 strain, JS3 strain, JS4 strain, LN2 strain, SD4 strain, Zhao strain, and SPL035A strain. Those skilled in the art can select the variants targeted by the guide strand and passenger strand, respectively, taking into account the spread of variants in each country or region.

[0030] In one embodiment, the guide strand and passenger strand each independently have a length of 21-23 mer. In one preferred embodiment, the guide strand is a 23 mer and the passenger strand is a 21 mer, and when the guide strand and the passenger strand hybridize to form a duplex, the 3' end of the guide strand overhangs by a 2 mer.

[0031] In certain embodiments, the guide strand and passenger strand can each have a length of a 21-mer and a 23-mer, a 21-mer and a 22-mer, or a 21-mer and a 21-mer; a 22-mer and a 23-mer, a 22-mer and a 22-mer, or a 22-mer and a 21-mer; or a 23-mer and a 23-mer, a 23-mer and a 22-mer, or a 23-mer and a 21-mer.

[0032] In one embodiment, the guide strand and passenger strand are, respectively, SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, SEQ ID NOs: 25 and 26, SEQ ID NOs: 27 and 28, SEQ ID NOs: 29 and 30, SEQ ID NOs: 31 and 32, SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, SEQ ID NOs: 49 and 50, SEQ ID NOs: 51 and 52, SEQ ID NOs: 53 and 54, SEQ ID NOs: 55 and 56, SEQ ID NOs: 57 and 58, SEQ ID NOs: 59 and 60, SEQ ID NOs: 61 and 62, SEQ ID NOs: 63 and 64, SEQ ID NOs: 65 and 66, SEQ ID NOs: 67 and 68, SEQ ID NOs: 69 and 70, SEQ ID NOs: 71 and 72, SEQ ID NOs: 73 and 74, SEQ ID NOs: 75 and 76, SEQ ID NOs: 77 and 78, SEQ ID NOs: 79 and 80, Sequence numbers 81 and 82, SEQ ID NOs: 83 and 84, SEQ ID NOs: 85 and 86, SEQ ID NOs: 87 and 88, SEQ ID NOs: 89 and 90, SEQ ID NOs: 91 and 92, SEQ ID NOs: 93 and 94, SEQ ID NOs: 95 and 96, SEQ ID NOs: 97 and 98, SEQ ID NOs: 99 and 100, SEQ ID NOs: 101 and 102, SEQ ID NOs: 103 and 104, SEQ ID NOs: 105 and 106, SEQ ID NOs: 107 and 108, SEQ ID NOs: 109 and 110, SEQ ID NOs: 111 and 112, SEQ ID NOs: 113 and 114, SEQ ID NOs: 115 and 116, SEQ ID NOs: 117 and 118, sequence nos. Nos. 119 and 120, SEQ ID NOs: 121 and 122, SEQ ID NOs: 123 and 124, SEQ ID NOs: 125 and 126, SEQ ID NOs: 127 and 128, SEQ ID NOs: 129 and 130, SEQ ID NOs: 131 and 132, SEQ ID NOs: 133 and 134, SEQ ID NOs: 135 and 136, SEQ ID NOs: 137 and 138, SEQ ID NOs: 139 and 140, SEQ ID NOs: 141 and 142, SEQ ID NOs: 143 and 144, SEQ ID NOs: 145 and 146, SEQ ID NOs: 147 and 148, SEQ ID NOs: 149 and 150, SEQ ID NOs: 151 and 152, SEQ ID NOs: 153 and 154,The siRNA may have any one or more selected from the group consisting of SEQ ID NOs: 155 and 156, 157 and 158, 159 and 160, 161 and 162, and 163 and 164. An exemplary siRNA is a guide strand and passenger strand that targets a position one to several bases (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases) upstream or downstream from the site targeted by the siRNA having the guide strand and passenger strand. In one aspect, the siRNA may be a guide strand and passenger strand that targets a position one to two bases, one to three bases, one to four bases, one to five bases, one to six bases, one to seven bases, one to eight bases, one to nine bases, or one to ten bases upstream or downstream from the site targeted by the siRNA having the guide strand and passenger strand.

[0033] In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 1 and 2, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 3 and 4, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 101 and 102, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 103 and 104, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 105 and 106, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 107 and 108, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 109 and 110, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 111 and 112, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 113 and 114, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 115 and 116, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 117 and 118, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 119 and 120, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 121 and 122, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 123 and 124, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 125 and 126, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 127 and 128, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 129 and 130, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 131 and 132, respectively. In some embodiments, the passenger strand and the guide strand may have SEQ ID NOs: 133 and 134, respectively. In one embodiment, the passenger strand and guide strand can have SEQ ID NOs: 135 and 136, respectively.In a preferred embodiment, the passenger strand and the guide strand may each have one or more selected from the group consisting of SEQ ID NOs: 101 and 102, SEQ ID NOs: 109 and 110, SEQ ID NOs: 113 and 114, SEQ ID NOs: 119 and 120, SEQ ID NOs: 121 and 122, SEQ ID NOs: 127 and 128, SEQ ID NOs: 129 and 130, SEQ ID NOs: 131 and 132, and SEQ ID NOs: 133 and 134. siRNAs having a guide strand and a passenger strand that target a position one to several bp (e.g., 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, or 10 bp) upstream or downstream of the site targeted by the siRNA having the guide strand and passenger strand are also exemplary siRNAs. In one aspect, the siRNA can be an siRNA having a guide strand and a passenger strand that targets a position 1 to 2 bases, 1 to 3 bases, 1 to 4 bases, 1 to 5 bases, 1 to 6 bases, 1 to 7 bases, 1 to 8 bases, 1 to 9 bases, or 1 to 10 bases upstream or downstream of the site targeted by the siRNA having the guide strand and passenger strand.

[0034] In a preferred embodiment, the knockdown nucleic acid of the present disclosure can reduce the SFTSV genome amount 72 hours after infection of Vero9013 cells in a culture system with the SFTSV-SPL010 strain (0.02 FFU / cell) to preferably 50% or less, more preferably 40% or less, even more preferably 30% or less, even more preferably 20% or less, and especially preferably 10% or less, compared to a negative control. In one embodiment, siRNA transfection can be performed 24 hours before infection. In one embodiment, the siRNA concentration is 25 nM. Transfection can be performed by conventional methods, preferably by lipofection. For example, Lipofectamine can be used as a transfection reagent. TMRNAiMAX Transfection Reagent (ThermoFisher Scientific) can be used. Eagle's Minimum Essential Medium (EMEM) containing heat-inactivated bovine serum can be used as a culture medium. In one embodiment, the knockdown nucleic acid of the present disclosure is used at 25 nM and lipofected (e.g., using Lipofectamine) into Vero9013 cells 24 hours before infection. TM Using RNAiMAX Transfection Reagent (ThermoFisher Scientific), the SFTSV genome amount was measured 72 hours after infection of Vero9013 cells with the SFTSV-SPL010 strain (0.02 FFU / cell) in a culture system, and the reduction in genome amount was evaluated compared to the negative control.

[0035] The knockdown nucleic acid of the present disclosure may contain a modified nucleic acid. The modification may include, but is not limited to, one or more modifications selected from the above-mentioned modifications, for example, one, two, or all modifications selected from the group consisting of phosphorothioate modification, 2'-OMe, and 2'-F modification. In a preferred embodiment, the 5'-end of the sense strand and / or the 5'-end of the antisense strand of the knockdown nucleic acid of the present disclosure may be modified with a phosphate group. In a more preferred embodiment, the 5'-end of the sense strand and / or the 5'-end of the antisense strand of the knockdown nucleic acid containing the modified nucleic acid may be modified with a phosphate group. In an even more preferred embodiment, the 5'-end of the antisense strand of the knockdown nucleic acid containing the modified nucleic acid may be modified with a phosphate group.

[0036] In some embodiments, the guide strand and passenger strand each contain a modified nucleic acid and each exert a knockdown effect. Typically, the passenger strand can be chemically modified to prevent silencing activity in order to suppress off-targets. However, when targeting an RNA virus as in the present disclosure, the passenger strand can also be designed to exert silencing activity (see Wei et al., PLoS One, 4(4):e5382, 2009). In some preferred embodiments, chemical modifications can be designed so that in the double-stranded RNA formed by hybridization of the guide strand and passenger strand, bases having 2'-F modifications at the sugar moiety do not pair with bases having 2'-F modifications. In some embodiments, the guide strand is a 23-mer, and the passenger strand is a 21-mer or 23-mer. In one embodiment, the guide strand is a 23-mer and the passenger strand is a 22-mer or 23-mer, and each strand has one or more, preferably all, selected from the group consisting of the 1st, 7th, 11th, 13th, 15th, 17th, 19th, 21st, and 23rd bases from the 5' end having a 2'-OMe modification, and one or more, preferably all, selected from the group consisting of the 2nd, 6th, 14th, and 16th bases having a 2'-F modification. In one embodiment, the guide strand and passenger strand have complementary sequences. In one embodiment, the guide strand and passenger strand have complementary sequences and target the minus and plus strands of the SFTSV genome, respectively (i.e., they have complementary sequences to the minus and plus strands of SFTSV, or exert a silencing effect on these strands, respectively). In some embodiments, the guide strand and passenger strand are complementary except for a mismatch (e.g., one or more mismatches) between the guide strand and passenger strand, and preferably, the guide strand and passenger strand are capable of duplex formation even when the mismatch is present.In one embodiment, the guide strand and passenger strand are complementary except for mismatches (e.g., one or more mismatches), and each strand targets the minus and plus strands of a different strain of SFTSV (i.e., each strand has a sequence complementary to the minus and plus strands of a different strain of SFTSV, or exerts a silencing effect on these strands, respectively). For example, when SFTSV has sequence variation between strains, the guide strand and passenger strand can be designed to target each strain. For example, the passenger strand can target a relatively major strain and the guide strand can target a relatively minor strain, or the passenger strand can target a relatively minor strain and the guide strand can target a relatively major strain. Also, for example, the passenger strand can target a relatively highly virulent strain and the guide strand can target a relatively less virulent strain. Alternatively, the passenger strand can be used to target a relatively less virulent strain, while the guide strand can be used to target a relatively more virulent strain. The guide and passenger strands are typically designed to target the same site in the RNA genome. This allows the siRNA to maintain its duplex formation ability while conferring knockdown potential to multiple strains with different sequences.

[0037] Conjugates of the Present Disclosure The present disclosure provides a conjugate of a targeting molecule (preferably an antibody or its antigen-binding fragment) and a knockdown nucleic acid. The targeting molecule (preferably an antibody or its antigen-binding fragment) can bind to an antigen expressed on the surface of a target cell. It has been found that the majority of SFTSV-infected cells are B-cell lymphocytes (Suzuki et al., J Clin Invest. (2020) 3;130(2):799-812). Therefore, the targeting molecule can be an antigen expressed on the surface of B-cell lymphocytes. The antigen expressed on B-cell lymphocytes can preferably be one or more selected from the group consisting of CD20, CD38, CD71 (transferrin receptor 1), CD79a, CD163, and MUM1. The targeting molecule (preferably an antibody or its antigen-binding fragment) and the knockdown nucleic acid are preferably linked via a linker. The link between the targeting molecule (preferably an antibody or its antigen-binding fragment) and the linker can be a covalent bond. The linker and the knockdown-type nucleic acid may be linked by a covalent bond. In a preferred embodiment, the antigen-binding fragment may be a Fab fragment or a Fab' fragment.

[0038] In the conjugates of the present disclosure, full-length antibodies and antigen-binding fragments thereof can be used as the antibody. In a preferred embodiment, the antibody binds to one or more selected from the group consisting of CD20, CD38, CD71, CD79a, CD163, CD19, CD21, CD22, CD24, CD27, CD25, CD80, CD95, and MUM1. In a preferred embodiment, the antigen-binding fragment may be a Fab fragment or a Fab' fragment. Examples of anti-CD19 antibodies include blinatumomab and tafasitamab, as well as antibodies having a heavy chain variable region having the amino acid sequences of the three heavy chain CDRs 1 to 3 of either of these antibodies and a light chain variable region having the amino acid sequences of the three light chain CDRs 1 to 3. Examples of anti-CD20 antibodies include rituximab, ibritumomab, tositumomab, ocrelizumab, and veltuzumab, as well as antibodies having a heavy chain variable region with the amino acid sequences of three heavy chain CDRs 1-3 of any of these antibodies and a light chain variable region with the amino acid sequences of three light chain CDRs 1-3. Examples of anti-CD25 antibodies include basiliximab and daclizumab, as well as antibodies having a heavy chain variable region with the amino acid sequences of three heavy chain CDRs 1-3 of any of these antibodies and a light chain variable region with the amino acid sequences of three light chain CDRs 1-3. Examples of anti-CD38 antibodies include daratumumab, isatuximab, felzalutamab, and mezagitamab, as well as antibodies having a heavy chain variable region with the amino acid sequences of three heavy chain CDRs 1-3 of any of these antibodies and a light chain variable region with the amino acid sequences of three light chain CDRs 1-3.

[0039] Further, for example, anti-CD71 antibodies include antibodies disclosed in the following publications, the entireties of which are incorporated herein by reference: JP2024503609A (US20240117356A), JP2023540746A (US20230330562A), JP2021532195A (US20220378934A), WO2018124121A (US10759864B), JP2018520143A (US11584793B), US20210261679A, JP5980202B (US9598496B), US8409573B, US9611323B, and US9994641B.

[0040] For example, JP2024503609A (US20240117356A) discloses, as anti-CD71 antibodies, 3-A4 antibody and variant antibodies thereof (e.g., antibodies having an N54T mutation, antibodies having an N54S mutation, etc.), 3-M12 and variant antibodies thereof, 5-H12 and variant antibodies thereof (e.g., antibodies having a C33Y mutation, antibodies having a C33D mutation, etc.), clone 8 and variant antibodies thereof, as well as antibodies (particularly humanized antibodies) having the heavy chain CDR1-3 and light chain CDR1-3 of these antibodies as heavy chain CDR1-3 and light chain CDR1-3, respectively. JP2024503609A (US20240117356A) also discloses, for example, an antibody that binds to amino acids 90-96 of human TfR1. JP2024503609A (US20240117356A) also discloses an antibody that binds to an epitope including, for example, one or more of residues Y222, T227, K231, H234, T367, S368, S370, T376, and S378 of human TfR1. JP2024503609A (US20240117356A) also discloses an antibody that binds to an epitope including, for example, one or more of residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1.

[0041] For example, JP2023540746A (US20230330562A) discloses as anti-CD71 antibodies OKT9 disclosed in US4364934B; clones M11, M23, M27, and B84 disclosed in US9994641B; 7A4, 8A2, 15D2, 10D11, 7B10, 15G11, 16G5, 13C3, 16G4, 16F6, 7G7, 4C2, 1B12, and 13D4 disclosed in US9708406B; 8D3 disclosed in US2010 / 077498A; OX26 disclosed in Cellular microbiology 16:1806-1821, 2014; and Plant J 48:757-770, 2006, DF1513; commercially available 1A1B2, 66IG10, MEM-189, JF0956, 29806, TFRC / 1818.1E6, TFRC / 1059, Q1 / 71, 23D10, 13E4, TFRC / 1149, ER-MP21, YTA74.4, BU54, 2B6, RI7 217, RI7 217.1.3, R17 217, R17 217.1.3, 5E9C11, OKT9 (BE0023 Examples include Saleta BA120g disclosed in US 2011 / 0311544A; Saleta LUCA31 disclosed in US 7,572,895B; Saleta B3 / 25 and T58 / 30 disclosed in Nature, 296, pp. 171-173, 1981; and Saleta BK19.9, B3 / 25, T56 / 14, and T58 / 1 disclosed in J Clin Pathol 36(5):539-545, 1983. In the present disclosure, antibodies (particularly humanized antibodies) having the heavy chain CDR1-3 and light chain CDR1-3 of any of these antibodies as the heavy chain CDR1-3 and light chain CDR1-3, respectively, may be used.

[0042] For example, WO2018124121A (US10759864B) discloses an anti-CD71 antibody (particularly a humanized antibody) having the heavy chain CDR1-3 and light chain CDR1-3 of antibody No. 3 as heavy chain CDR1-3 and light chain CDR1-3, respectively. WO2018124121A (US10759864B) also discloses antibodies Nos. 3-2, 3-3, and 3-4 as anti-CD71 antibodies. WO2018124121A (US10759864B) also discloses antibody No. 3N as an anti-CD71 antibody.

[0043] For example, JP2018520143A (US11584793B) discloses mAb128.1, clone 299, clone 494, and variant antibodies of any of these as anti-CD71 antibodies. A humanized version of clone 299 antibody is also disclosed, which may preferably have VH23 and VL9, respectively.

[0044] For example, US20210261679A exemplifies anti-CD71 antibodies such as 13E4_VH1, 13E4_VH2, 13E4_VH3, and 13E4_VH4, as well as 13E4_VL1, 13E4_VL2, 13E4_VL3, and 13E4_VL4. US20210261679A also discloses combinations of these antibodies in TABLE 5. The CDR sequences of these antibodies are disclosed in TABLES 1 and 2 of US20210261679A.

[0045] Furthermore, for example, JP5980202B discloses, as anti-CD71 antibodies, TfR001 antibody, TfR002 antibody, TfR003 antibody, TfR004 antibody, TfR005 antibody, TfR006 antibody, TfR007 antibody, TfR008 antibody, TfR009 antibody, TfR010 antibody, TfR011 antibody, TfR012 antibody, TfR013 antibody, TfR014 antibody, TfR015 antibody, TfR016 antibody, TfR017 antibody, TfR018 antibody, TfR019 antibody, and TfR020 antibody, as well as antibodies having heavy chain CDR1 to 3 and light chain CDR1 to 3 thereof.

[0046] Although not limited to the above antibodies, the anti-CD71 antibodies exemplified above can also be preferably used in the present disclosure.

[0047] Those skilled in the art can appropriately determine CDRs using well-known conventional techniques. For example, those skilled in the art can find CDRs by applying the appropriate numbering system to the antibody amino acid sequence database compiled by Kabat et al. ("Sequence of Proteins of Immunological Interest," U.S. Dept. Health and Human Services, 1983) and examining the homology. Antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv (single-chain Fv), diabody, sc(Fv)2 (single-chain (Fv)2), and half-molecule Ig, and may be, for example, Fab or Fab'. In a preferred embodiment, the antibody or antigen-binding fragment thereof has internalization activity. The antibody may also be an antibody having six CDRs of any of the above antibodies, or an antibody having CDRs with 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the CDRs. The antibody may also be an antibody having six CDRs of any of the above antibodies, or an antibody having CDRs with a sequence containing 1, 2, 3, 4, or 5 amino acid mutations selected from the group consisting of addition, insertion, substitution, and deletion in one or more of the CDRs.

[0048] A conjugate of the present disclosure can have, for example, the following formula (L): Ab-(L-NA) ｎ...(L) {Here, Ab represents a targeting molecule (preferably an antibody or an antigen-binding fragment thereof), L represents a linker, NA represents a knockdown-type nucleic acid, and n represents the number of -(L-NA) units bound to the targeting molecule (preferably an antibody or an antigen-binding fragment thereof) (in one embodiment, the drug-antibody ratio (DAR)), which depends on the number of amino acid residues (e.g., the number of cysteine ​​residues) to which each of the units in the targeting molecule (preferably an antibody or an antigen-binding fragment thereof) is bound, and varies depending on the targeting molecule (preferably an antibody or an antigen-binding fragment thereof), but may be, for example, an integer from 1 to 8, and "-" represents a covalent bond.} In one embodiment, Ab can be a full-length antibody (referred to as "Ig"), and in one embodiment, can be Fab or Fab'. In one embodiment, Ab can be Fab'.

[0049] In one aspect, the number of -(L-NA) units bound to a targeting molecule (preferably an antibody or antigen-binding fragment thereof) depends on the amino acid residues to which each of the units is bound (e.g., the number of cysteine ​​residues (particularly the number (m) of cysteine ​​residues forming inter-chain disulfide bonds)) present in the targeting molecule (preferably an antibody or antigen-binding fragment thereof). For example, the number of bonds (n) can be an integer from 1 to m, where m is the number of cysteine ​​residues forming inter-chain disulfide bonds of the targeting molecule (preferably an antibody or antigen-binding fragment thereof). For example, with respect to human antibodies, m is 8 for IgG1, 12 for IgG2, 14 for IgG3, and 8 for IgG4. For example, when Ab is a full-length IgG1 antibody or F(ab')2, n can be an integer from 1 to 8. Furthermore, for example, when Ab is a Fab fragment, n can be an integer of 1 or 2. For example, when Ab is a Fab' fragment, n can be an integer from 1 to 4 (e.g., 1 or 2). In this way, n can be set to a maximum value equal to the number (m) of sulfur atoms involved in interchain disulfide bonds of IgG.

[0050] In the conjugates of the present disclosure, a non-cleavable linker may be preferably used as the linker. Furthermore, in the conjugates of the present disclosure, a cleavable linker may be preferably used as the linker. The non-cleavable linker is a stable linker that does not cleave after administration into the body, while the cleavable linker is a linker that contains a moiety that is cleaved within a cell, such as a disulfide bond that is cleaved in a reducing environment, or a ketal, hydrazone, carbamate ester, or valine-citrulline that is cleaved by cathepsin in a low pH environment. The moiety other than the cleavable moiety of the cleavable linker and the non-cleavable linker may be a chemically stable, inert group.

[0051] A conjugate of an antibody and a knockdown-type nucleic acid can be prepared by a person skilled in the art using a method well known as a method for preparing an antibody-knockdown-type nucleic acid conjugate (ADC), as well as a conjugate of an antigen-binding fragment of an antibody and a knockdown-type nucleic acid.

[0052] The present invention provides a method for producing a conjugate of a targeting molecule (preferably an antibody or an antigen-binding fragment thereof) and a knockdown nucleic acid, the method comprising introducing a group for introducing a thiol-reactive group into the knockdown nucleic acid or a carbon chain having a substituent bound to the knockdown nucleic acid.The present invention provides a method for producing a conjugate of a targeting molecule (preferably an antibody or an antigen-binding fragment thereof) and a knockdown nucleic acid, the method comprising introducing a group for introducing a thiol-reactive group into the knockdown nucleic acid or a carbon chain having a substituent bound to the knockdown nucleic acid, and reacting the thiol group of the targeting molecule (preferably an antibody or an antigen-binding fragment thereof) with the knockdown nucleic acid or the thiol-reactive group introduced into the knockdown nucleic acid.

[0053] When the knockdown-type nucleic acid is a double-stranded nucleic acid such as siRNA, the thiol-reactive group, the group that introduces a thiol-reactive group, or the carbon chain having a substituent can be introduced by linking via a spacer (e.g., a spacer consisting of one to several nucleic acid bases) introduced into the siRNA. The linker can be linked, for example, to the 5'-end or 3'-end of the passenger strand of the siRNA, or the 5'-end or 3'-end of the guide strand. The linker can preferably be linked to the 3'-end of the passenger strand of the siRNA.

[0054] When the knockdown-type nucleic acid is a single-stranded nucleic acid such as ASO, the thiol-reactive group, the group that introduces a thiol-reactive group, or the carbon chain having a substituent can be introduced by linking via a spacer (e.g., a spacer consisting of a nucleic acid of one to several bases) introduced into the ASO.

[0055] Conjugates of the present invention having a linker can be produced by a method known per se, for example, by binding a carbon chain having a substituent (preferably a carbon chain having an amino group at the end of the carbon chain) to a knockdown-type nucleic acid, then binding the amino group to a group for introducing a thiol-reactive group, and further reacting the thiol-reactive group with a targeting molecule having a thiol group (preferably an antibody or an antigen-binding fragment thereof).

[0056] The carbon chain having the above-mentioned substituent has 1 to 10 carbon atoms, preferably 2 to 8 carbon atoms, more preferably 3 to 6 carbon atoms, for example, 4, 5, or 6 carbon atoms, and can be, for example, 6 carbon atoms.

[0057] The substituent on the carbon chain in the substituted carbon chain includes an amino group, a thiol group, an azide group, an alkynyl group, etc., and is preferably an amino group. Note that an azide and an alkyne react by a click reaction to produce a triazole.

[0058] As the carbon chain having the above substituent, an alkyl chain having 6 carbon atoms and an amino group at the terminal (sometimes referred to as a C6 amino chain in this specification) can be particularly preferably used.

[0059] When a nucleic acid is used as the knockdown nucleic acid, the knockdown nucleic acid can be linked to a carbon chain having a substituent by a nucleic acid synthesis reaction using a commercially available 3'- or 5'-amino modification carrier or amidite reagent (for example, but not limited to, 3'-PT-Amino-Modifier C6 CPG or 5'-DMS(O)MT-Amino-Modifier C6 (Glen Research)).

[0060] When a carbon chain having a substituent and an amino group at the end is used, the amino group can then be bonded to a group that introduces a thiol-reactive group.

[0061] The thiol-reactive group refers to a functional group that is reactive with a specific site (thiol group) of a targeting molecule (preferably an antibody or its antigen-binding fragment). Examples of the thiol-reactive group include a maleimide group, a bromoacetamide group, a pyridyldithio group, a vinylsulfone group, and an iodoacetamide group.

[0062] The group that introduces a thiol-reactive group refers to a reagent having a thiol-reactive group, and examples of such reagents include bonds; maleimidocaproyl (mc); maleimidocaproyl-p-aminobenzylcarbamate; maleimidocaproyl-peptide-aminobenzylcarbamate (e.g., maleimidocaproyl-L-phenylalanine-L-lysine-p-aminobenzylcarbamate and maleimidocaproyl-L-valine-L-citrulline-p-aminobenzylcarbamate (vc)); N-maleimidocaproyl-valyl-citrullyl-p-aminobenzylcarbamate p-nitrophenyl ester (mc-Val-Cit-PABC-PNP); N-succinimidyl 3-(2-pyridyldithio)proprionate (N-succinimidyl 4-(2-pyridyldithio)pentanoate); N-succinimidyl 4-Methyl-4-(2-pyridyldithio)pentanate; 4-Succinimidyl-oxycarbonyl-2-methyl-2-(2-pyridyldithio)-toluene (SMPT); N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP); N-Succinimidyl 4-(2-pyridyldithio)butyrate (SPDB); 2-Iminothiolane; S-Acetylsuccinic Anhydride; Disulfide Benzyl Carbamate; Carbonate; Hydrazone Linker; N-(α-Maleimidoacetoxy)succinimide Ester; N-[4-(p-Azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (AMAS); N-Succinimidyl 3-Maleimidopropionate (BMPS); [N-ε-maleimidocaproyloxy]succinimide ester (EMCS); N-[γ-maleimidobutyryloxy]succinimide ester (GMBS); succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate] (LC-SMCC); succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate (LC-SPDP); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl [4-iodoacetyl]aminobenzoate (SIAB); succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC);N-Succinimidyl 3-[2-pyridyldithio]-propionamide (SPDP); [N-ε-maleimidocaproyloxy]sulfosuccinimide ester (sulfo-EMCS); N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (sulfo-GMBS); 4-Sulfosuccinimidyl-6-methyl-α-(2-pyridyldithio)toluamido]hexanoate) (sulfo-LC-SMPT); Sulfosuccinimidyl 6-(3'-[2-pyridyldithio]-propionamido)hexanoate (sulfo-LC-SPDP); m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS); N-Sulfo succinimidyl [4-iodoacetyl]aminobenzoate (sulfo-SIAB); sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC); sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate (sulfo-SMPB); ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) (EGS); disuccinimidyl tartrate (DST); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); diethylenetriamine-pentaacetic acid (DTPA); N-maleimidocaproyl-valyl-citrullyl-p-aminobenzylcarbamate p-nitrophenyl ester (mc-Val-Cit-PABC-PNP); and thiourea. The linker may have a structure obtained by reacting these groups with a targeting molecule (preferably an antibody or antigen-binding fragment thereof);

[0063] The linker may comprise a peptide, which may be or may include any of Val-Cit (VC), GGFG (SEQ ID NO: 166), FK, VK, GFK, FFK, AK, VR, FC, FR, LC, IC, WC, FA, ALAL, and GFLG (SEQ ID NO: 167) (amino acids in peptide sequences are written in single letter notation throughout this document, with C representing citrulline, but other amino acids having the usual meanings).

[0064] Homobifunctional crosslinkers (e.g., DSP, DTSSP, DSS, BS, DSG, DSC, DMA, DMP, DMS, DTBP, DPDPB, BMH, DFDNB, DFDNPS, BASED, formaldehyde, glutaraldehyde, etc.) and heterobifunctional linkers (e.g., sPDP, LC-sPDP, sulfo-LC-sPDP, sMPT, sulfo-LC-sMPT, sMCC, sulfo-sMCC, MBs, sulfo-MBs, sIAB, sulfo-sIAB, sMPB, sulfo-sMPB, GMBs, sulfo-GMBs, sIAX, sIAX X, sIAC, sIACX, NPIA, MPBH, M2C2H, PDPH, NHs-AsA, sulfo-NHs-AsA, sulfo-NHs-LC-AsA, sAsD, HsAB, sulfo-HsAB, sANPAH, sulfo-sANPAH, ANB-NOs, sAND, sADP, sulfo-sADP, sulfo-sAPB, sAED, sulfo-sAMCA, pNPDP, PNP-DTP, AsIB, APDP, benzophenone-4-iodoacetamide, benzophenone-4-maleimide, ABH, AsBA, APG, etc.) can also be introduced into the linker.

[0065] The group for introducing a thiol reactive group is preferably SPDP, BMPS, GMBS, SMCC, EMCS, or mc-Val-Cit-PABC-PNP.

[0066] The bond between the amino group and the group that introduces a thiol-reactive group can be carried out, for example, by condensation reaction, addition reaction, etc. More specifically, for example, an excess amount of BMPS is added to a knockdown nucleic acid to which a carbon chain having an amino group is bound, and the mixture is incubated at room temperature for about 2 to 4 hours. After the amino group has completely reacted, unreacted BMPS is removed by ultrafiltration or the like.

[0067] In certain embodiments of the present invention, a group for introducing a thiol-reactive group can be introduced into vesicles encapsulating knockdown nucleic acids. For example, in the case of vesicles using lipids having an amino group at their termini, the amino group and the group for introducing a thiol-reactive group can be bonded by condensation reaction, addition reaction, or the like. More specifically, this can be achieved, for example, by adding an excess amount of BMPS to the knockdown nucleic acid, incubating at room temperature for approximately 2 to 4 hours, and after the amino group has completely reacted, removing unreacted BMPS by dialysis, ultrafiltration, or the like. Alternatively, this can be achieved by a post-insertion method in which a lipid having a thiol-reactive group is separately synthesized along with the lipids that are components of the vesicles, and this lipid or a lipid reacted with a target ligand is inserted into the lipid membrane after the vesicles are prepared; or a premix method in which vesicles are prepared from raw materials such as lipids containing thiol-reactive lipids or lipids to which a target ligand has been bound.

[0068] When a targeting molecule (preferably an antibody or an antigen-binding fragment thereof) has a -SH group, the thiol-reactive group can be bound to the -SH group by, for example, an oxidation-reduction reaction, a condensation reaction, an addition reaction, or the like. More specifically, for example, the binding can be carried out by mixing approximately equal amounts of a targeting molecule (preferably an antibody or an antigen-binding fragment thereof) and a knockdown nucleic acid into which a thiol-reactive group has been introduced, and incubating at room temperature for 4 to 16 hours. The thiol-reactive group may react with amino groups, particularly those with low pKa, by the same reaction mechanism that reacts with thiols. Excess reactants can be removed by chromatography.

[0069] According to the present invention, when the knockdown-type nucleic acid has a thiol-reactive group, there is provided a method for producing a conjugate of a targeting molecule (preferably an antibody or an antigen-binding fragment thereof) and the knockdown-type nucleic acid, the method comprising reacting the targeting molecule (preferably an antibody or an antigen-binding fragment thereof) with the thiol-reactive group of the knockdown-type nucleic acid.

[0070] The conjugate of the targeting molecule (preferably an antibody or an antigen-binding fragment thereof) and the knockdown nucleic acid can be separated from unreacted substances such as the targeting molecule (preferably an antibody or an antigen-binding fragment thereof) and the knockdown nucleic acid using a separation technique such as HPLC, including size exclusion chromatography and hydrophobic chromatography.

[0071] The nucleic acid in the conjugate can be a knockdown nucleic acid as described above. According to the present disclosure, the nucleic acid in the conjugate targets, for example, the S segment of the RNA genome and has an antiviral effect against SFTSV. In a preferred embodiment, the nucleic acid in the conjugate targets the N-encoding region of the S segment. Alternatively, in certain preferred embodiments, the nucleic acid in the conjugate targets the region of the S segment between positions 1-882 (i.e., the region encoding N), 1-870, 1-860, 1-850, 1-840, 1-838, 1-830, or 1-820, and more preferably the region of the S segment between positions 50-882, 60-882, 70-882, 80-882, 91-882, 50-870, 60-870, 70-870, 80-870, 91-870, ... region, 60-860 region, 70-860 region, 80-860 region, 93-860 region, 50-850 region, 60-850 region, 70-850 region, 80-850 region, 91-850 region, 90-840 region, 50-840 region, 60-840 region, 70-840 region, 80-840 region, 91-838 region, 90-840 region, 50-838 region, 60-838 region, 70-838 region, 80-838 region, 44-781, or 91-838 region.

[0072] More preferably, the nucleic acid in the conjugate targets positions 150-300, 450-650, or 780-830 of the S segment. In a preferred embodiment, the nucleic acid in the conjugate reduces the amount of S segment RNA in a cell by 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. In a preferred embodiment, the nucleic acid in the conjugate reduces the amount of SFTSV genomic RNA in a cell by 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. The cells may preferably be Vero9013 cells (e.g., Japanese Collection of Research Bioresources Cell Bank, accession number: JCRB9013).

[0073] The nucleic acid in the conjugate is, for example, an siRNA. The siRNA preferably has a sequence complementary to the target RNA and can reduce the amount of the target RNA and / or protein expression from the target RNA. The siRNA is a double-stranded RNA, and each strand has a length of, for example, about 19-25 mer, but is not particularly limited as long as it achieves effective knockdown. The 5' end of the nucleic acid may have a phosphate. The nucleic acid in the conjugate is, for example, an shRNA. An shRNA typically has a structure in which the two strands of siRNA are linked by a hairpin.

[0074] The nucleic acid in the conjugate may contain a modified nucleic acid. The modification may include, but is not limited to, one or more modifications selected from the above-mentioned modifications, for example, one, two, or all modifications selected from the group consisting of phosphorothioate modification, 2'-O methyl modification, and 2'-F modification. In a preferred embodiment, the nucleic acid in the conjugate may contain a modified nucleic acid in one to several bases (e.g., one to three bases) at its 5'-end and one to several bases (e.g., one to three bases) at its 3'-end. In a preferred embodiment, the nucleic acid in the conjugate may further contain a modified nucleic acid in a region other than one to several bases (e.g., one to three bases) at its 5'-end and one to several bases (e.g., one to three bases) at its 3'-end.

[0075] <Medicinal Uses of Knockdown Nucleic Acids and Conjugates of the Present Disclosure> The knockdown nucleic acids of the present disclosure can be incorporated into cells, for example, in vitro or in vivo, to reduce the amount of SFTSV infecting the cells. Micelles, liposomes, or lipid nanoparticles (LNPs) containing the knockdown nucleic acids of the present disclosure are suitable for delivering the nucleic acids into cells. Furthermore, conjugates of the present disclosure containing the knockdown nucleic acids of the present disclosure are suitable for delivering the nucleic acids into cells. Therefore, micelles, liposomes, or lipid nanoparticles (LNPs) containing the knockdown nucleic acids of the present disclosure can be incorporated into cells, for example, in vitro or in vivo, to reduce the amount of SFTSV infecting the cells. Furthermore, the conjugates of the present disclosure can be incorporated into cells, for example, in vitro or in vivo, to reduce the amount of SFTSV infecting the cells. Thus, the present disclosure provides a composition comprising a knockdown nucleic acid of the present disclosure for use in reducing the amount of SFTSV that has infected a cell in vitro or in vivo. The present disclosure also provides a composition (preferably a pharmaceutical composition) comprising a micelle, liposome, or lipid nanoparticle (LNP) comprising a knockdown nucleic acid of the present disclosure for use in reducing the amount of SFTSV that has infected a cell in vitro or in vivo. The present disclosure also provides a composition (preferably a pharmaceutical composition) comprising a conjugate of the present disclosure for use in reducing the amount of SFTSV that has infected a cell in vitro or in vivo. The present disclosure further provides a composition (preferably a pharmaceutical composition) comprising a micelle, liposome, or lipid nanoparticle (LNP) comprising a knockdown nucleic acid of the present disclosure for use in treating SFTS in a subject. The present disclosure also provides a composition (preferably a pharmaceutical composition) comprising a conjugate of the present disclosure for use in treating SFTS in a subject.

[0076] The lipid nanoparticles are not particularly limited, and examples thereof include lipid nanoparticles described in U.S. Pat. Nos. 9,364,435B, 8,822,668B, 8,802,644B, and 8,058,069B2. Alternatively, mRNA may be encapsulated in a polyion complex micelle or a polyion complex polymersome (Miyata et al., Chem. Soc. Rev., 2012, 41, 2562-2574). Lipid nanoparticles can deliver encapsulated nucleic acids to, for example, B cells (e.g., Loomis et al., Exp. Mol. Pahol., 88(2):238-249, 2010).

[0077] The lipid nanoparticles include, but are not limited to, one or more, or preferably all, selected from the group consisting of ionized lipids (e.g., DLin-MC3-MDA, ALC-0315, and SM-102), PEG lipids (e.g., DMG-PEG (2000), and ALC-0159), phospholipids (e.g., 1,2-DSPC), and cholesterol. DLin-MC3-MDA represents (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid, ALC-0315 represents (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), and SM-102 represents (heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6 -(undecyloxy)hexyl)amino)octanoate}, 1,2-DSPC represents 1,2-distearoyl-sn-glycero-3-phosphocholine, DMG-PEG(2000) represents alpha-(3'-{[1,2-di(myristyloxy)propanoxy]carbonylamino}propyl)-ω-methoxy, polyoxyethylene, and ALC-0159 represents 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. Ionized lipids are neutral at physiological pH but cationic in an acidic environment.

[0078] The lipid nanoparticles preferably contain an ionized lipid (e.g., DLin-MC3-MDA, ALC-0315, and SM-102), a PEG lipid (e.g., DMG-PEG (2000), and ALC-0159), a phospholipid (e.g., 1,2-DSPC), and cholesterol, and the lipid molar ratio (ionized lipid / PEG lipid / phospholipid / cholesterol) may be 45-50 / 5-15 (preferably 8-10) / 35-45 (preferably 37-42) / 1-2 (provided that the total is 100 or less). For details of lipid nanoparticles, see, for example, Int. J. Pharm., 601:120586, 2021.

[0079] For example, Patisiran (Onpattro TM ) uses lipid nanovesicles containing siRNA as an active ingredient and contains Lin-MC3-DMA / 1,2-DSPC / DMG-PEG(2000) / cholesterol in a molar ratio of 50 / 10 / 38.5 / 1.5. Lipid nanoparticles containing lipids in this molar ratio may also be used in the present disclosure.

[0080] In one aspect, lipid nanoparticles comprising Lin-MC3-DMA, 1,2-DSPC, DMG-PEG(2000), and cholesterol are provided, which comprise a knockdown nucleic acid of the present disclosure, and the lipid nanoparticles preferably contain Lin-MC3-DMA / 1,2-DSPC / DMG-PEG(2000) / cholesterol in a molar ratio of 45-50 / 5-15 (preferably 8-10) / 35-45 (preferably 37-42) / 1-2 (provided that the total is 100 or less).

[0081] In one aspect, lipid nanoparticles comprising Lin-MC3-DMA, 1,2-DSPC, DMG-PEG(2000), and cholesterol are provided, comprising a knockdown nucleic acid of the present disclosure, wherein the lipid nanoparticles contain Lin-MC3-DMA / 1,2-DSPC / DMG-PEG(2000) / cholesterol, preferably in a molar ratio of 50 / 10 / 38.5 / 1.5.

[0082] The lipid nanoparticles can be obtained, for example, by preparing an alcohol solution containing a lipid mixture and an aqueous solution containing nucleic acid, and then mixing the two solutions using a microfluidic device, etc. The lipid nanoparticles can include, for example, but are not limited to, micelles (e.g., reverse micelles) in which nucleic acid is encapsulated by ionized lipid and cholesterol inside an outer shell formed by PEG lipid and cholesterol.

[0083] The present disclosure provides a method for treating severe fever with thrombocytopenia syndrome (SFTS) in a subject, the method comprising administering to the subject an effective amount of a knockdown nucleic acid of the present disclosure. The present disclosure also provides a method for treating SFTS in a subject, the method comprising administering to the subject an effective amount of a conjugate of the present disclosure. The present disclosure provides a method comprising administering to the subject an effective amount of a knockdown nucleic acid of the present disclosure or a conjugate of the present disclosure. In some aspects, the subject may be a subject not infected with SFTSV. In some aspects, the subject may be a subject infected with SFTSV. In some aspects, the subject may be a subject infected with SFTSV and who has developed SFTS. In some aspects, the subject may be a subject infected with SFTSV and who has not developed SFTS. By administering a knockdown nucleic acid or conjugate of the present disclosure to a subject, the severity of SFTS due to SFTSV infection in the subject may be reduced, the worsening of SFTS may be inhibited, the worsening of SFTS may be halted, and / or the symptoms of SFTS may be improved.

[0084] The present disclosure provides use of a knockdown nucleic acid of the present disclosure in the manufacture of a composition or medicament for use in reducing the amount of SFTSV that has infected a cell.The present disclosure provides use of a conjugate of the present disclosure in the manufacture of a composition or medicament for use in reducing the amount of SFTSV that has infected a cell.The present disclosure provides use of a conjugate of the present disclosure in the manufacture of a medicament for use in treating SFTS in a subject.

[0085] Experimental methods Reagents and experimental methods Cultured cells: HCT116 (American Type Culture Collection (ATCC), registration number: CCL-247) were cultured in McCoy's 5A medium (Gibco) supplemented with 10% heat-inactivated bovine serum; K562 cells (Japanese Collection of Research Bioresources (JCRB), registration number: JCRB0019) were cultured in 10% heat-inactivated bovine serum / RPMI-1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd.); Vero76 (JCRB, registration number: JCRB9007) and Vero 9013 (JCRB, registration number: JCRB9013) were cultured in EMEM (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 5% heat-inactivated bovine serum; and Huh7.5.1-8 cells (Jpn J Infect Dis. 2015;68(2):81-8) were cultured in DMEM medium (Fujifilm Wako Pure Chemical Industries) supplemented with 10% heat-inactivated bovine serum.

[0086] Reagents The reagents and experimental equipment used in this example are as follows: Plate reader: EnSight multimode plate reader (Perkin Elmer) Flow cytometer: BCM Analysis equipment: FACS Verse, FACS AriaII, FACSymphony A3 (BD ​​Biosciences) Transfection reagents: FuGENE 6 (Promega), Lipofectamine RNAi Max (Invitrogen) Transfection equipment (electroporator): NEPA21 (Nepgene)

[0087] siRNA / Chemically Modified siRNA. The sequences of siRNA targeting each segment of the severe fever with thrombocytopenia syndrome virus (SFTSV) genomic RNA are listed in Tables 1 to 5. The sequences of the negative control siRNA (siNC2), which does not target the host mRNA, and the siRNAs targeting the host are listed in Table 6. In Tables 1 to 6, the 5' end is on the left and the 3' end is on the right. N(M) indicates a 2'-OMe-modified base N, and N(F) indicates a 2'-F-modified base N. N^N indicates that the internucleotide phosphate group has been replaced with a phosphorothioate (N is A, U, G, or C). The anti-SFTSV siRNA numbers (represented by a "#" followed by a number) listed in Tables 1 to 6 correspond to the numbers in Figures 3A to 3D, 4A to 4G, 5A to 5F, and 6.

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094] - Plasmid for evaluating knockdown activity (psiCEHCK2, Promega) The S segment (AB817999), M segment (AB817991), and L segment (AB817983) of SFSTV were fused downstream of the Renilla luciferase gene cDNA of psiCHECK2.

[0095] The intracellular SFTSV genomic RNA was quantified as follows. Total intracellular RNA was collected using FABRK 001-2 (FAVORGEN). Reverse transcription quantitative PCR was performed using the THUNDERBIRD™ Probe One-step qRT-PCR Kit (TOYOBO) and the primer set listed in Table 7. This probe detects the positive and negative strands of the S segment of the SPL010 strain-derived SFTSV genomic RNA (AB817999.1).

[0096]

[0097] ・Evaluation of siRNA knockdown activity HCT116 cells were placed in a T-150 flask at 6 × 10 6 Cells were seeded at 20 mL / well and cultured at 37°C. The day after seeding, transfection with psiCHECK2 (25 nM) was performed. 110 μg of psiCHECK2, 99 μL of FuGENE6 (Promega), and 5.5 mL of Opti-MEMI (Gibco) were mixed and left to stand for 15 minutes. 5 mL of this solution was added to the seeded cells and cultured at 37°C. The day after psiCHECK2 transfection, siRNA was transfected. Equal volumes of a solution containing 5 pmol of siRNA and 15 μL of opti-MEMI were mixed with 0.3 μL of Lipofectamine RNAi Max (Thermo Fisher Scientific) and left to stand at room temperature for 20 minutes. The mixture was added at 20 μL / well to a 96-well plate, and 1 x 10 cells transfected with psiCHECK2 were added to the same well. 4 The cells were seeded at 100 μL per well and cultured at 37° C. The day after siRNA transfection, the activities of two types of luciferase were measured using the Dual-Luciferase® Reporter Assay System (Promega).

[0098] The viruses used in this application were obtained from SFTS patients at the National Institute of Infectious Diseases, and stocks were prepared as follows: The SFTSV-SPL010 strain was inoculated into Huh7.5.1-8 cells (Jpn J Infect Dis. 2015;68(2):81-8), and the culture supernatant was collected 5 days later. The collected SFTSV-SPL010 strain was serially diluted 10-fold and then inoculated into Huh7.5.1-8 cells. The formed foci were quantified, and the titer was determined (FFU / mL). The infectious titer (FFU / mL) was determined as follows: 2 x 10 Huh7.5.1-8 cells were inoculated into a 96-well plate. 4 The prepared SFTSV was seeded at 10 to 10 cells / well and cultured at 37°C for 24 hours. 11 The cells were infected with 100 mL of the diluted anti-SFTSV NP rabbit polyclonal antibody (J Infect Dis. 2014Mar 15;209(6):816-827) diluted 1:1,000 in PBS at 50 mL / well and incubated at 37°C for 2 hours. After 10 washes with tap water, 50 mL / well of Alexa Fluor 488-labeled anti-Rabbit pAb (Invitrogen) diluted 1:1,000 in 0.4% Evans blue was added and incubated at 37°C for 2 hours. After 10 washes with tap water, the number of SFTSV-positive cell foci was counted under a fluorescence microscope, and the FFU / mL was calculated based on the dilution factor.

[0099] Evaluation of siRNA antiviral activity The antiviral activity of siRNA was evaluated by transfecting with siRNA and then infecting with a virus. siRNA (25 pmol; transfection concentration: 25 nM) was mixed with 1.5 μL of Lipofectamine RNAi Max (Thermo Fisher Scientific) and 50 μL of opti-MEM (Gibco) and left to stand at room temperature for 20 minutes. The siRNA was diluted with opti-MEM (Gibco) to the amount to be added, and added at 50 μL / well. Vero9013 cells (JCRB Cell Bank, cell number: JCRB9013) were cultured at 5 × 10 4 After seeding at 500 μL / well, the cells were cultured at 37°C. 24 hours after transfection, the cells were infected with SFTSV-SPL010 strain (0.02 FFU / cell) at 37°C for 1 hour, washed three times with medium, and cultured at 37°C. 72 hours after SFTSV infection, total intracellular RNA was collected, and SFTSV genomic RNA was quantified by reverse transcription-quantitative PCR.

[0100] Experimental Results Example 1: Establishment of an SFTSV Infection Evaluation System and Evaluation of Susceptibility of Cultured Cells to Viral Infection. The susceptibility of Vero9013 cells (JCRB Cell Bank, cell number: JCRB9013), Vero76 cells (JCRB Cell Bank, cell number: JCRB9007), and human hematopoietic / lymphoid K562 cells (JCRB, JCRB0019) to SFTSV infection was evaluated. SFTSV infection susceptibility was compared with that of the positive control, human liver-derived Huh7.5.1-8 cells (Jpn J Infect Dis. 2015;68(2):81-8). As shown in Figures 1(a) and (b), Vero9013 cells had approximately five-fold higher SFTSV genomic RNA copy numbers both intracellularly and extracellularly than Vero76 cells. When SFTSV was infected into K562 cells, the number of SFTSV genome RNA copies in the cells was one-tenth of that in the positive control, and in the culture supernatant was equivalent to that of the control cells, indicating sufficient virus production ability.

[0101] Example 2: In silico selection of siRNA candidate sequences and multi-alliance analysis of viral nucleotide sequences. SFTSV genotypes are known to exist in China and Japan, with five genotypes in China and three genotypes in Japan (Tomoki et al., J Infect Dis. (2015) 15;212(6):889-98). Nucleotide sequence information for one strain from each source (Figure 2a) was obtained, and the homology of the nucleotide sequences between the Chinese and Japanese strains was evaluated. The homology between the strains in each segment was found to be high, at over 95%.

[0102] Selection of siRNA target sites: The homology of each strain was confirmed using multi-alliance analysis, and 50 siRNAs were selected whose seed regions, important for sequence specificity of knockdown activity, were conserved across strains (Fig. 2b). The siRNA sequences were designed so that they did not match the base sequence of host-derived mRNA (Fig. 2b). For example, the sequences were designed so that human mRNA would not be found in a Blast search (default parameters).

[0103] Example 3: Evaluation of siRNA knockdown effect using the psiCHECK system - Construction of psiCHECK constructs cDNAs derived from each segment of SFTSV were fused downstream of the Renilla luciferase cDNA of psiCHECK2 (Promega). The full-length S and M segments were inserted, and the L segment was divided into three parts, to construct psiCHECK2-based constructs.

[0104] ・siRNA design, synthesis, and knockdown activity evaluation The knockdown activity of the synthesized SFTSV-targeting siRNA (siSFTSV) was evaluated using the psiCHECK2 system. Experimental results (Figures 3A-3D) show that all siRNAs reduced gene expression to less than 20% of the non-targeting siNC2 control siRNA. Based on these results, all 50 of the designed siRNAs (Tables 1-3) had knockdown activity in the psiCHECK2 system.

[0105] Example 4: Verification of antiviral activity of candidate siRNAs against SFTSV infection - Development of a lipofection method for siRNA delivery in K562 cells. The antiviral activity of siSFTSV was examined in Vero cells, for which conditions for efficient lipofection transfection have already been established. As shown in Figure 4A(a), the knockdown efficiency of SFTSV genomic RNA by siRNA delivery in Vero9013 cells was less than 1% compared to the siNC2 control group for both siSFTSV-SPL010-rS2 (hereinafter referred to as "siSFTSV#2") and siSFTSV-SPL010-Su7r7a7 (hereinafter referred to as "siSFTSV#3") (right panel). To confirm that siSFTSV is also effective in human suspension cells, lipofection was performed on K562 cells, a human cultured cell line derived from a patient with chronic myeloid leukemia, despite the known low transfection efficiency of lipofection in suspension cell systems. In K562 cells, the transfection efficiency was 27% for siSFTSV#2 and 14% for siSFTSV#3 compared to the PBS control group (left panel). Based on these results, the antiviral activity of siRNA alone was effective in both suspension and adherent cells, so we decided to use Vero9013 cells, which are often used in virus research.

[0106] Antiviral activity screening of siSFTSV in Vero cells. The antiviral activity of the selected 50 siRNAs was evaluated by infecting siRNA-transfected Vero9013 cells with SFTSV and measuring intracellular SFTSV RNA copy number after 3 days. Figure 4A(b) shows that 5 of 7 S-segment siRNAs, 11 of 13 M-segment siRNAs, and 22 of 30 L-segment siRNAs reduced SFTSV genomic RNA copy number. These results demonstrated significant differences compared with siNC2, which does not target any specific target sequences, confirming their antiviral activity. Among these, two siRNAs targeting the S-segment (siSFTSV#2 and siSFTSV#3) demonstrated knockdown of SFTSV genomic RNA by up to 1%.

[0107] Next, to further investigate the antiviral activity of loci surrounding the two siRNAs with high antiviral activity, 16 additional siRNAs were synthesized and evaluated for their antiviral effects. The knockdown activity of the 16 synthesized siRNAs was confirmed using the psiCHECK2 system. Figures 4B–4D confirmed that all siRNAs knocked down expression levels to 80% or less, so the antiviral activity of each siRNA was evaluated. Figure 4E confirmed that siRNAs within the range of 91–838 of the S segment significantly reduced SFTSV genomic RNA copy number compared to the negative control siNC2. Among these, siSFTSV#2, siSFTSV#3, and siSFTSV-SPL010-Sre3-2 (hereafter simply referred to as "siSFTSV#Sre3-2") reduced SFTSV genomic RNA copy number to 10% or less. The region 91–838 of the S segment is located in the region encoding the nonstructural protein (N). These results demonstrate that targeting the N-encoding region provides particularly effective antiviral activity against SFTSV. Furthermore, we designed two siRNAs targeting not only the normal locus but also the complementary strand (inverted locus) of the SFTSV genomic RNA in AB817999.1. One is siSFTSV-Sr#2, which covers 579-601, and the other is siSFTSV-Sr#3, which covers 201-223. Each locus is based on AB817999.1. The knockdown activity of each siRNA was evaluated using psiCHECK. The results in panel (F) of Figure 4 confirm a concentration-dependent knockdown effect of siSFTSV-Sr#2 and #3. Next, we evaluated the antiviral activity of each siRNA. Figure 4(G) shows that siSFTSV-Sr#2 reduced SFTSV genomic RNA copy number by 27%, and siSFTSV-Sr#3 reduced it to less than 1%. These results demonstrate that siRNAs targeting the N protein coding region in both normal (antisense strand; guide strand) and inverted (sense strand; passenger strand) positions have antiviral effects. Although SFTSV is a minus-strand RNA virus, it synthesizes plus-strands during genome replication, which is why inverted siRNAs are thought to have a knockdown effect as described above.

[0108] Example 6: Chemical modification of nucleic acids and chemical modification design of siRNA. The chemical modification patterns of 2'-F or 2'-OMe sugars were examined for siSFTSV#2 and siSFTSV#3, which showed high antiviral effects. The knockdown and antiviral activities of siRNAs with six different chemical modification patterns (Figure 5A) were evaluated.

[0109] Evaluation of knockdown activity of chemically modified siRNAs. The knockdown activity of siRNAs with each chemical modification pattern against the SFTSV_S segment was evaluated using the psiCHECK2 system, as in Example 2. As shown in Figures 5B and 5C, knockdown activity was reduced to 20% or less at 10 nM for all chemical modifications. The concentration-dependent knockdown efficiency was similar for all chemically modified siRNAs. For non-chemically modified siRNAs, the knockdown activity against the SFTSV#S segment was also evaluated using the psiCHECK2 system, as in Example 2. As shown in Figures 5B and 5C, knockdown activity was reduced to 20% or less at 10 nM for all chemical modifications. This suggests that chemical modification of siRNA contributes to stabilization in blood, rather than to the effects of experiments using transfection reagents.

[0110] Evaluation of antiviral activity of chemically modified siRNAs. The antiviral activity of siRNAs with each chemical modification pattern was evaluated by lipofection of Vero9013 cells with siRNA and subsequent infection with SFTSV, as in Example 4, and by measuring the intracellular SFTSV genomic RNA copy number. The results, shown in the upper panel of Figure 5D, showed that all patterns reduced the SFTSV genomic RNA copy number to 20% or less compared to the siNC2 control. With siSFTSV#2, the SFTSV genomic RNA copy number was reduced to 10% or less compared to the siNC2 control except for chemical modification pattern E, with no significant difference between the chemical modification patterns. With siSFTSV#3, the SFTSV genomic RNA copy number was reduced to 30% or less compared to the siNC2 control. In all cases, the SFTSV genomic RNA copy number was reduced to 90% or less compared to the siNC2 control, with no significant difference between the chemical modification patterns (lower panel of Figure 5D).

[0111] The antiviral activity of non-chemically modified siRNA (siRNA consisting only of RNA) was also evaluated in the same manner as in Example 4. Vero9013 cells were lipofected with siRNA, then infected with SFTSV, and the intracellular SFTSV genomic RNA copy number was measured. As shown in Figures 5E and 5F, the SFTSV genomic RNA copy number was reduced by 6% for non-chemically modified siSFTSV#2 and by less than 1% for non-chemically modified siSFTSV#3 compared to the siNC2 group. These results confirm that the antiviral effect is independent of chemical modification.

[0112] Evaluation of antiviral activity of siRNA against infection with other SFTSV strains. 25 pmol of siRNA and 1.5 μL of Lipofectamine RNAi Max (Thermo Fisher Scientific) were mixed in 50 μL of Opti-MEM (Gibco) and left to stand at room temperature for 20 minutes. The mixture was further diluted with Opti-MEM (Gibco) to the desired siRNA amount, and the resulting dilution was added to each well at 50 μL / well. Vero9013 cells (JCRB Cell Bank, cell number: JCRB9013) were cultured at 5 × 10 4 Cells were seeded into each well at 500 μL / well and cultured at 37°C. Twenty-four hours after transfection, SFTSV-YG1 and SFTSV-HB29 strains (0.02 FFU / cell) were added to each well and infected at 37°C for 1 hour. After washing three times with medium, the cells were further cultured at 37°C. Seventy-two hours after SFTSV infection, total intracellular RNA was collected, and SFTSV genomic RNA was quantified by reverse transcription-quantitative PCR. As shown in Figure 6 , siSFTSV#2 reduced the SFTSV genomic RNA copy number to less than 20% compared to the siNC2 control. siSFTSV#3 reduced the SFTSV genomic RNA copy number to less than 40% compared to the siNC2 control.

[0113] In vitro therapeutic experiments: K562 cell clones were isolated. mCD71 was knocked into K562 cells. K562 cells were seeded at 3,000 cells / well and infected with SFTSV-SPL010 strain at 0.02 FFU / cell. The day after infection, CD71-binding Fab'-siRNA (RI7-siSFTSV#2) was added to the cells at a final siRNA concentration of 100 nM and the cells were cultured at 37°C. Three days after the addition of Fab'-siRNA (RI7-siSFTSV#2), the intracellular SFTSV genomic RNA copy number was measured by reverse transcription-quantitative PCR. The results are shown in Figure 6. Figure 7 shows that CD71-binding Fab'-siRNA (RI7-siSFTSV#2) reduced SFTSV RNA by more than 100-fold.

[0114] In vivo therapeutic experiments: Mice (AG129 mice) doubly deficient in interferon α, β, and γ receptors were intramuscularly inoculated with 2, 20, or 200 FFU of SFTSV-SPL010, and body weights were measured over time. The endpoints were as follows: mice were observed and weighed every 1–2 days after inoculation. Blood was collected from the heart under anesthesia (euthanasia) upon (i) a 20% or greater weight loss (or a 15% or greater weight loss within 1–2 days) or (ii) the development of SFTSV infection symptoms, such as paralysis or lameness. Survival rates are shown in Figure 8A, and weight changes over time are shown in Figure 8B. Mice died approximately 5–6 days after infection.

[0115] Next, we examined whether this was affected by administration of siSFTSV-Ab, which binds to CD71. Specifically, AG129 mice were administered siSFTSV-Ab (10 mg / kg / mouse as siRNA) via the tail vein at 125 μL / mouse. As a positive control, Favipiravir (60 mg / kg / mouse) was administered intraperitoneally at 500 μL / mouse. The following day, mice were inoculated intramuscularly with 2 FFU of SFTSV-SPL010 strain, and body weight was measured over time. The endpoints were as follows: mice were observed and weighed every 1–2 days after inoculation. At the time of (i) a 20% or greater weight loss (or a 15% or greater weight loss within 1–2 days) or (ii) symptoms of SFTSV infection, such as paralysis or difficulty walking, they were euthanized by cardiac blood sampling under anesthesia. Survival rates are shown in Figure 9A, and weight changes over time are shown in Figure 9B. The group administered Fab'-siRNA that binds to CD71 ("AOC(siSFTSV)") showed a significant prolongation of survival, demonstrating greater efficacy than Favipiravir, which was used as a positive control.

[0116] These results demonstrate that both targeting CD71 with antibody-siRNA and the delivered siRNA are effective in treating SFTSV.

[0117] Discussion: When designing antiviral siRNA in silico, it is possible to select sequences that do not match endogenous mRNA. Considering final administration to the living body, chemical modification of siRNA is essential. However, there is no in silico method for predicting effective siRNAs, including chemical modifications. Therefore, we synthesized chemically modified siRNAs and identified those with knockdown ability through screening. In virus infection experiments using cells, antiviral effects were confirmed in both suspension and adherent cells, but the target locus of the effective siRNA was limited to the S segment of SFTSV.

[0118] All documents cited herein are incorporated by reference in their entirety.

Claims

1. A knockdown nucleic acid that targets the S segment of the negative-strand single-stranded RNA genome of severe fever with thrombocytopenia syndrome virus (SFTSV) or the antisense strand (positive strand) of said region, and that exerts an antiviral effect against intracellular SFTSV after infection of cells.

2. The nucleic acid of claim 1, which targets the region 91 to 838 of the S segment of the RNA genome or the antisense strand (plus strand) of said region.

3. The nucleic acid of claim 1 or 2, which targets the region 150 to 300 of the S segment of the RNA genome or the antisense strand (plus strand) of said region; the region 450 to 650 of the S segment of the RNA genome or the antisense strand (plus strand) of said region; or the region 780 to 830 of the S segment or the antisense strand (plus strand) of said region.

4. The nucleic acid according to any one of claims 1 to 3, which is an siRNA.

5. A conjugate of a targeting molecule and a knockdown nucleic acid, wherein the targeting molecule and the knockdown nucleic acid are linked via a linker, the knockdown nucleic acid targets the RNA genome of severe fever with thrombocytopenia syndrome virus (SFTSV), and exerts an antiviral effect against intracellular SFTSV after infecting a cell.

6. The conjugate of claim 5, wherein the knockdown nucleic acid targets the S segment of the RNA genome.

7. The conjugate of claim 5 or 6, wherein the knockdown nucleic acid targets the region 91 to 838 of the S segment of the RNA genome.

8. The conjugate according to any one of claims 5 to 7, wherein the knockdown-type nucleic acid targets the region 200 to 240 of the S segment of the RNA genome or the antisense strand (plus strand) of said region; the region 400 to 750 of the S segment of the RNA genome or the antisense strand (plus strand) of said region; or the region 780 to 820 of the S segment of the RNA genome or the antisense strand (plus strand) of said region.

9. The conjugate according to any one of claims 5 to 8, wherein the knockdown-type nucleic acid is siRNA.

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