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WO2025216277A1 - Nouvel arn double brin basé sur la séquence d'arn du rsv-a et utilisation associée - Google Patents

Nouvel arn double brin basé sur la séquence d'arn du rsv-a et utilisation associée

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Publication number
WO2025216277A1
WO2025216277A1 PCT/JP2025/014296 JP2025014296W WO2025216277A1 WO 2025216277 A1 WO2025216277 A1 WO 2025216277A1 JP 2025014296 W JP2025014296 W JP 2025014296W WO 2025216277 A1 WO2025216277 A1 WO 2025216277A1
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Prior art keywords
sequence
rsv
double
rna
stranded rna
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PCT/JP2025/014296
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English (en)
Japanese (ja)
Inventor
徹彦 吉田
菜穂子 ベイリー小林
和雄 高山
里菜 橋本
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Toagosei Co Ltd
Kyoto University NUC
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Toagosei Co Ltd
Kyoto University NUC
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Publication of WO2025216277A1 publication Critical patent/WO2025216277A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • This disclosure relates to double-stranded RNA that inhibits the proliferation of respiratory syncytial virus (RSV), and its use.
  • RSV respiratory syncytial virus
  • RSV is a virus that infects humans. It causes symptoms such as fever, runny nose, and cough, and in severe cases can lead to bronchitis, pneumonia, asthma, and other conditions. RSV is distributed throughout the world, with almost no geographical or climatic bias. RSV infection poses a low risk of becoming severe in older children and adults. However, there is a high risk of the infection becoming severe in those with immunodeficiencies and in infancy.
  • RSV causes repeated infection and disease. It is believed that most infants are infected with RSV during infancy. There is no specific cure for RSV infection. Therefore, treatment generally involves symptomatic therapy such as oxygen administration, intravenous drip (or blood transfusion), or respiratory management. Therefore, there is a need for an effective treatment for RSV infection.
  • symptomatic therapy such as oxygen administration, intravenous drip (or blood transfusion), or respiratory management. Therefore, there is a need for an effective treatment for RSV infection.
  • Japanese Patent Application Laid-Open No. 2022-065140 discloses an RSV antibody pharmaceutical composition.
  • nucleic acid drugs can be mass-produced using organic synthesis, making it easy to control consistent quality.
  • the present disclosure therefore aims to provide double-stranded RNA that inhibits the proliferation of RSV.
  • the double-stranded RNA disclosed herein has a first strand and a second strand complementary to the first strand.
  • the first strand has a main sequence consisting of 19 to 23 bases, the 5'-terminal base of which is guanine (G) or cytosine (C), and an additional sequence consisting of 2 to 4 bases added to the 3'-terminal side of the main sequence.
  • the main sequence is a part of a base sequence encoding a fusion protein of RSV-A, and includes at least a part of a base sequence encoding the signal peptide region of the fusion protein.
  • the above-mentioned double-stranded RNA can function as at least small interfering RNA (siRNA). That is, such double-stranded RNA is predicted to induce RNA interference (RNAi).
  • RNAi RNA interference
  • the inventors speculated that RSV proliferation could be inhibited by suppressing the expression of specific RSV proteins through RNA interference (RNAi).
  • RNAi RNA interference
  • the inventors focused on the signal peptide region as the target sequence for RNAi. Because the signal peptide region is the part that directs the transport and localization of proteins, it can be said to be one of the regions essential for proteins to properly perform their inherent functions. This effect results in the suppression of RSV proliferation, as at least the expression of the F protein is suppressed.
  • RSV proliferation can be significantly suppressed by using double-stranded RNA having a base sequence that includes at least a portion of the base sequence encoding the signal peptide region of an RSV protein.
  • the inventors also discovered that the natural immune response is extremely low when the double-stranded RNA disclosed herein is introduced into cells.
  • the second strand has a main sequence complementary to the first strand and an additional sequence consisting of 2 to 4 bases added to the 3' end of the complementary main sequence.
  • Such double-stranded RNA can function favorably as siRNA, thereby more reliably suppressing the proliferation of RSV-A.
  • At least three of the seven bases on the 3' end of the main sequence are adenine (A) and/or uracil (U). This more fully suppresses the expression of the fusion protein, thereby inhibiting the proliferation of RSV-A.
  • the base sequence comprising at least a part of the base sequence encoding the signal peptide region of the fusion glycoprotein is the following base sequence: GUUGCUAAUCCUCAAAGCA (SEQ ID NO: 1); CUCAAAGCAAAUGCAAUUA (SEQ ID NO: 2); GCAAUUACCACAAUCCUCA (SEQ ID NO: 3); CCUCACUGCAGUCACAUUU (SEQ ID NO: 4); CACAUUUUGUUUUGCUUCU (SEQ ID NO: 5); GCUUCUGGUCAAAACAUCA (SEQ ID NO: 6); GUCAAACAUCACUGAAGA (SEQ ID NO: 7); GUCAAACAUCACUGAAGAAU (SEQ ID NO: 8);
  • Such double-stranded RNA more specifically suppresses the expression of the fusion protein. Furthermore, such double-stranded RNA can suppress the innate immune response when introduced into cells.
  • the base sequence constituting the additional sequence is thymine-thymine (TT). This improves the stability of the double-stranded RNA.
  • the present disclosure provides a composition for inhibiting the proliferation of RSV-A.
  • One embodiment of the composition disclosed herein contains the double-stranded RNA of the present disclosure.
  • the present disclosure provides a method for treating RSV infection in animals other than humans.
  • One aspect of the treatment method disclosed herein includes a step of administering a composition of the present disclosure to the animal other than humans.
  • FIG. 1 is a schematic diagram showing the major domains of the RSV-A F protein.
  • FIG. 2 is a graph showing the amount of RNA of nuclear proteins contained in extract samples from cells transfected with the double-stranded RNA of this embodiment and infected with RSV-A.
  • FIG. 3 is a graph showing the amount of interferon ⁇ RNA contained in extract samples from cells transfected with the double-stranded RNA of this embodiment and infected with RSV-A.
  • FIG. 4 is a graph showing the amount of interferon ⁇ RNA contained in extract samples from cells transfected with the double-stranded RNA of this embodiment and infected with RSV-A.
  • FIG. 1 is a schematic diagram showing the major domains of the RSV-A F protein.
  • FIG. 2 is a graph showing the amount of RNA of nuclear proteins contained in extract samples from cells transfected with the double-stranded RNA of this embodiment and infected with RSV-A.
  • FIG. 3 is a
  • FIG. 5 is a graph showing the amount of RNA of interferon-stimulated gene 15 contained in extract samples from cells transfected with the double-stranded RNA of this embodiment and infected with RSV-A.
  • FIG. 6 is a graph showing the amount of myxovirus resistance protein 1 RNA contained in extract samples from cells transfected with the double-stranded RNA of this embodiment and infected with RSV-A.
  • FIG. 7 is a graph showing the virus copy number after 3 days when RSV-A infected cells were transfected with the double-stranded RNA of this embodiment.
  • polynucleotide refers to a polymer in which multiple (two or more) nucleotides are linked by phosphodiester bonds, and is not limited by the number of nucleotides.
  • polynucleotides herein also include those containing both deoxyribonucleotides and nucleotides.
  • artificially designed polynucleotides refer to polynucleotides whose nucleotide chains (full length) do not exist alone in nature, but are artificially synthesized by chemical synthesis or biosynthesis (i.e., production based on genetic engineering).
  • first strand and second strand refer to one being the sense strand (or coding strand or passenger strand) and the other being the antisense strand (or template strand or non-coding strand or guide strand). That is, if the first strand is the sense strand, the second strand refers to the antisense strand.
  • the first strand and the second strand may be fully complementary to each other, or may be at least partially complementary. That is, they may be capable of hybridizing at least under physiological conditions.
  • the left side of a base sequence always indicates the 5'-end and the right side indicates the 3'-end.
  • amino acid residue includes the N-terminal amino acid and C-terminal amino acid of a peptide chain, unless otherwise specified.
  • the left side always indicates the N-terminal side and the right side indicates the C-terminal side.
  • RSV respiratory syncytial virus
  • respiratory syncytial virus is also referred to as respiratory syncytial virus, but is meant to encompass all synonyms, including, but not limited to, naturally occurring RSV as well as variants thereof, unless otherwise specified.
  • RSV is a human virus classified in the Pneumovirus genus of the Paramyxoviridae family. It is an enveloped RNA virus that replicates in the cytoplasm and matures by budding at the plasma membrane of host cells.
  • the RSV genome is a nonsegmented, negative-sense, single-stranded RNA.
  • the RSV genome is approximately 15 kbp and encodes 11 proteins, including nine structural proteins and two nonstructural proteins.
  • RSV possesses surface proteins, including an F protein (fusion protein), a G protein (attachment glycoprotein), and an SH protein (low molecular weight hydrophobic protein). Of these, the G protein and F protein are known to induce neutralizing antibodies.
  • the G protein and F protein are major transmembrane surface glycoproteins that regulate the initial stage of host cell infection.
  • RSV attaches to host cells via the G protein and enters the cell by fusing with the host cell membrane via the F protein. After RSV invades a host cell, RNA replication occurs via the N protein (nucleoprotein) and other components.
  • RSV is divided into two subtypes, type A and type B (RSV-A and RSV-B), based on differences in the reactivity of the G protein with monoclonal antibodies. These subtypes can be further classified into several genotypes, for example, based on the gene sequence of the C-terminal or second hypervariable region of the G protein.
  • Known genotypes of type A RSV i.e., RSV-A
  • RSV-A include GA1, GA2, GA3, GA4, GA5, GA6, GA7, SAA1, NA1, NA2, and ON1.
  • known genotypes of type B RSV include GB1, GB2, GB3, GB4, BA1, BA2, BA3, BA4, BA5, BA6, BA7, BA8, BA9, BA10, SAB1, SAB2, SAB3, SAB4, and URU1-2.
  • subtypes are classified into several genotypes depending on the gene sequence of the C-terminal end of the G protein or the second hypervariable region. Unlike the G protein, the gene sequence of the F protein tends to be conserved. Therefore, if double-stranded RNA targeting the signal peptide region can inhibit the proliferation of RSV-A, it is expected that it will also have a proliferation-inhibitory effect on other genotypes or strains of RSV-A.
  • the F protein is a type I glycoprotein that promotes fusion between the virus (here, RSV-A) and the host cell membrane.
  • the F protein consists of approximately 574 amino acid residues.
  • Figure 1 is a schematic diagram showing the major domains of the RSV-A F protein. As shown in Figure 1, the F protein is mainly composed of a signal peptide region, an extracellular domain, a transmembrane domain, and a cytoplasmic tail from the N-terminus. From the extracellular domain to the cytoplasmic tail, the F2 subunit, the pep27 region, and the F1 subunit are divided from the N-terminus.
  • the F protein has different structures before fusion with the host cell membrane (pre-F) and after fusion (post-F). More specifically, the F protein has an inactive precursor (F0). After the signal peptide is cleaved from F0, the pep27 region is cleaved by a furin-like protease, splitting it into F1 and F2. F1 and F2 form pre-F via a disulfide bond. Pre-F exists as a trimer. Furthermore, pre-F has several epitopes. However, pre-F is unstable and easily converts to post-F, which has a stable structure. This structural change allows the F protein to be inserted into the host cell membrane. However, this structural change results in the loss of some epitopes.
  • pre-F which has many epitopes
  • post-F does not have as many epitopes as pre-F, so antibodies are limited.
  • the double-stranded RNA of this embodiment can inhibit the expression of the F protein, for example, by RNAi, thereby inhibiting RSV-A membrane fusion and more reliably controlling the initial fusion process.
  • RSV is a single-stranded negative-strand virus.
  • the nucleotide sequence of RSV-A can be obtained from international databases.
  • international databases include the National Center for Biotechnology Information (NCBI), the European Nucleotide Archive (ENA), the DNA Data Bank of Japan (DDBJ), UniProt, Ensembl, etc.
  • NCBI National Center for Biotechnology Information
  • ENA European Nucleotide Archive
  • DDBJ DNA Data Bank of Japan
  • UniProt Ensembl
  • the nucleotide sequence of RSV-A is provided by NCBI under accession number KT992094.1, etc.
  • the amino acid sequence of the F protein is provided by UniProt under accession number P03420, etc.
  • Information on the signal peptide region of the F protein, etc. can also be obtained from the above-mentioned international databases.
  • the genome sequence of RSV-A is represented by thymine (T) instead of uracil (U).
  • T can be read as U, taking into consideration that RSV is a single-stranded negative-sense virus.
  • the amino acid sequence shown in SEQ ID NO: 9 is the entire amino acid sequence of the RSV-A F protein.
  • the amino acid sequence shown in SEQ ID NO: 10 consists of 25 amino acid residues and represents the amino acid sequence of the F protein signal peptide.
  • the base sequence shown in SEQ ID NO: 11 consists of 75 bases and represents the base sequence of the RSV-A signal peptide.
  • the double-stranded RNA of the present disclosure is a double-stranded RNA having a first strand and a second strand complementary to the first strand.
  • the first strand will be referred to as the sense strand and the second strand as the antisense strand, as will be described in detail.
  • the sense strand has a main sequence consisting of 19 to 23 bases, the 5'-terminal base of which is guanine (G) or cytosine (C), and an additional sequence consisting of 2 to 4 bases added to the 3'-terminal side of the main sequence.
  • the main sequence is a portion of a base sequence encoding RSV-A, and includes at least a portion of a base sequence encoding the signal peptide region of RSV-A.
  • the main sequence is typically composed of a polynucleotide, which is a polymer of ribonucleotides.
  • the main sequence is composed of RNA. That is, the base sequence of the main sequence is typically represented by the four letters A (adenine), U (uracil), G (guanine), and C (cytosine), or the four letters a, u, g, and c.
  • uracil can also be represented by T (thymine).
  • the main sequence of the sense strand can be, for example, a base sequence that includes a portion of the base sequence encoding the signal peptide region of RSV-A. This allows the double-stranded RNA to function as an siRNA (small interfering RNA) that targets RSV-A. Furthermore, because the base sequence of the RSV-A signal peptide region is located upstream of the mRNA, when the double-stranded RNA functions as an siRNA, it can effectively suppress the expression of RSV-A.
  • RNAi RNA interference
  • siRNAi is a gene silencing process in which short double-stranded RNAs such as siRNAs suppress gene expression in a sequence-specific manner.
  • siRNAs When siRNAs are introduced into cells, they form a complex with intracellular proteins called RISC (RNA-induced silencing complex). RISC binds to homologous sequences in mRNA transcribed from the target gene (here, the RSV-A gene) and specifically cleaves the mRNA, thereby inhibiting translation.
  • RISC RNA-induced silencing complex
  • the main sequence is preferably selected to include the signal peptide region of RSV-A or a base sequence encoding the signal peptide region of RSV-A, but one or more bases (e.g., two bases) may be substituted, deleted, and/or added (inserted) to other bases as long as the effects of the present technology are achieved.
  • one or more bases e.g., two bases
  • the proportion of the RSV-A signal peptide region or the base sequence encoding the RSV-A signal peptide region in the main sequence is preferably 5% or more, but may also be 10% or more, 15% or more, 90% or more, or 100% or more.
  • the 5' end of the main sequence is preferably guanine or cytosine. Because guanine and cytosine have stronger binding strength with complementary strands than adenine and uracil, the 5' end of the sense strand (i.e., the 3' end of the antisense strand) is more stable. In other words, the 5' end of the antisense strand is relatively less stable. While the details of this mechanism are unclear, RISC, an RNAi-related protein, tends to preferentially incorporate the sense strand or antisense strand, whichever has the more energetically unstable 5' end.
  • the antisense strand can be more easily incorporated into RISC, more effectively inducing RNAi. This allows the double-stranded RNA to function effectively as siRNA.
  • the antisense strand is more easily taken up by RISC, allowing for more effective induction of RNAi.
  • the GC content of the entire main sequence (the total proportion of G and C in the entire base sequence constituting the main sequence) is not particularly limited, but may be, for example, 20% to 60%, preferably 30% to 50%, or may be 30% to 45%.
  • the GC content is a parameter related to the binding strength between the antisense strand taken up by RISC and RNA comprising the main sequence, the ease of cleavage of the RNA, etc. With this GC content, the effect of RNAi can be efficiently exerted.
  • the main sequence can be selected from 19 to 23 bases from G or C of the gene encoding RSV-A.
  • the main sequence can be the following base sequence: GUUGCUAAUCCUCAAAGCA (SEQ ID NO: 1); CUCAAAGCAAAUGCAAUUA (SEQ ID NO: 2); GCAAUUACCACAAUCCUCA (SEQ ID NO: 3); CCUCACUGCAGUCACAUUU (SEQ ID NO: 4); CACAUUUUGUUUUGCUUCU (SEQ ID NO: 5); GCUUCUGGUCAAAACAUCA (SEQ ID NO: 6); GUCAAACAUCACUGAAGA (SEQ ID NO: 7); GUCAAACAUCACUGAAGAAU (SEQ ID NO: 8);
  • the base sequences shown in SEQ ID NOs: 1 to 8 are all composed of RNA.
  • double-stranded RNA When double-stranded RNA is introduced into cells, there is a possibility that nonspecific expression inhibition or nonspecific cell growth inhibition due to interferon response or the like may occur.
  • double-stranded RNA when double-stranded RNA is applied clinically, depending on the base sequence of the double-stranded RNA to be introduced, it may bind to various RNA receptors in cells and promote innate immune responses.
  • double-stranded RNAs whose main sequences are the base sequences shown in SEQ ID NOs: 1 to 8 cause very few side reactions (e.g., innate immune responses) when introduced into cells. Therefore, clinical applications are highly anticipated.
  • the base sequence shown in SEQ ID NO: 1 is the 6th to 24th bases of the base sequence encoding the RSV-A F protein (i.e., the sequence from the start codon to the stop codon).
  • the base sequence shown in SEQ ID NO: 2 is the 16th to 34th bases of the base sequence encoding the F protein.
  • the base sequence shown in SEQ ID NO: 3 is the 28th to 46th bases of the base sequence encoding the F protein.
  • the base sequence shown in SEQ ID NO: 4 is the 42nd to 60th bases of the base sequence encoding the F protein.
  • the base sequence shown in SEQ ID NO: 5 is the 54th to 72nd bases of the base sequence encoding the F protein.
  • the base sequence shown in SEQ ID NO: 6 is the 67th to 85th bases of the base sequence encoding the F protein.
  • the base sequences shown in SEQ ID NOs: 1 to 5 are base sequences selected from the base sequence encoding the signal peptide region of the RSV-A F protein.
  • SEQ ID NOs: 6 to 8 are portions of the base sequence encoding the RSV-A F protein, and include at least a portion of the base sequence encoding the signal peptide region of the F protein.
  • the nucleotide sequence from positions 1 to 6 of SEQ ID NO: 6 is a nucleotide sequence that encodes the signal peptide region.
  • nucleotide sequence from positions 7 to 19 of SEQ ID NO: 6 is a nucleotide sequence outside the signal peptide region.
  • the nucleotide sequence from positions 1 to 2 of SEQ ID NO: 7 is a nucleotide sequence that encodes the signal peptide region.
  • the nucleotide sequence from positions 3 to 19 of SEQ ID NO: 7 is a nucleotide sequence outside the signal peptide region.
  • SEQ ID NO: 8 is the nucleotide sequence of SEQ ID NO: 7 extended by two bases on the C-terminal side.
  • Double-stranded RNA consisting of the main sequences shown in SEQ ID NOs: 1 to 8 can suppress or inhibit the proliferation of RSV-A by supplying it to cells infected with RSV-A.
  • the RSV-A F protein is not expressed in uninfected, normal cells. Therefore, even if the double-stranded RNA disclosed herein is supplied to uninfected cells, it is believed that the double-stranded RNA will have little effect on normal cells.
  • the sense strand of the double-stranded RNA disclosed herein may have an additional sequence consisting of 2 to 4 bases added to the 5'-end or 3'-end of the main sequence.
  • the additional sequence is added to the 3'-end of the main sequence.
  • the additional sequence is composed of polynucleotides (dimers, trimers, or tetramers).
  • the polynucleotides that make up the additional sequence may be composed of only ribonucleotides, only deoxynucleotides, or both ribonucleotides and deoxynucleotides.
  • the sense strand and antisense strand may be entirely RNA, or may be chimeric polynucleotides of RNA and DNA.
  • the additional sequence may also contain modified deoxyribonucleotides, modified ribonucleotides, other known nucleotide analogs, etc.
  • the base sequence constituting the additional sequence is not particularly limited, but preferably contains at least one base: adenine, uracil, or thymine. Furthermore, from the perspective of improving the stability of the double-stranded RNA, it is more preferable that the base sequence constituting the additional sequence is TT (thymine-thymine).
  • the sense strand is composed of, for example, a base sequence of 21 to 27 bases, and may be composed of 21 to 25 bases, or 21 to 23 bases. In a preferred example, the sense strand is composed of 21 to 23 bases, consisting of a main sequence of 19 to 21 bases and an additional sequence of 2 bases. In such an example, RNAi can be effectively induced.
  • the antisense strand has a base sequence complementary to the main sequence of the sense strand. This allows the antisense strand to hybridize with the sense strand, forming a double-stranded structure.
  • the base sequence of the antisense strand may also be partially complementary to the main sequence of the sense strand. That is, one or more bases (e.g., two bases) of the antisense strand may be substituted, deleted, and/or added (inserted) with other bases. If the sense strand and the antisense strand can hybridize at least under physiological conditions, they can function as siRNA.
  • the complementary base sequence portion is typically composed of a ribonucleotide polymer (RNA).
  • the sense strand or antisense strand is typically composed of chemically unmodified ribonucleotides (RNA).
  • the double-stranded RNA of the present disclosure may also contain DNA, chemically modified DNA or RNA, other known nucleotide analogs, etc., to the extent that it does not significantly impair the technology of the present disclosure. That is, one or more bases (e.g., two bases) in the sense strand or antisense strand may be substituted with chemically modified RNA (or DNA) such as methylated or pseudouridylated.
  • chemically modified RNA include pseudouridine, N1-methylpseudouridine, 5-methylcytosine, and inosine.
  • one or more bases (e.g., two bases) of uridine in the double-stranded RNA of the present disclosure can be substituted with pseudouridine.
  • the antisense strand may have a main sequence complementary to the sense strand and an additional sequence consisting of 2 to 4 bases added to the 5' or 3' end of the complementary main sequence.
  • the additional sequence is preferably added to the 3' end of the complementary base sequence.
  • the additional sequence of the antisense strand is added to the 3' end of the complementary base sequence.
  • the configuration of the additional sequence in the antisense strand may be the same as the configuration of the additional sequence in the sense strand described above.
  • the base sequence of the additional sequence in the antisense strand is the same as the additional sequence in the sense strand to which it hybridizes, but it may also be a different base sequence.
  • the antisense strand is composed of a base sequence of, for example, 21 to 27 bases, and can be composed of 21 to 25 bases, or 21 to 23 bases.
  • the antisense strand is composed of a base sequence of the same length as the sense strand, and all or part of the base sequence, excluding the additional sequence, is composed of a base sequence that is complementary to the main sequence of the sense strand.
  • the antisense strand is composed of a base sequence of the same length as the sense strand, and all of the base sequence, excluding the additional sequence, is composed of a base sequence that is complementary to the main sequence of the sense strand.
  • the sense and antisense strands constituting the double-stranded RNA disclosed herein can be produced according to common chemical synthesis methods. For example, they can be synthesized using a commercially available DNA/RNA automatic synthesizer. Alternatively, the sense and antisense strands may be synthesized in vitro or in vivo based on genetic engineering techniques. The synthesized sense and antisense strands are preferably purified, and can be purified, for example, by HPLC.
  • the double-stranded RNA disclosed herein can be produced, for example, by annealing (hybridizing) a sense strand and an antisense strand.
  • Annealing can be performed in accordance with conventional methods.
  • annealing can be performed by mixing equal amounts of the sense strand and antisense strand in a solvent, heating at 90°C for 1 to 5 minutes, and then cooling to 4°C to room temperature.
  • solvents that can be used include distilled water, pure water, ultrapure water, and buffers (e.g., HEPES-KOH buffer at pH 7.4, PBS, etc.).
  • buffers e.g., HEPES-KOH buffer at pH 7.4, PBS, etc.
  • double-stranded RNA also includes those in which the first strand and the second strand form a locally double-stranded structure via a loop structure.
  • the double-stranded RNA can also be used as shRNA (short hairpin RNA) in another embodiment.
  • shRNA is an RNA in which a main sequence and its complementary sequence exist on a single strand, and a loop sequence exists to form these loop structures.
  • the shRNA has a loop structure, which allows the main sequence and its complementary sequence to hybridize and form a locally double-stranded structure. This allows the shRNA to be processed by Dicer, an enzyme present in cells, to form the siRNA of this embodiment.
  • the structure of the shRNA may be the same as that of conventionally known shRNA.
  • the length of the shRNA may be, for example, 50 to 70 bases.
  • the length of the loop sequence may be, for example, 19 to 29 bases.
  • the shRNA may be incorporated into a vector (e.g., a lentiviral expression vector). Use of the shRNA can stably induce RNAi in cells, thereby stably suppressing viral proliferation.
  • compositions disclosed herein contain the double-stranded RNA described above.
  • the compositions disclosed herein may contain one or more of the double-stranded RNAs described above.
  • the compositions may contain various pharmaceutically acceptable carriers depending on the intended use.
  • Preferred carriers include those commonly used in pharmaceuticals as diluents, excipients, etc.
  • the carriers vary depending on the intended use and form of the composition. Typical examples include water, physiological buffer solutions, various organic solvents, etc.
  • the carriers may also include aqueous solutions of alcohol (e.g., ethanol) at appropriate concentrations, glycerol, non-drying oils such as olive oil, or liposomes.
  • secondary components that may be contained in pharmaceutical compositions include various fillers, extenders, binders, humectants, surfactants, dyes, fragrances, etc.
  • the compositions may also contain carriers used in conventional drug delivery systems (DDS).
  • DDS drug delivery systems
  • compositions disclosed herein are not particularly limited. Typical composition forms include solutions, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, and ointments. Furthermore, for injections and the like, the compositions may be freeze-dried or granulated, so that they can be dissolved in physiological saline or an appropriate buffer solution (e.g., PBS) immediately before use to prepare a medicinal solution. Furthermore, the process of preparing various forms of drugs (compositions) using double-stranded RNA (main component) and various carriers (minor components) may conform to conventionally known methods. Since such formulation methods do not characterize the present disclosure, detailed explanations are omitted. A source of detailed information regarding formulations is, for example, *Comprehensive Medicinal Chemistry*, edited by Corwin Hansch, published by Pergamon Press (1990).
  • compositions disclosed herein inhibit at least the proliferation of RSV-A. Accordingly, methods for treating RSV-A infection are provided.
  • the treatment methods include administering the compositions to humans and/or non-human animals.
  • RSV-A is a virus that can infect not only humans but also non-human animals. Examples of such animals include mammals such as monkeys, cows, sheep, and goats.
  • composition disclosed herein includes, in addition to the double-stranded RNA described above, a peptide fragment (cell-penetrating peptide, CPP) with cell membrane permeability that allows it to pass through the cell membrane from outside the cell and introduce foreign substances into the cytoplasm.
  • the peptide fragment is directly or indirectly bound (linked) to the double-stranded RNA disclosed herein to construct a construct of the peptide fragment and double-stranded RNA.
  • double-stranded RNA is negatively charged and therefore cannot pass through the cell membrane.
  • the construct of the peptide fragment and the double-stranded RNA can be introduced into the cytoplasm.
  • the number of amino acid residues in the peptide fragment is not limited as long as cell membrane permeability is not impaired.
  • linker is placed between the peptide fragment and the double-stranded RNA.
  • the type of linker is not particularly limited. Typically, it is a peptidic linker, a non-peptidic linker, or the like.
  • the method for binding the peptide fragment and the double-stranded RNA is not particularly limited, and can be carried out according to various conventionally known scientific techniques.
  • composition disclosed herein comprises a peptide fragment and the double-stranded RNA disclosed herein.
  • the double-stranded RNA does not have to be bound to the N- or C-terminus of the peptide fragment.
  • the double-stranded RNA and the peptide fragment may form a complex, for example, through electrical or molecular interaction. This complex is more easily introduced into eukaryotic cells, allowing for efficient introduction of the double-stranded RNA.
  • Nucleic acids such as double-stranded RNA are typically negatively charged. Therefore, the peptide fragment used preferably has a high proportion of basic amino acids and is positively charged. Furthermore, the proportion of the peptide fragment in this case may be 5 to 100 times the molar amount of the double-stranded RNA, preferably 40 to 60 times.
  • the present disclosure may provide a method for inhibiting the proliferation of RSV-A using the composition disclosed herein, which includes the steps of preparing the composition disclosed herein and delivering the composition to a target cell.
  • composition disclosed herein may be prepared by a conventionally known method, for example, as described above.
  • the composition disclosed herein is supplied to at least RSV-A infected cells in vivo or outside the body in vitro.
  • the animal species to which the cells are supplied is not particularly limited, and may be, for example, mammals, birds, amphibians, reptiles, fish, etc. Note that, although cells other than RSV-A infected cells may be present at the destination of the composition, the composition may be supplied only to the target cells (i.e., RSV-A infected cells).
  • the method of administering the composition is not particularly limited and may be similar to methods conventionally used in animal treatment.
  • the composition can be administered in vivo in a manner and dosage appropriate for its form and purpose.
  • a liquid it can be administered in the desired amount to the affected area (e.g., malignant tumor tissue, virus-infected tissue, inflammatory tissue, etc.) of a patient or animal (i.e., a living organism) by intravenous, intralymphatic, intramuscular, subcutaneous, intradermal, or intraperitoneal injection.
  • a solid form such as a tablet, or a gel or aqueous jelly such as an ointment
  • a solid form such as a tablet, or a gel or aqueous jelly such as an ointment
  • the affected area e.g., the affected area of a tissue or organ containing tumor cells, inflammatory cells, etc.
  • a solid form such as a tablet can be administered orally.
  • encapsulation or the application of a protective (coating) material is preferred to prevent degradation by digestive enzymes in the digestive tract.
  • the amount of the composition to be supplied is not particularly limited.
  • the lower limit of the amount of double-stranded RNA per kg of animal may be 0.01 mg or more, 0.05 mg or more, or 0.1 mg or more.
  • the upper limit of the amount of double-stranded RNA per kg of animal may be, for example, 10 mg or less, 5 mg or less, or 1 mg or less.
  • the amount of the composition to be supplied is not particularly limited.
  • the lower limit of the double-stranded RNA concentration may be, for example, 1 nM or more, 5 nM or more, or 10 nM or more.
  • the upper limit of the double-stranded RNA concentration in such culture medium may be, for example, 10 ⁇ M or less, 5 ⁇ M or less, 2 ⁇ M or less, 1 ⁇ M or less, or 100 nM or less.
  • compositions disclosed herein can be delivered to the interior of target cells using known transfection methods. Examples include chemical gene transfer methods using cationic molecules (such as commercially available transfection reagents), physical transfer methods such as microinjection and electroporation, and biological gene transfer methods using viruses. Furthermore, as mentioned above, the compositions may also be delivered to the interior of cells using cell membrane-permeable peptide fragments.
  • the sense strand of the siRNA in Sample 1 is composed of a main sequence consisting of SEQ ID NO: 1 (a base sequence including a portion of the base sequence encoding the signal peptide region of the RSV-A F protein) and an additional sequence consisting of TT added to the 3' end of the main sequence.
  • the sense strands of the siRNA in Samples 2 to 8 shown in Table 1 are composed of a main sequence consisting of SEQ ID NOs: 2 to 8 (a base sequence including a portion of the base sequence encoding the signal peptide region of the F protein) and an additional sequence consisting of TT added to the 3' end of the main sequence.
  • the antisense strand in each example is composed of a sequence complementary to the main sequence and an additional sequence consisting of TT added to the 3' end of the sequence. Note that in Control 1 and Control 3, described below, AccuTarget Negative Control siRNA (SN-1012, manufactured by BIONEER) was used as the siRNA.
  • HEp-2 cells a human laryngeal carcinoma-derived cell line, were used as the cells to be infected with the virus.
  • the culture medium for HEp-2 cells was Dulbecco's minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS).
  • DMEM Dulbecco's minimal essential medium
  • FBS 10% fetal bovine serum
  • the prepared siRNA was added to the wells using the transfection reagent Lipofectamine® RNAiMAX (Thermo Fisher Scientific) to a concentration of 50 nM in the medium in the wells, and then cultured at 37°C in a 5% CO environment for 6 hours.
  • Lipofectamine® RNAiMAX Thermo Fisher Scientific
  • Samples 2 to 6 were prepared in the same manner as Sample 1, except that the siRNA in Sample 1 was changed to Samples 2 to 6 shown in Table 1.
  • Control 1 was transfected with a control siRNA that did not target a specific gene, except for the above, which was the same as Sample 1. That is, Control 1 represents a test example in which no siRNA targeting a specific gene was transfected.
  • Control 2 was the same as Sample 1 except that no virus was introduced. That is, Control 1 represents a test example without RSV infection.
  • N protein is a protein that binds to the RSV genome. Its sequence is highly conserved and constitutively expressed. Therefore, low levels of N protein RNA indicate suppression of RSV-A proliferation (increase in genomic RNA).
  • SYBR Green Real-Time PCR Master Mix (Thermo Fisher Scientific) was used as the qRT-PCR reagent, and the same PCR machine as above was used. Primers targeting the internal region of the N protein (SEQ ID NO:28 and SEQ ID NO:29) were used (see Table 2).
  • qRT-PCR targeting human GAPDH was performed to standardize the qRT-PCR (primers SEQ ID NO:38 and SEQ ID NO:39).
  • Figure 2 shows relative values, with Control 1 set to 1.
  • Figures 3 to 6 show relative values, with Control 2 set to 1. 2 to 6, the smaller the value on the vertical axis, the lower the expression level of the target gene.
  • the amount of N protein RNA was significantly reduced in Samples 1 to 6 compared to Control 1. This indicates that Samples 1 to 6 inhibit the proliferation of RSV-A. Furthermore, the amount of N protein RNA in Samples 1 and 4 to 6 was reduced to approximately one-tenth of that in Control 1, demonstrating a significantly high inhibitory effect on RSV-A proliferation.
  • ⁇ Amount of human IFNA1 RNA> The amount of human IFNA1 RNA in cell extract samples was quantified by qRT-PCR. The quantification method was the same as for the N protein RNA amount described above, except that the primers used were changed to SEQ ID NO: 30 and SEQ ID NO: 31.
  • IFNA1 refers to interferon ⁇ , a type of type I interferon.
  • IFNA1 is a cytokine induced by viral infection, etc.
  • IFNA1 expression is involved in the innate immune response. Therefore, low levels of IFNA1 RNA indicate suppression of the cellular innate immune response. As shown in Figure 3, IFNA1 RNA levels were significantly reduced in Samples 1 to 6 compared to Control 1.
  • Samples 1 to 6 were shown to reduce the innate immune response induced by viral infection.
  • IFNA1 RNA levels were significantly elevated in Control 1 compared to Control 2, which was not infected with a virus. This is thought to be due to the induction of IFNA1 by viral infection.
  • the amount of IFNA1 RNA was reduced to approximately one-fourth of that in Control 1, demonstrating a significantly high inhibitory effect on the innate immune response.
  • the amount of IFNA1 RNA was reduced to the same level as in Control 2, which was not infected with a virus.
  • ⁇ Amount of human IFNB1 RNA> The amount of human IFNB1 RNA in cell extract samples was quantified by qRT-PCR. The quantification method was the same as for the N protein RNA amount described above, except that the primers used were changed to SEQ ID NO: 32 and SEQ ID NO: 33.
  • IFNB1 refers to interferon beta, a type of type I interferon.
  • IFNB1 is a cytokine induced by viral infection, etc.
  • IFNB1 expression is involved in the innate immune response. Therefore, low levels of IFNB1 RNA indicate suppression of the cellular innate immune response. As shown in Figure 4, IFNB1 RNA levels were significantly reduced in Samples 1 to 6 compared to Control 1.
  • Samples 1 to 6 were shown to reduce the innate immune response induced by viral infection.
  • IFNB1 RNA levels were significantly elevated in Control 1 compared to Control 2, which was not infected with a virus. This is thought to be due to the induction of IFNB1 by viral infection.
  • the IFNB1 RNA level was reduced to approximately one-fifth compared to Control 1, demonstrating a significantly high inhibitory effect on the innate immune response.
  • the IFNB1 RNA level was reduced to the same level as in Control 2, which was not infected with a virus.
  • ISG15 refers to interferon-stimulated gene 15. ISG15 is induced by viral infection, etc. ISG15 expression is involved in the innate immune response. Therefore, low levels of ISG15 RNA indicate suppression of the cellular innate immune response. As shown in Figure 5, ISG15 RNA levels were significantly reduced in Samples 1 to 6 compared to Control 1. Therefore, Samples 1 to 6 were shown to reduce the cellular innate immune response.
  • Control 1 showed significantly increased ISG15 RNA levels compared to Control 2, which was not infected with a virus. Furthermore, ISG15 RNA levels were reduced to approximately one-sixth of those in Control 1 in Samples 1 and 2 and Samples 4 to 6, demonstrating a significantly enhanced suppression of the innate immune response. Furthermore, in samples 1 and 2 and samples 4 to 6, the amount of ISG15 RNA was reduced to the same level as in control 2, which was not infected with a virus.
  • ⁇ Amount of human MX1 RNA> The amount of human MX1 RNA in cell extract samples was quantified by qRT-PCR. The quantification method was the same as for the N protein RNA amount described above, except that the primers used were changed to SEQ ID NO: 36 and SEQ ID NO: 37.
  • MX1 encodes MxA. MxA refers to myxovirus resistance protein 1. MxA expression is induced by viral infection, etc. Therefore, a low level of MX1 RNA indicates that viral proliferation is suppressed. As shown in Figure 6, the amount of MX1 RNA was significantly increased in Control 1 compared to Control 2, which was not infected with a virus. The amount of MX1 RNA was significantly reduced in Samples 1 to 6 compared to Control 1. Furthermore, the amount of MX1 RNA in Samples 1 to 6 was reduced to the same level as in Control 2, which was not infected with a virus. Therefore, Samples 1 to 6 were shown to suppress RSV-A proliferation.
  • the siRNAs in samples 1 to 6 have the effect of suppressing the proliferation of RSV-A. Furthermore, it is believed that the siRNAs in samples 1 to 6 have the effect of suppressing the natural immune response in viral infection of cells. Therefore, the siRNAs in samples 1 to 6 induce a small natural immune response, and are expected to be suitable for clinical application.
  • the virus used was human RSV, RSV/Sendai/28-30 (subgroup A).
  • Human iPS cell-derived lung organoids were used as the cells to be infected with the virus.
  • Dulbecco's minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS) was used as the culture medium.
  • DMEM Dulbecco's minimal essential medium
  • FBS fetal bovine serum
  • the prepared siRNA was added to the wells using the transfection reagent Lipofectamine® RNAiMAX (Thermo Fisher Scientific) to a concentration of 50 nM in the medium in the wells, and then cultured at 37°C in a 5% CO environment for 6 hours.
  • Lipofectamine® RNAiMAX Thermo Fisher Scientific
  • the human iPS cell-derived lung organoids cultured in the wells were infected with RSV-A at 0.1 MOI.
  • the wells were then washed with PBS, and the culture medium was added and further cultured for 3 days.
  • the medium (supernatant) in the wells was then collected.
  • the collected supernatant was then mixed with an equal volume of 2x RNA lysis buffer (0.4 ⁇ L SUPERase I® RNase Inhibitor (Thermo Fisher Scientific), 2% Triton X-100, 50 mM KCl, 100 mM TCl-HCl (pH 7.4), 40% glycerol) and allowed to stand at room temperature for 10 minutes.
  • Control 3 represents a test example in which no siRNA targeting a specific gene was introduced.
  • the amount of RSV-A N protein RNA in the supernatant samples prepared above was quantified by qRT-PCR.
  • the One Step TB green PrimeScript PLUS RT-PCR kit (Perfect Real Time) (Takara Bio Inc.) was used as the qRT-PCR reagent, and the PCR machine was the same as above. Primers targeting the internal region of the N protein (sequence numbers 40 and 41) were used (see Table 3).
  • the probe used here had the base sequence shown in sequence number 42.
  • the probe was modified with FAM (5-Carboxyfluorescein) at the 5'-end and TAMRA (5-Carboxytetramethylrhodamine) at the 3'-end.
  • FIG. 7 is a graph showing the virus copy number three days after transfection of RSV-A infected cells with the siRNA of this embodiment. In Figure 7, a smaller value on the vertical axis indicates a smaller amount of RSV-A in the supernatant sample. Compared to control 3, the siRNA in samples 7 and 8 significantly reduced the virus copy number. Therefore, it is believed that the siRNA in samples 7 and 8 has the effect of suppressing the proliferation of RSV-A.
  • Item 1 A double-stranded RNA having a first strand and a second strand complementary to the first strand, wherein the first strand has a main sequence consisting of 19 to 23 bases, the 5'-terminal base of which is guanine (G) or cytosine (C), and an additional sequence consisting of 2 to 4 bases added to the 3'-terminal side of the main sequence, wherein the main sequence is a portion of a base sequence encoding a fusion protein of RSV-A and includes at least a portion of a base sequence encoding a signal peptide region of the fusion protein.
  • G guanine
  • C cytosine
  • Item 2 The double-stranded RNA described in Item 1, wherein the second strand has a main sequence complementary to the first strand and an additional sequence consisting of 2 to 4 bases added to the 3' end of the complementary main sequence.
  • Item 3 The double-stranded RNA according to Item 1 or 2, wherein at least three of the seven bases on the 3'-terminal side of the main sequence are adenine (A) and/or uracil (U).
  • A adenine
  • U uracil
  • the base sequence comprising at least a part of the base sequence encoding the signal peptide region of the fusion glycoprotein is the following base sequence: GUUGCUAAUCCUCAAAGCA (SEQ ID NO: 1); CUCAAAGCAAAUGCAAUUA (SEQ ID NO: 2); GCAAUUACCACAAUCCUCA (SEQ ID NO: 3); CCUCACUGCAGUCACAUUU (SEQ ID NO: 4); CACAUUUUGUUUUGCUUCU (SEQ ID NO: 5); GCUUCUGGUCAAAACAUCA (SEQ ID NO: 6); GUCAAACAUCACUGAAGA (SEQ ID NO: 7); GUCAAACAUCACUGAAGAAU (SEQ ID NO: 8); Item 4.
  • the double-stranded RNA according to any one of Items 1 to 3, comprising:
  • Item 5 The double-stranded RNA according to any one of Items 1 to 4, wherein the base sequence constituting the additional sequence is thymine-thymine (TT).
  • TT thymine-thymine
  • Item 6 A composition for inhibiting the proliferation of RSV-A, comprising the double-stranded RNA described in any one of Items 1 to 5.
  • Item 7 A method for treating RSV infection, comprising administering the composition described in Item 6 to an animal other than a human.

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Abstract

La présente invention concerne un ARN double brin présentant un premier brin et un second brin complémentaire du premier brin. Le premier brin présente : une séquence principale qui présente 19 à 23 nucléotides, le nucléotide en position 5' étant la guanine (G) ou la cytosine (C) ; et une séquence supplémentaire ajoutée à l'extrémité 3' de la séquence principale, la séquence supplémentaire comprenant 2 à 4 nucléotides. La séquence principale comprend au moins une partie d'une séquence nucléotidique codant pour une protéine de fusion de RSV-A, cette partie étant une partie de la séquence nucléotidique codant pour une région peptidique signal de la protéine de fusion.
PCT/JP2025/014296 2024-04-12 2025-04-10 Nouvel arn double brin basé sur la séquence d'arn du rsv-a et utilisation associée Pending WO2025216277A1 (fr)

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