WO2025143000A1 - Novel double-stranded rna based on c3 rna sequence and use thereof - Google Patents

Abstract

A 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 that comprises 19-23 bases including guanine (G) or cytosine (C) as a base at the 5' end, and an additional sequence that comprises 2-4 bases added to the 3' end side of the main sequence. The main sequence includes at least a portion of a base sequence encoding a complement component C3, said portion encoding the signal peptide region of the complement component C3.

Description

C3. Novel double-stranded RNA based on RNA sequence and its use

The present disclosure relates to double-stranded RNA and compositions containing the double-stranded RNA, and methods of using the same. Specifically, the present disclosure relates to double-stranded RNA used to suppress or inhibit tumor cell proliferation or metastasis, and compositions comprising the double-stranded RNA. This application claims priority based on Japanese Patent Application No. 2023-221596, filed on December 27, 2023, the entire contents of which are incorporated herein by reference.

The complement system is a part of the innate immune system that protects the body from infection by pathogens such as bacteria and viruses. It is made up of dozens of interacting proteins. Complement system proteins are mainly produced in the liver and circulate in the blood and extracellular fluid. Most of the complement system proteins are normally inactive, but are activated by infection with bacterial and viral pathogens. There are three complement activation pathways: the classical pathway (first pathway), the lectin pathway (mannose-binding lectin pathway), and the alternative pathway (second pathway). Activation of the complement system leads to opsonization, migration of phagocytes and lymphocytes, and elimination of pathogens by the membrane attack complex (MAC). However, excessive activation and inappropriate regulation of complement have been suggested to be involved in autoimmune diseases, inflammatory diseases, and tumor cell proliferation and metastasis.

The early components of each of the above pathways act locally, but all of the pathways converge on the activation of complement component C3 (hereinafter simply referred to as "C3"). In the activation of C3, C3 is degraded into C3b and C3a. C3b binds covalently to the surface of the pathogen and attracts cleavage fragments of C2 and C4 or complement factor B, etc. to form a complex. The complex with C3b cleaves the late component C5 into C5a and C5b. The cleaved C5b binds to C6, 7, 8, and 9 to form MAC. C3a and C5a act alone as diffusible signals, attracting phagocytes and lymphocytes to the site of infection and promoting inflammatory reactions. As described above, C3 plays a central role in the complement activation pathway, and therefore pharmaceutical compositions that inhibit the activation of C3 and the expression of C3 have been disclosed. Published Japanese Translation of PCT International Publication No. 2004-520287 discloses an antibody pharmaceutical composition that targets C3. In addition, JP 2009-521234 A discloses siRNA that targets C3.

Special Publication No. 2009-520287 Special Publication No. 2009-521234

Antibody drugs and the like are expensive, and it is difficult to maintain consistent quality. Nucleic acid drugs can be mass-produced through organic synthesis, making it easy to control the consistency of quality. The inventors focused on nucleic acid drugs, and in particular on the signal peptide region of C3, which plays a central role in the complement system.

The main objective of this disclosure is to provide a technology for suppressing or inhibiting cell proliferation involving gene expression of complement component C3.

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. Here, the main sequence is a part of the base sequence encoding complement component C3, and is determined from a base sequence that includes at least a part of the base sequence encoding the signal peptide region of complement component C3.

The above double-stranded RNA can function at least as small interfering RNA (siRNA). In other words, such double-stranded RNA is predicted to induce RNA interference (RNAi). This effect is to inhibit the proliferation of cells in which complement components are involved, at least by suppressing the expression of complement component C3.

In one embodiment of the double-stranded RNA disclosed herein, 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. This makes it possible to more reliably inhibit the proliferation of cells in which complement components are involved.

In one embodiment of the double-stranded RNA disclosed herein, at least three of the five bases on the 3' end of the main sequence are adenine (A) and/or uracil (U). This more fully suppresses the expression of complement component C3, thereby inhibiting the proliferation of cells in which complement components are involved.

In one embodiment of the double-stranded RNA disclosed herein, the base sequence encoding the complement component C3 is the following base sequence:
GCCTGCTGCTCCTGCTTACT (SEQ ID NO: 1);
CCTGCTGCTCCTGCTA CTA (SEQ ID NO: 2);
CTGCTGCTCCTGCTACTAA (SEQ ID NO:3);
CTCTGGGGAGTCCCATGTA (SEQ ID NO: 4);
Such double-stranded RNA more specifically inhibits the expression of complement component C3, thereby making it possible to inhibit the proliferation of cells in which the expression level of complement component C3 is increased.

In one embodiment of the double-stranded RNA disclosed herein, the base sequence constituting the additional sequence is thymine-thymine (TT). This can improve the stability of the double-stranded RNA.

The present disclosure provides a composition capable of inhibiting the proliferation of at least one type of cell. One embodiment of the composition disclosed herein comprises a first strand and a second strand complementary to the first strand, the first strand having a main sequence of 19 to 23 bases, the 5'-terminal base of which is guanine (G) or cytosine (C), and an additional sequence of 2 to 4 bases added to the 3'-terminal side of the main sequence. Here, the main sequence is a part of a base sequence encoding complement component C3, and includes double-stranded RNA determined from a base sequence including at least a part of a base sequence encoding a signal peptide region of complement component C3. When such a composition is supplied to a cell, the expression of at least complement component C3 is suppressed, thereby inhibiting the proliferation of cells in which the complement component is involved.

In one embodiment of the composition disclosed herein, the cell type whose proliferation is inhibited by the composition is a tumor cell. This allows for more reliable inhibition of cell proliferation.

One aspect of the composition disclosed herein includes a peptide fragment that has cell membrane permeability and can pass through the cell membrane from the outside of the cell to introduce a foreign substance into the cytoplasm. This makes it easier to introduce the double-stranded RNA into the target cell.

The present disclosure provides a method for inhibiting the proliferation of at least one type of cell. One aspect of the method disclosed herein includes the steps of (1) preparing a composition disclosed herein, and (2) supplying the composition to a target cell in vitro. This makes it possible to inhibit the proliferation of cells in which complement component C3 is involved.

In one embodiment of the method disclosed herein, the biological species of the cells is the same as the biological species containing complement component C3. This makes it possible to more reliably inhibit the proliferation of cells in which complement component C3 is involved.

FIG. 1 is a schematic diagram outlining the complement pathway. 1 is a graph showing cell viability of neuroblastoma cells in Samples 1 to 5 and a comparative example. 1 is a graph showing the cell viability of neuroblastoma at different addition amounts for Samples 1 to 4 and a comparative example. 1 is a graph showing cell viability of breast cancer cells in Samples 1 to 4 and a comparative example. 1 is a graph showing cell viability of lung cancer cells in Sample 2 and the comparative example.

<Definition of terms>
The technology disclosed herein will be described in detail below. Matters other than those specifically mentioned in this specification (e.g., the structure of double-stranded RNA) that are necessary for carrying out this technology (e.g., general matters such as methods for synthesizing polynucleotides, cell culture techniques, constructs mainly composed of peptides or nucleic acids, etc.) can be understood as design matters of a person skilled in the art based on conventional techniques in the fields of cell engineering, physiology, medicine, pharmacology, organic chemistry, biochemistry, genetic engineering, protein engineering, molecular biology, genetics, etc. The technology disclosed herein can be carried out based on the contents disclosed in this specification and the technical common sense in the field.

The term "polynucleotide" as used herein 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. For example, a "polynucleotide" as used herein also includes a polymer that contains both deoxyribonucleotides and nucleotides as nucleotides. In addition, as used herein, an "artificially designed polynucleotide" refers to a polynucleotide whose nucleotide chain (full length) does not exist alone in nature, but is artificially synthesized by chemical synthesis or biosynthesis (i.e., production based on genetic engineering).

In this specification, the terms "first strand" and "second strand" refer to one being a sense strand (or a coding strand or a passenger strand) and the other being an antisense strand (or a template strand or a non-coding strand or a guide strand). That is, when the first strand is a sense strand, the second strand refers to an antisense strand. Also, when the second strand is a sense strand, the first strand refers to an antisense strand. The first strand and the second strand may be completely complementary to each other, or may be at least partially complementary to each other. That is, they may be capable of hybridizing at least under physiological conditions.

In the present specification, unless otherwise specified by "5'" and "3'", the left side of a base sequence always indicates the 5'-terminus and the right side indicates the 3'-terminus. Furthermore, in the present specification, the term "amino acid residue" includes the N-terminal amino acid and the C-terminal amino acid of a peptide chain, unless otherwise specified. Furthermore, in the amino acid sequences described in the present specification, the left side always indicates the N-terminus and the right side indicates the C-terminus.

In this specification, the term "tumor" is interpreted broadly and refers to tumors in general (typically malignant tumors), including carcinomas and sarcomas, or lesions of blood or hematopoietic tissues (leukemia, lymphoma, etc.). Furthermore, "tumor cells" are synonymous with "cancer cells" and refer to cells that form such tumors, typically cells that have reached the stage of abnormal proliferation independent of surrounding normal tissue (so-called cancerous cells). Therefore, unless otherwise specified, cells that are classified as tumor cells (cancer cells) rather than normal cells are referred to as tumor cells, regardless of the origin or properties of the cells.

In this specification, when a numerical range is stated as "A to B (where A and B are arbitrary numerical values)," it means "greater than A and less than B," and also includes the meanings of "greater than A and less than B," "greater than A and less than B," and "greater than A and less than B."

<Complement components>
In this specification, "complement components" refers to proteins involved in the complement pathway and its activation. Examples of complement components include C1 (C1r, C1s, C1p), C2, C3, C4, C5, C6, C7, C8, C9, CFB, CFD, MBL, and MASP. FIG. 1 is a schematic diagram showing an outline of complement activation. In the classical pathway, complement components C1, C2, C4, and the like are mainly involved. The classical pathway begins with the binding of IgM or IgG antibody molecules to complement component C1q. C1r and C1s are activated, and C2 and C4 are degraded into C2a and C2b, and C4a and C4b, respectively. Then, C4b and C2a form a complex. The C4bC2a complex acts as a C3 convertase and degrades C3 into C3a and C3b. The alternative pathway begins with the hydrolysis of C3 on the cell membrane of a microorganism. Complement factor B (CFB) binds to the hydrolyzed C3 to form a complex. Complement factor D (CFD) acts on this complex, acting as a C3 convertase and decomposing C3 into C3a and C3b. The lectin pathway begins with the activation of mannose-binding lectin (MBL), a type of serum lectin, by binding to sugar chains such as mannose on the surface of a pathogen. MASP, a type of serine protease, is activated, and C2 and C4 are decomposed into C2a and C2b, and C4a and C4b, respectively. Then, as in the classical pathway, C4b and C2a form a complex. The C4bC2a complex acts as a C3 convertase and decomposes C3 into C3a and C3b. In this way, each pathway of the complement system converges on the activation of C3. Therefore, inhibiting the expression of C3 is effective in suppressing excessive activation and inappropriate regulation of complement.

<Complement component C3>
In this specification, "complement component C3 (also simply referred to as "C3")" is a type of protein involved in the complement system. However, it is meant to include all synonyms, including naturally occurring C3 and its variants. The species from which C3 is derived is not particularly limited, but is preferably the same as the animal species of the cells to which the double-stranded RNA or composition disclosed herein is to be supplied. For example, when the double-stranded RNA or composition disclosed herein is to be supplied to cells derived from humans, a base sequence based on the base sequence of human C3 may be used as the main sequence. Note that, although human-derived C3 is described here as a preferred example, the present technology can also be applied to C3 derived from species including mammals other than humans and other animal species.

C3 is known to be overexpressed in various diseases and disorders. For example, it has been suggested that the expression level of C3 is involved in macular degeneration (retinal detachment, chorioretinal degeneration, retinal degeneration, photoreceptor degeneration, RPE degeneration, mucopolysaccharidosis, rod-cone dystrophy, cone-rod dystrophy, cone degeneration), age-related macula, cancer, Stargardt's disease, Best's disease, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, rheumatoid arthritis, and neurodegenerative diseases. It is also involved in inflammatory responses and cell death associated with these diseases and disorders. Specifically, it is known that the expression of complement system proteins is increased on the surface of tumor cells. That is, the double-stranded RNA and composition disclosed herein act favorably on cells in which the expression level of C3 is increased due to the above-mentioned diseases and disorders, and can inhibit their proliferation. In addition, in the activation of the complement system, the complement pathway converges on the activation of C3. Therefore, suppressing the expression of C3 has the advantage of reliably suppressing cell proliferation. It can also suppress inflammatory responses and anaphylaxis caused by C3a.

<Signal peptide region>
The base sequence of C3 can be obtained from an international database. For example, the international database is NCBI (National Center for Biotechnology Information), ENA (European Nucleotide Archive), DDBJ (DNA Data Bank of Japan), UniPlot, Ensembl, etc. Specifically, the base sequence of human C3 is provided by NCBI under the accession number NM_000064.4, etc. Information on the signal peptide region of C3 can also be obtained from the above-mentioned international database.

C3 consists of approximately 1,663 amino acid residues. The amino acid sequence shown in SEQ ID NO:5 consists of 22 amino acid residues and shows the amino acid sequence of the signal peptide of human C3. The base sequence shown in SEQ ID NO:6 consists of 66 bases and shows the base sequence of the signal peptide of human C3.

<Double-stranded RNA>
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. Here, the first strand is referred to as a sense strand, and the second strand is referred to as an antisense strand, and will be described in detail below. 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 part of a base sequence encoding complement component C3, and is determined from a base sequence including at least a part of a base sequence encoding a signal peptide region of complement component C3.

The main sequence is typically composed of a polynucleotide, which is a polymer of ribonucleotides. In other words, 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. However, in the attached sequence table, uracil may be represented by T (thymine).

The main sequence of the sense strand can be, for example, a part of the base sequence encoding the signal peptide region of C3. This allows the double-stranded RNA to function as an siRNA (small interfering RNA) targeting C3. In addition, since the base sequence of the signal peptide region of C3 is located upstream of the mRNA, when the double-stranded RNA functions as an siRNA, it can effectively suppress the expression of C3.

The double-stranded RNA disclosed herein can at least function as an siRNA. In other words, such double-stranded RNA is predicted to induce RNA interference (RNAi). RNAi is a gene silencing process that uses short double-stranded RNA such as siRNA to suppress gene expression in a sequence-specific manner. When siRNA is introduced into a cell, it forms a complex with intracellular proteins called RISC (RNA-induced silencing complex). RISC binds to the homologous sequence of mRNA transcribed from the target gene (here, the C3 gene) and specifically cleaves the mRNA. This inhibits translation.

The main sequence is preferably selected from the base sequence encoding the signal peptide region of C3, but one or more bases (e.g., two bases) may be replaced with other bases, deleted, and/or added (inserted) within the scope of the effect of the present technology.

When the entire main sequence is taken as 100%, the proportion of the base sequence encoding the C3 signal peptide region in the main sequence is, for example, preferably 45% or more, and may be 60% or more, 75% or more, 90% or more, or 100% or more.

The 5' end of the main sequence is preferably guanine or cytosine. Since guanine and cytosine have a stronger binding strength with a complementary strand than adenine and uracil, the stability of the 5' end of the sense strand (i.e., the 3' end of the antisense strand) is higher. In other words, the stability of the 5' end of the antisense strand is relatively lower. Although the details of the mechanism are not clear, RISC, which is an RNAi-related protein, tends to preferentially incorporate the strand whose 5' end is more energetically unstable between the sense strand and the antisense strand. Therefore, by having guanine or cytosine at the 5' end of the main sequence, the antisense strand can be more easily incorporated into RISC, and RNAi can be more suitably induced. This allows the double-stranded RNA to function suitably as siRNA.

It is preferable that 60% or more (i.e., 3 or more) of the 5 bases on the 3' end of the main sequence are adenine and/or uracil, 80% or more (i.e., 4 or more), or even 100% (i.e., 5 bases). This makes the 5' end of the antisense strand relatively less stable than the 3' end. As a result, the antisense strand is more easily taken up by RISC, and RNAi can be more suitably induced.

The GC content of the entire main sequence (the total percentage 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 incorporated into RISC and RNA having the main sequence, the ease of cleavage of RNA, etc. With the above GC content, the effect of RNAi can be efficiently exerted.

The main sequence can be selected from 19 to 23 bases starting from G or C of the gene encoding the signal peptide region of human C3. For example, the main sequence can be the following base sequence:
GCCUGCUGCUCUCCUGCUACU (SEQ ID NO: 18);
CCUGCUGCUCUCCUGCUACUA (SEQ ID NO: 19);
CUGCUGCUCCUGCUACUAA (SEQ ID NO:20);
CUCUGGGGAGUCCCAUGUA (SEQ ID NO:21);
The base sequences shown in SEQ ID NOs: 18 to 21 are all composed of RNA. The base sequences shown in SEQ ID NOs: 18 to 21 are all specific to the C3 gene, and can avoid the risk of inhibiting the translation of mRNA in a host cell having a base sequence similar to the target sequence (so-called off-target effect). In addition, double-stranded RNA having the base sequences shown in SEQ ID NOs: 18 to 21 as its main sequence significantly suppresses the proliferation of abnormally proliferating cells even at low concentrations, and can avoid non-specific expression inhibition, non-specific cell proliferation inhibition, stress on cells, and the like.

The base sequence shown in SEQ ID NO:1 (the DNA sequence corresponding to the RNA sequence of SEQ ID NO:18) is the 23rd to 41st bases of the base sequence encoding human C3 (i.e., the sequence from the start codon to the stop codon). The base sequence shown in SEQ ID NO:2 (the DNA sequence corresponding to the RNA sequence of SEQ ID NO:19) is the 24th to 42nd bases of the base sequence encoding human C3. The base sequence shown in SEQ ID NO:3 (the DNA sequence corresponding to the RNA sequence of SEQ ID NO:20) is the 25th to 43rd bases of the base sequence encoding human C3. The base sequence shown in SEQ ID NO:4 (the DNA sequence corresponding to the RNA sequence of SEQ ID NO:21) is the 59th to 77th bases of the base sequence encoding human C3. The base sequences shown in SEQ ID NO:1 to 3 are part of the base sequence of the signal peptide region of human C3. The base sequence shown in SEQ ID NO:4 includes part of the base sequence of the signal peptide region of human C3.

Double-stranded RNA composed of the main sequences shown in SEQ ID NOs: 18 to 21 can suppress or inhibit the proliferation of at least one type of cell by supplying it to the cell. Typically, by supplying it to tumor cells (e.g., neuroblastoma, breast cancer, lung cancer, etc.), the proliferation of the tumor cells can be suppressed or inhibited. C3 is expressed at low levels in normal cells other than tumor cells, but is overexpressed in tumor cells. Therefore, even if the double-stranded RNA disclosed herein is supplied to normal cells, the amount of C3 present in normal cells is relatively small, so it is considered that the double-stranded RNA will have almost no effect.

<Additional sequence>
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. Preferably, the additional sequence is added to the 3'-end of the main sequence. By adding the additional sequence, RNAi can be induced more effectively.

The additional sequence is composed of a polynucleotide (dimer, trimer, or tetramer). The polynucleotide constituting the additional sequence may be composed of only ribonucleotides, only deoxynucleotides, or both ribonucleotides and deoxynucleotides. In other words, the sense strand and the 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 analogues, etc.

The base sequence constituting the additional sequence is not particularly limited, but preferably contains at least one base, such as adenine, uracil, or thymine. From the viewpoint 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).

Sense and antisense strands
The sense strand may be, for example, composed of a base sequence of 21 to 27 bases, 21 to 25 bases, or 21 to 23 bases. In a preferred example, the sense strand is composed of 21 bases, consisting of a 19-base main sequence and 2-base additional sequence. 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 to form 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 replaced with other bases, deleted and/or added (inserted). 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).

In the double-stranded RNA of the present disclosure, the sense strand or antisense strand is typically composed of chemically unmodified ribonucleotides (RNA). However, the double-stranded RNA of the present disclosure may contain DNA, chemically modified DNA or RNA, other known nucleotide analogs, etc., to the extent that the technology of the present disclosure is not significantly impaired. That is, one or more bases (e.g., two bases) of the sense strand or antisense strand may be replaced with chemically modified RNA (or DNA) such as methylated or pseudouridylated. Examples of chemically modified RNA include pseudouridine, N1-methylpseudouridine, 5-methylcytosine, or inosine. For example, one or more bases (e.g., two bases) of uridine in the double-stranded RNA of the present disclosure may be replaced with pseudouridine.

In the double-stranded RNA of the present disclosure, 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. From the viewpoint of improving the function as siRNA, the additional sequence may be added to the 3' end of the complementary base sequence. In a preferred example, when the additional sequence of the sense strand is added to the 3' end of the main 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 of the sense strand described above. Typically, the base sequence of the additional sequence of the antisense strand is the same as the additional sequence of the sense strand to which it hybridizes, but it may be a different base sequence.

The antisense strand is composed of a base sequence of, for example, 21 to 27 bases, and may 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 complementary to the main sequence of the sense strand. In a preferred example, 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 complementary to the main sequence of the sense strand.

<Method of Producing Double-Stranded RNA>
The sense strand and antisense strand constituting the double-stranded RNA disclosed herein can be produced according to a general chemical synthesis method. For example, they can be synthesized using a commercially available DNA/RNA automatic synthesizer. In addition, the sense strand and antisense strand can be synthesized in vitro or in vivo based on genetic engineering techniques. In addition, the synthesized sense strand and antisense strand are preferably purified, and can be purified, for example, by HPLC or the like.

The double-stranded RNA disclosed herein can be produced, for example, by annealing (hybridizing) a sense strand and an antisense strand. The annealing method may be any conventional method. In one example, annealing can be performed by mixing equal amounts of a sense strand and an antisense strand in a solvent, heating at 90°C for 1 to 5 minutes, and then cooling to 4°C to room temperature. Examples of such a solvent include distilled water, pure water, ultrapure water, and buffers (e.g., HEPES-KOH buffer at pH 7.4, PBS, etc.). In order to prevent active RNase (RNA degrading enzyme) from being mixed into the solvent, it is preferable to use a solvent that has been treated with, for example, DEPC or autoclaved.

<Composition>
The composition disclosed herein includes the above-mentioned double-stranded RNA. In addition to the above-mentioned double-stranded RNA, the composition may include various medicamentally (pharmacologically) acceptable carriers depending on the form of use. As the carrier, for example, a carrier generally used in medicine as a diluent, excipient, etc. is preferable. Such a carrier varies appropriately depending on the use and form of the composition. Typically, water, physiological buffer solutions, various organic solvents, etc. are included. In addition, such a carrier may be an aqueous solution of alcohol (ethanol, etc.) of an appropriate concentration, glycerol, a non-drying oil such as olive oil, or a liposome. Examples of secondary components that can be contained in the pharmaceutical composition include various fillers, extenders, binders, moisturizers, surfactants, dyes, fragrances, etc. In addition, the composition may include carriers used in conventionally known drug delivery systems (DDS).

The form of the composition disclosed herein is not particularly limited. For example, typical forms of the composition include liquids, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, and ointments. In addition, for use in injections, etc., the composition may be freeze-dried or granulated to be dissolved in physiological saline or an appropriate buffer solution (e.g., PBS) immediately before use to prepare a medicinal solution. In addition, the process itself of preparing various forms of drugs (compositions) using double-stranded RNA (main component) and various carriers (secondary components) as materials may be in accordance with conventionally known methods, and such formulation methods themselves do not characterize the present disclosure, so detailed explanations are omitted. For example, Comprehensive Medicinal Chemistry, edited by Corwin Hansch, published by Pergamon Press (1990) is an example of a detailed source of information on prescriptions.

The composition disclosed herein inhibits the proliferation of at least one type of cell. The cells whose proliferation is inhibited are cells in which C3 expression is involved, such as tumor cells (e.g., neuroblastoma, breast cancer cells, lung cancer, lymphoma, etc.), liver cells, eye cells, etc. Among these, the composition disclosed herein preferably inhibits the proliferation of tumor cells. In other words, the double-stranded RNA and composition disclosed herein can be preferably used as an antitumor agent (anticancer agent) that suppresses the proliferation of tumor cells.

One aspect of the composition disclosed herein includes, in addition to the double-stranded RNA described above, a peptide fragment (cell-penetrating peptide, CPP) that has cell membrane permeability and can pass through the cell membrane from outside the cell to introduce a foreign substance 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 the double-stranded RNA. Generally, double-stranded RNA has a negative charge and cannot pass through the cell membrane. However, for example, by directly or indirectly binding (linking) the double-stranded RNA disclosed herein to the N-terminus and/or C-terminus of the peptide fragment, the construct of the peptide fragment and the double-stranded RNA can be introduced into the cytoplasm. The number of amino acid residues of the peptide fragment is not limited as long as the cell membrane permeability is not impaired.

When the peptide fragment and the double-stranded RNA are indirectly bound, for example, a 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. In addition, the method of binding the peptide fragment and the double-stranded RNA is not particularly limited, and can be carried out according to various scientific methods known in the art.

One embodiment of the composition disclosed herein includes a peptide fragment and the double-stranded RNA of the present disclosure. However, the double-stranded RNA does not have to be bound to the N-terminal or C-terminal side of the peptide fragment. In such an embodiment, the double-stranded RNA and the peptide fragment may form a complex, for example, by electrical or molecular interaction. Such a complex is easily introduced into eukaryotic cells, and therefore the double-stranded RNA may be efficiently introduced. 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. In addition, the proportion of the peptide fragment in this case may be 5 to 100 times that of the double-stranded RNA in molar terms, and preferably 40 to 60 times.

<Method of producing the composition disclosed herein and its use>
The present disclosure may provide a method for inhibiting the proliferation of at least one type of cell using the composition disclosed herein, the method comprising the steps of preparing a composition disclosed herein and delivering the composition to a cell of interest.

In the preparation step, for example, the composition disclosed herein may be prepared by a conventionally known method as described above.

In the supplying step, the composition disclosed herein is supplied to at least one type of cell (e.g., tumor cells, etc.) in a living body (in vivo) or outside the living body (in vitro). The animal species of the cells to be supplied is not particularly limited, and may be, for example, mammals, birds, amphibians, reptiles, fish, etc. Preferably, the animal species from which C3, which is the basis of the main sequence of the double-stranded RNA contained in the composition, is derived is the same as the animal species of the target cells. The type of the target cells is also not particularly limited, but is preferably tumor cells, more preferably neuroblastoma, breast cancer, or lung cancer. Note that, although cells other than tumor cells may be present at the destination of the composition, the composition may be supplied only to the target cells (i.e., tumor cells).

The method of administration of the composition is not particularly limited, and may be similar to the method conventionally used for the treatment of animals. The composition may be used in vivo in a manner and dosage appropriate to its form and purpose. For example, as a liquid formulation, it can be administered in a desired amount to the affected area (e.g., malignant tumor tissue, virus-infected tissue, inflammatory tissue, etc.) of a patient or an individual animal (i.e., a living body) by intravenous, intralymphatic, intramuscular, subcutaneous, intradermal, or intraperitoneal injection. Alternatively, a solid form such as a tablet or a gel or aqueous jelly such as an ointment can be administered directly to a specific tissue (e.g., an affected area such as a tissue or organ containing tumor cells, inflammatory cells, etc.). Alternatively, a solid form such as a tablet can be administered orally. In the case of oral administration, it is preferable to encapsulate or apply a protective (coating) material to suppress decomposition by digestive enzymes in the digestive tract.

The amount of the composition to be supplied in vivo is not particularly limited. For example, the lower limit of the amount of double-stranded RNA per kg of an 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 an 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 in vitro is not particularly limited. In the culture medium of the subject to be supplied, such as cells, 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 a culture medium may be, for example, 10 μM or less, 5 μM or less, 2 μM or less, 1 μM or less, or 100 nM or less.

The compositions disclosed herein can be delivered to the inside of target cells by 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. As described above, the compositions may also be delivered to the inside of cells using peptide fragments that have cell membrane permeability.

Below, we will explain some test examples related to the technology disclosed herein, but we are not intended to limit the technology disclosed herein to those shown in these test examples.

<Preparation of double-stranded RNA>
Polynucleotides having the base sequences shown in SEQ ID NOs: 7 to 21 were artificially synthesized. The base sequences of each polynucleotide are shown in Table 1. In each polynucleotide, "TT" (additional sequence) on the 3' end is DNA, and the other sequence (main sequence) is composed of RNA. The obtained polynucleotides were annealed with a sense strand and an antisense strand having complementary sequences to prepare double-stranded RNAs used in Samples 1 to 5 shown in Table 1. Each of the double-stranded RNAs shown in Samples 1 to 5 was dissolved in PBS to give an RNA concentration of 2 mM, and an RNA solution was prepared.

As shown in Table 1, the sense strand of the double-stranded RNA of sample 1 is composed of a main sequence (a part of the base sequence encoding the signal peptide region of C3) consisting of SEQ ID NO: 18 and an additional sequence consisting of TT added to the 3' end of the main sequence. Similarly, the sense strand of the double-stranded RNA of samples 2 to 4 shown in Table 1 is composed of a main sequence (a sequence including the base sequence encoding the signal peptide region of C3) consisting of SEQ ID NO: 19 to 21 and an additional sequence consisting of TT added to the 3' end of the main sequence. The sense strand of the double-stranded RNA of sample 5 is composed of a main sequence (a randomly artificially created sequence) consisting of SEQ ID NO: 7 and an additional sequence consisting of TT added to the 3' end of the main sequence. The antisense strand of 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.

<Cell proliferation test of human neuroblastoma cells>
The SK-N-SH strain, which is a human neuroblastoma cell, was used as the tumor cell. The SK-N-SH cells were pre-cultured in a culture medium of 10% FBS (fetal bovine serum) + E-MEM (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Cat No. 051-07615) + 1% MEM non-essential amino acid solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Cat No. 139-15651). Note that 0.5% penicillin-streptomycin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Cat No. 168-23191) was added to the culture medium only during pre-culture, but was not added during the following culture and evaluation.

On the first day, the SK-N-SH cells that had adhered to the culture plate were washed with PBS, and then a 0.25% trypsin/EDTA solution was added and incubated at 37°C for 2 minutes. After the incubation, the above culture medium was added to inactivate the trypsin. Then, the cells were precipitated by centrifugation at 150×g for 5 minutes. After removing the supernatant generated by centrifugation, the above culture medium was added to the precipitate (cell pellet) to prepare a cell suspension of approximately 5×10 ⁴ cells/mL. A commercially available 96-well plate was prepared, and the cell suspension was seeded in each well at 5×10 ³ cells/100 μL/well, and incubated overnight at 37°C under 5% CO ₂ .

(Sample 1)
On the second day, 3 μL of RNA solution prepared to 2 mM with PBS was mixed with 75 μL of Opti-MEM (trademark) to prepare solution A. Also, 4.5 μL of Lipofectamine (trademark) RNAiMAX was mixed with 75 μL of Opti-MEM (trademark) to prepare solution B. Next, equal amounts of solution A and solution B were mixed to prepare solution C, which was then incubated at room temperature for 5 minutes. The prepared solution C was added to the wells in which SK-N-SH cells were cultured, at 11 μL/well (final concentration of double-stranded RNA was 4 μM). The mixture was then incubated at 37° C. under 5% CO ₂ for 3 days.

Cell proliferation was evaluated using Cell Counting Kit-8 (CCK-8, Dojin Kagaku Kenkyusho). On the fifth day (three days after the addition of siRNA), the 96-well plate in which SK-N-SH cells had been cultured was removed, 10 μL of CCK-8 was added to each well, and the plate was incubated at 37° C. under 5% CO ₂ for 1.5 hours. The absorbance of each well was measured at 450 nm. The absorbance was the average value of three wells. In addition, a well containing only the culture medium and CCK-8 reagent was provided as a blank. The value obtained by subtracting the absorbance of the blank from the absorbance of sample 1 was used as the measured value of sample 1.

(Samples 2 to 5)
Samples 2 to 5 were prepared in the same manner as Sample 1, except that the double-stranded RNA in Sample 1 was replaced with the double-stranded RNA in Samples 2 to 5 shown in Table 1.

Comparative Example
The comparative example was the same as sample 1, except that a PBS solution was used instead of the RNA solution in sample 1. That is, in the comparative example, no double-stranded RNA was introduced.

Untreated wells were prepared in the same manner as Sample 1, except that no RNA solution or Lipofectamine (trademark) RNAiMAX was added. The cell viability in each test example is shown in Figure 2 as a percentage of the measured value for the untreated well, which is set at 100%. Figure 2 shows the results of a test using the double-stranded RNA shown in Table 1, that is, double-stranded RNA with a main sequence that includes a base sequence encoding the C3 signal peptide region (SEQ ID NOs: 1 to 4) in neuroblastoma cells.

As shown in Figure 2, the cell viability of samples 1 to 4 was decreased, and was significantly lower than that of the comparative example. Sample 2 (Sequence No. 2) had the lowest cell viability. From the above test results, it is considered that the double-stranded RNA of samples 1 to 4 has the function of inhibiting the proliferation of tumor cells (neuroblastoma cells). Furthermore, the base sequence of Sequence No. 7 is a random sequence with an ATCG content almost equivalent to that of the base sequences of Sequence Nos. 1 to 4. Sample 5, which has Sequence No. 7 as the main sequence, did not affect cell proliferation. Therefore, these sequences of Sequence Nos. 1 to 4 are specific to the C3 gene. For this reason, the double-stranded RNA of samples 1 to 4 can avoid off-target effects and do not affect other organs, and are fully expected to be used in clinical applications.

<Cell proliferation test of human neuroblastoma cells using low concentrations of double-stranded RNA>
The double-stranded RNA used in Samples 1 to 4 shown in Table 1 was prepared. The double-stranded RNA shown in Samples 1 to 4 was dissolved in PBS so that the RNA concentration was 2 mM, and an RNA solution was prepared. The RNA solution was then further diluted 10-fold with PBS to prepare a low-concentration RNA solution with an RNA concentration of 200 μM. A test was performed in the same manner as in the cell proliferation test of human neuroblastoma cells, except that the low-concentration RNA solution was used. That is, the final concentration of the double-stranded RNA added to the wells in which SK-N-SH cells were cultured was set to 0.4 μM. The cell viability in each test example was expressed as a percentage when the measured value of the untreated well was set to 100%.

Figure 3 is a graph comparing the cell viability when the final concentration of added double-stranded RNA was 4.0 μM and 0.4 μM. As shown in Figure 3, the cell viability of samples 1 to 4 decreased. Furthermore, the cell viability of samples 1 to 4 was significantly lower than that of the comparative example. This shows that the double-stranded RNA of samples 1 to 4 had the function of inhibiting the proliferation of tumor cells (neuroblastoma cells) even at low concentrations. Furthermore, the double-stranded RNA of samples 1 to 4 had the same or greater cell inhibition function even at one-tenth the concentration. Therefore, the double-stranded RNA of samples 1 to 4 has a sufficient tumor cell proliferation inhibition function even at low concentrations, which can avoid non-specific expression inhibition and non-specific cell proliferation inhibition, and is fully expected to be used in clinical applications.

<Cell proliferation test of human breast cancer cells>
The same procedure was used as in the cell proliferation test of human neuroblastoma cells, except that human breast cancer cells, MDA-MB-231 strain, were used as tumor cells. The cell viability in each test example is shown as a percentage relative to the measured value of the untreated well, which is taken as 100%, and is shown in Figure 4.

As shown in Figure 4, the cell viability of samples 1 to 4 was reduced, and was significantly lower than that of the comparative example. From this, it is believed that the double-stranded RNA of samples 1 to 4 has the function of inhibiting the proliferation of tumor cells (breast cancer cells).

<A549 strain cell proliferation test>
The human lung cancer cell line A549 was used as the tumor cell. The cell proliferation was evaluated by removing the 96-well plate in which the A549 cells were cultured on the fourth day (two days after the addition of siRNA), adding 10 μL of CCK-8 to each well, and incubating for 2.0 hours at 37° C. under 5% CO2. The rest of the experiment was the same as in the cell proliferation test of human neuroblastoma cells. The cell viability in each test example is expressed as a percentage of the measured value of the untreated well, which is taken as 100%, and is shown in FIG. 5.

As shown in Figure 5, the cell viability of sample 2 was reduced, and was significantly lower than that of the comparative example. This suggests that the double-stranded RNA of sample 2 has the function of inhibiting the proliferation of tumor cells (lung cancer cells).

The above provides detailed examples of the technology disclosed herein, but these are merely examples and do not limit the scope of the claims. The technology described in the claims includes various modifications and variations of the specific examples provided above.

In the technology disclosed herein, the components and processes mentioned herein may be omitted or combined as appropriate, unless a particular problem arises. This specification also includes the disclosures described in the following sections.

Item 1: A double-stranded RNA having a first strand and a second strand complementary to the first strand, the first strand having 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 being a part of a base sequence encoding complement component C3 and including at least a part of a base sequence encoding a signal peptide region of complement component C3.

Item 2: The double-stranded RNA according to Item 1, wherein the second strand is composed of a main sequence complementary to the first strand and an additional sequence 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, in which at least three of the five bases on the 3'-terminal side of the main sequence are adenine (A) and/or uracil (U).

Item 4: The base sequence encoding the signal peptide region of the complement component C3 is the following base sequence:
GCCTGCTGCTCCTGCTTACT (SEQ ID NO: 1);
CCTGCTGCTCCTGCTA CTA (SEQ ID NO: 2);
CTGCTGCTCCTGCTACTAA (SEQ ID NO:3);
CTCTGGGGAGTCCCATGTA (SEQ ID NO: 4);
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).

Item 6: A composition that inhibits the proliferation of at least one type of cell, comprising the double-stranded RNA described in any one of Items 1 to 5.

Item 7: The composition according to Item 6, wherein the cells are tumor cells.

Item 8: The composition according to item 6 or 7, which contains a peptide fragment having cell membrane permeability that can pass through the cell membrane from the outside of the cell and introduce a foreign substance into the cytoplasm.

Item 9: A method for inhibiting proliferation of at least one type of cell, comprising:
A step of preparing a composition according to any one of items 6 to 8;
and providing said composition to said cell in vitro or in vivo.

Item 10: The method according to Item 9, wherein the biological species of the cells is the same as the biological species containing the complement component C3.

As described above, the double-stranded RNA disclosed herein can inhibit (or suppress) cell proliferation. Therefore, by using the double-stranded RNA, it is possible to provide a composition (e.g., an antitumor agent) that inhibits the proliferation of at least one type of cell (e.g., tumor cells).

Claims (10)

A double-stranded RNA having a first strand and a second strand complementary to the first strand,
The first strand comprises a main sequence consisting of 19 to 23 bases, the 5'-terminal 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,
Here, the main sequence is
A double-stranded RNA comprising a portion of a base sequence encoding complement component C3, the portion comprising at least a portion of a base sequence encoding the signal peptide region of complement component C3. The double-stranded RNA according to claim 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. The double-stranded RNA according to claim 1, wherein at least three of the five bases on the 3'-terminal side of the main sequence are adenine (A) and/or uracil (U). The base sequence encoding the complement component C3 is the following base sequence:
GCCTGCTGCTCCTGCTTACT (SEQ ID NO: 1);
CCTGCTGCTCCTGCTA CTA (SEQ ID NO: 2);
CTGCTGCTCCTGCTACTAA (SEQ ID NO:3);
CTCTGGGGAGTCCCATGTA (SEQ ID NO: 4);
The double-stranded RNA according to claim 1, which consists of any one of the following: The double-stranded RNA according to claim 1, wherein the base sequence constituting the additional sequence is thymine-thymine (TT). A composition that inhibits the proliferation of at least one type of cell, comprising the double-stranded RNA according to any one of claims 1 to 5. The composition of claim 6, wherein the cells are tumor cells. The composition according to claim 7, comprising a peptide fragment having cell membrane permeability capable of passing through the cell membrane from the outside of the cell and introducing a foreign substance into the cytoplasm. 1. A method for inhibiting proliferation of at least one cell, comprising:
providing a composition according to claim 6;
and providing said composition to said cell in vitro. The method according to claim 9, wherein the biological species of the cells is the same as the biological species containing the complement component C3.

Images