WO2024186166A1 - Double-stranded rna having innate immune response-inducing effect, and use thereof - Google Patents

Abstract

The present invention relates to a double-stranded RNA having innate immune response-inducing effect, and use thereof, and, more specifically, to: a double-stranded RNA comprising a nucleotide into which an innate immune activation motif and a chemical modification are introduced; a composition for enhancing innate immunity, comprising the double-stranded RNA; and a pharmaceutical composition comprising the double-stranded RNA.

Description

Double-stranded RNA having an innate immune response-inducing effect and uses thereof

The present invention relates to a double-stranded RNA having an innate immune response inducing effect and a use thereof, and more particularly, to a double-stranded RNA comprising a nucleotide into which an innate immune activation motif and a chemical modification have been introduced.

Induction of innate/adoptive immune responses plays an important role in the treatment of various diseases. Among these, the innate immune response is a non-specific immune response of the body, and it acts as the first line of defense of the body to recognize external factors and respond first. Since it has been known that pathological conditions such as cancer, pathogenic infection, and hereditary diseases that are difficult to treat can be improved by enhancing or activating the innate immune response, research on therapeutics using the innate immune response has been actively conducted. To this end, research has been conducted on the mechanism of action that can activate or regulate the innate immune response. For example, it has been reported that when activating the innate immune response as an anticancer treatment, the recruitment of macrophages or dendritic cells to the cancer cell site is activated through chemokine production and promotion of inflammation, and the production of cytokines such as interferon can be increased to induce a normal state.

In addition, some innate immune receptors specialized in detecting foreign or damaged nucleic acids have been identified. Pattern recognition receptors (PRRs) are proteins present in the cell membrane or cytoplasm of cells, which recognize pattern recognition molecules and mediate innate immune responses. Pattern recognition receptors are distributed in dendritic cells, macrophages, monocytes, neutrophils, epithelial cells, etc., and recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Representative pattern recognition receptors include Toll-like receptors (TLR), C-type lectin receptors (CLR), retinoic acid-inducible gene-I-like receptors (RLR), nucleotide oligomerization domain-like receptors (NLR), and absence-in-melanoma-like receptors (ALR). Each receptor recognizes different types of ligands and contributes to the enhancement of the immune system in various ways.

Meanwhile, technologies utilizing the RNA interference (RNAi) phenomenon have been developed as RNA-based treatments, and this RNA interference is a method of suppressing target gene expression by inducing cleavage of target mRNA. Specifically, short interfering RNA (small interfering RNA) can be used as an RNAi agent, and in this case, the induction of an innate immune response by administration of an external substance, siRNA, is recognized as a non-specific response and rather as a factor that inhibits the efficacy of the RNAi agent. Accordingly, the introduction of modified RNA nucleotides, for example, the introduction of nucleotides with chemical modifications, has been suggested as a technology for reducing the innate immune response by administration of siRNA. However, RNA-based therapeutics can be applied not only to the aforementioned RNAi agents, but also to passive and active immunotherapy, vaccine agents, or genetic engineering in general. In addition, RNA molecules can also be used as therapeutics for replacement therapy, such as protein replacement therapy to replace missing or mutated proteins.

Under this technical background, the inventors of the present invention have conducted research efforts to develop a double-stranded RNA capable of specifically activating an innate immune response to enhance therapeutic efficacy, and as a result, have confirmed the effect of enhancing an innate immune response resulting from chemical modification of specific motifs and nucleotides, and have completed the present invention based on this.

The purpose of the present invention is to provide double-stranded RNA capable of specifically activating an innate immune response.

Another object of the present invention is to provide a composition for enhancing an innate immune response comprising the double-stranded RNA, a pharmaceutical composition comprising the double-stranded RNA, or a method for treating a disease or improving a pathological condition comprising a step of administering the pharmaceutical composition to a subject.

One aspect is a double-stranded RNA having 25 to 60 base pairs (bp) comprising a first strand; and a second strand complementarily binding to the first strand, wherein one strand of the double-stranded RNA contains at least 10 nucleotides to which a chemical modification has been introduced, and wherein the double-stranded RNA enhances an MDA5 (Melanoma differentiation-associated protein 5)-mediated innate immune response.

Another aspect provides a double-stranded RNA of 25 to 60 base pairs, comprising a first strand; and a second strand complementarily binding to said first strand, said double-stranded RNA comprising an innate immune activation motif of 10 to 30 base pairs, wherein one strand of said double-stranded RNA comprises at least 10 nucleotides to which a chemical modification has been introduced.

Another aspect provides a composition for enhancing innate immunity comprising the double-stranded RNA.

Another aspect provides a pharmaceutical composition comprising the double-stranded RNA as an active ingredient.

Another aspect provides a method of treating a disease comprising administering to a subject the double-stranded RNA.

Another aspect provides a medicinal use of said double-stranded RNA for the treatment or amelioration of a disease or pathological condition.

Double-stranded RNA according to one aspect can induce a high level of innate immune response by including an innate immune response Activating Motif and a chemically modified nucleotide.

Additionally, double-stranded RNA according to one aspect may enhance the efficacy of vaccine formulations or immunotherapeutic agents by enhancing innate immune responses.

Figure 1 is a schematic diagram showing the structure of a double-stranded RNA containing an innate immune activation motif according to one aspect.

Figure 2 shows the results of confirming the mRNA expression levels of IFIT1, CXCL10, and ISG15 after transfecting a T98G cell line with a double-stranded RNA containing an innate immune activation sequence of sequence number 6 according to one aspect.

Figure 3 shows the results of confirming the mRNA expression levels of TNF-α and IL-1β after transfecting a T98G cell line with a double-stranded RNA containing an innate immune activation sequence of sequence number 6 according to one aspect.

Figure 4 shows the results of confirming the mRNA expression levels of IFIT1, CXCL10, and ISG15 after transfecting a T98G cell line with a double-stranded RNA containing an innate immune activation sequence of sequence number 8 according to one aspect.

Figure 5 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting a double-stranded RNA containing an innate immune activation sequence of sequence number 6 according to one aspect into a wild-type cell line (WT) or a cell line lacking RIG-I (RIG-I KO).

Figure 6 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting a double-stranded RNA containing an innate immune activation sequence of sequence number 8 according to one aspect into a wild-type cell line (WT) or a cell line lacking RIG-I (RIG-I KO).

Figure 7 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting a double-stranded RNA containing an innate immune activation sequence of sequence number 2 or sequence number 4 according to one aspect into a wild-type cell line (WT) or a cell line lacking RIG-I (RIG-I KO).

Figure 8 is according to the daily aspect. This is the result of confirming the mRNA expression level of the Lamin A/C gene after transfecting the Huh7 cell line with a 30-bp double-stranded RNA containing the innate immune activation sequence of sequence number 2.

Figure 9 is according to the daily aspect. This is the result of confirming the activity level of interferon regulatory factor (IRF) after transfecting each of the double-stranded RNAs containing the innate immune activation sequence of sequence number 2, wherein the innate immune activation sequence within the double-stranded RNA is located at the 5' end of the second strand or the 3' end of the first strand, into a wild-type cell line (WT) and a cell line lacking RIG-I (RIG-KO).

Figure 10 is a diagram showing an embodiment in which a chemical modification is introduced into either the first strand or the second strand. This is the result of confirming the activity level of interferon regulatory factor (IRF) after transfecting double-stranded RNA into wild-type cell line (WT) and RIG-I deficient cell line (RIG-I KO), respectively.

Figure 11 is a diagram showing an embodiment of a method in which chemical modifications are introduced into both the first and second strands. This is the result of confirming the activity level of interferon regulatory factor (IRF) after transfecting double-stranded RNA into wild-type cell line (WT) and RIG-I deficient cell line (RIG-I KO), respectively.

Figure 12 shows an aspect in which 10 nucleotides into which chemical modifications are introduced are positioned at the 5' end or the 3' end of the first strand (#1 5'-1-10, #1 3'-1-10), or the position of the first nucleotide into which chemical modifications are introduced in the first strand is changed (#1 5'-1-10, #1 5'-2-10). This is the result of confirming the activity level of interferon regulatory factors after transfecting double-stranded RNA into wild-type cell line (WT) and RIG-I deficient cell line (RIG-I KO), respectively.

Figure 13 shows chemical modifications introduced into 8, 10, 12 or 15 nucleotides, respectively. This is the result of confirming the activity level of interferon regulatory factor (IRF) after transfecting double-stranded RNA into wild-type cell line (WT) and RIG-I deficient cell line (RIG-I KO), respectively.

Figure 14 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting chemically modified double-stranded RNA into a wild-type cell line (WT), a cell line lacking RIG-I (RIG-I KO), or a cell line lacking MDA5 (MDA5 KO), respectively.

Figure 15 shows the results of examining the activity level of interferon regulatory factor (IRF) over time after transfecting a human monocyte cell line (THP-1 Dual) with double-stranded RNA with chemical modifications.

Figure 16 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting RAW-Lucia ISG cell lines with double-stranded RNAs having different nucleotide lengths of 20 to 40 bp, in which the innate immune activation motif is located at the 5' end of the second strand and at least 10 chemical modifications are introduced into the first strand.

Figure 17 shows the results of examining the activity level of interferon regulatory factor (IRF) over time after transfecting a human monocyte cell line (THP-1 Dual) with double-stranded RNA containing or not containing an innate immune activation motif in a 30bp double-stranded RNA (#1 5'-2-15) according to one embodiment.

FIG. 18 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting RAW-Lucia ISG cell line with a double-stranded RNA including an innate immune activation sequence of SEQ ID NO: 2, an innate immune activation sequence of SEQ ID NO: 8 (20 nt), or an innate immune activation sequence of SEQ ID NO: 62 (30 nt) in a 30 bp double-stranded RNA (#1 5'-2-15) according to one embodiment.

FIG. 19 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting RAW-Lucia ISG cell lines with double-stranded RNA containing chemical modification of 2' -OCH3(-O-methyl) or chemical modification of 2' -F in 30 bp double-stranded RNA (#1 5'-2-15) according to one embodiment.

FIG. 20 shows the results of examining the activity level of interferon regulatory factor (IRF) over time after transfecting a human monocyte cell line (THP-1 Dual) with a double-stranded RNA containing a chemical modification of 2' -OCH3(-O-methyl) or a chemical modification of 2' -F in a 30 bp double-stranded RNA (#1 5'-2-15) according to one embodiment.

FIG. 21 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting RAW-Lucia ISG cell line with double-stranded RNA in which two adjacent nucleotide bonds at both ends of the first strand, the second strand, or both the first strand and the second strand are modified with phosphorothioate bonds in a 30 bp double-stranded RNA (#1 5'-2-15) according to one embodiment.

Each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of this application cannot be considered limited by the specific description described below.

One aspect provides a double-stranded RNA of 25 to 60 base pairs, comprising a first strand; and a second strand complementarily binding to the first strand, wherein the double-stranded RNA comprises an innate immune activation motif of 10 to 30 base pairs, wherein one strand of the double-stranded RNA comprises at least 10 nucleotides to which a chemical modification has been introduced.

double-stranded RNA

As used herein, the term "double-stranded RNA" means RNA having two strands (a first strand and a second strand) that complementarily bind to each other. The double-stranded RNA may be used interchangeably with "RNA-based immune enhancer/adjuvant", "RNA-based immunogenic composition", and "RNAi-inducing nucleic acid molecule", depending on the intended use.

As used herein, the terms “first strand” or “second strand” may be referred to as the antisense strand or the sense strand, respectively, when the double-stranded RNA comprises a region for RNA interference.

The double-stranded RNA may have a length of 25 to 60 base pairs, for example, 25 to 60, 25 to 55, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 26 to 60, 26 to 55, 26 to 50, 26 to 45, 26 to 40, 26 to 35, 26 to 30, 30 to 60, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 60, 35 to It may have a length of 55, 35 to 50, 35 to 45, or 35 to 40 base pairs.

As used herein, the terms "complementarity" or "complementary" refer to their generally accepted meanings in the art. The terms may generally refer to the formation or presence of hydrogen bond(s) between one nucleic acid sequence and another nucleic acid sequence, either by traditional Watson-Crick or other non-traditional types of binding as described herein. Perfect complementarity may mean that every adjacent residue in one nucleic acid sequence hydrogen bonds with the same number of adjacent residues in the other nucleic acid sequence. Partial complementarity can include various mismatches or non-based paired nucleotides within a nucleic acid molecule (e.g., more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches, e.g., 1 to 3 mismatches, non-nucleotide linkers, or non-base paired nucleotides). The partial complementarity can result in bulges, loops, overhangs, or blunt ends between the first and second strands of a double-stranded RNA, or between the first strand of a double-stranded RNA and a corresponding target nucleic acid molecule.

Innate immune activation motif

The term "Innate Immune Response Activating Motif" as used herein refers to a region in a double-stranded RNA that induces or enhances an innate immune response in a subject to which the double-stranded RNA has been administered, and refers to a double-stranded structure composed of an innate immune activating sequence and a sequence that forms a complementary bond with the sequence.

The term "innate immune response" as used herein may refer to an in vivo innate defense immune system induced from double-stranded RNA, which may contribute to improving the efficacy of treatment by enhancing the immunity of an individual from a therapeutic perspective. The innate immune response may be, for example, induced by interferon, or mediated by RIG-I (Retinoic acid-inducible gene I), or MDA5 (Melanoma differentiation-associated gene 5).

It is known that the innate immune response by double-stranded RNA depends on the terminal region structure of the double-strand. Therefore, as a technical means for enhancing the innate immune response, an approach of introducing 5'-triphosphate or introducing a modified terminal structure has been reported. In addition, it has been reported that the MDA5-mediated immune response is induced by long-length double-stranded RNA. Meanwhile, the present inventors have elucidated that the innate immune response can be induced or enhanced through the innate immune activation motif and any chemical modification even though the double-stranded RNA according to one embodiment has a relatively short length.

As used herein, the term "innate immune activation sequence" refers to a nucleotide sequence located on either strand of a double-stranded RNA that activates an innate immune response. For example, the innate immune activation sequence may comprise a structure capable of activating an innate immune response mediated by RIG-I. Specifically, the innate immune activation sequence may be a nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 62. In one specific embodiment, the innate immune activation sequence may comprise a variant of the nucleotide sequence having at least 80% sequence identity with any one of the nucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 62.

In one specific embodiment, the innate immune activation motif can be comprised of a nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 62, and a nucleotide sequence complementarily binding to said nucleotide sequence. The innate immune activation motif can be located at one terminal region of the double-stranded RNA, for example, can be located at the 3' end of the first strand or the 5' end of the second strand.

The innate immune activation motif comprises 10 to 30 nucleotides comprising the nucleotide sequence of SEQ ID NO: 2, for example, 10 to 30, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 30, 14 to It may consist of 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, or 14 to 16 nucleotides and another nucleotide sequence complementarily binding to said sequence.

The innate immune activation motif may have a length of 10 to 30 base pairs, for example, 10 to 30, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 30, 14 to 28, It may have a length of 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to 30, or 18 to 20 base pairs.

As used herein, "variants" refer to entities that exhibit substantial structural identity with a reference entity (e.g., a wild type sequence) but are structurally different from the reference entity in one or more respects. For example, a polynucleotide may differ from a reference polynucleotide by one or more differences in the nucleotide sequence and/or by one or more differences in the chemical moieties (e.g., carbohydrates, lipids, etc.) covalently linked to the polynucleotide backbone. The variants exhibit at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity with the reference polynucleotide.

As used herein, the term "identity" refers to the overall relatedness between polymer molecules, for example, between nucleic acids (e.g., DNA molecules and/or RNA molecules). For example, polynucleotide sequences are considered to be "substantially identical" to one another if their sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculating the percent identity of two polynucleotide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). Determining the percent identity between two sequences and comparing the sequences can be accomplished using mathematical algorithms. As is well known to those skilled in the art, nucleotide sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN.

In one embodiment, the innate immune activation motif is introduced into one terminal region of the double-stranded RNA, so that it can operate independently of other regions other than the innate immune activation motif.

Double-stranded RNA with chemical modifications

In the double-stranded RNA of the present specification, either the first strand or the second strand may include a nucleotide into which a chemical modification has been introduced.

In one specific embodiment, the nucleotide into which the chemical modification is introduced may comprise one or more chemical modifications selected from the group consisting of: replacement of the -OH group at the 2' carbon position of the sugar structure in the nucleotide with -H, -CH ₃ (methyl), -OCH ₃ (-O-methyl), -NH ₂ , -F, -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl.

In one specific embodiment, the nucleotides to which the chemical modification has been introduced may be included in either the first strand or the second strand, and the number of nucleotides to which the chemical modification has been introduced may be at least 10 or more. The number of nucleotides into which the chemical modification is introduced is, for example, 10 to 60, 10 to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 60, 20 to 55, 20 to 50, 20 to It can be 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, or 15 to 20.

In one specific embodiment, the nucleotides into which the chemical modification has been introduced may be arranged either continuously or discontinuously within either the first strand or the second strand.

In one specific embodiment, the nucleotide into which the chemical modification is introduced may be included in either the first strand or the second strand and may be located in a region within the innate immune activation motif and/or a region outside the innate immune activation motif.

In one specific embodiment, the nucleotides to which the chemical modification has been introduced are included in at least 10 nucleotides in the first strand, and may be arranged alternatively with nucleotides to which the chemical modification has not been introduced. For example, nucleotides to which the chemical modification has been introduced in which the -OH group at the 2' carbon position is substituted with -OCH ₃ (-O-methyl) and nucleotides to which the chemical modification has not been introduced may be arranged alternatively. In this case, 50% or less, for example, 50%, 45%, 40%, 35%, or 30% of the total nucleotides in the first strand may be nucleotides to which the chemical modification has been introduced.

In one specific embodiment, the nucleotides to which the chemical modification has been introduced are included in the second strand at least 10 times, and may be arranged alternately with nucleotides to which the chemical modification has not been introduced, for example, nucleotides to which the chemical modification has been introduced in which the -OH group at the 2' carbon position is substituted with -OCH ₃ (-O-methyl) and nucleotides to which the chemical modification has not been introduced may be arranged alternately. In this case, 50% or less, for example, 50%, 45%, 40%, 35%, or 30% of the total nucleotides of the second strand may be nucleotides to which the chemical modification has been introduced.

The nucleotide to which the chemical modification has been introduced can induce or enhance an innate immune response. Specifically, the nucleotide to which the chemical modification has been introduced can induce or enhance an MDA5-mediated innate immune response. In addition, the RNA duplex according to the present invention comprising the nucleotide to which the chemical modification has been introduced can be for inducing or enhancing an RIG-I-mediated innate immune response and/or an MDA5-mediated innate immune response.

As used herein, the term "MDA5 (Melanoma differentiation-associated gene 5)" is a RIG-I-like receptor, a cytoplasmic RNA helicase that functions as a pattern recognition receptor protein to recognize foreign invaders. The structures of RIG-I and MDA5 are similar and share the N-tandem amino-terminal caspase activation and recruitment domains (CARDs) structure and the C-terminal domain (CTD). It is known that when the CTD recognizes double-stranded RNA, the CARDs are released, and the subsequent continuous signal activates interferon regulatory factors (IRFs). The above MDA5 and RIG-I bind to foreign RNA, i.e., double-stranded RNA, and initiate signal transduction. However, unlike RIG-I, which prefers attachment to short dsRNA, it is known that MDA5 initiates signal transduction by relatively long dsRNA.

In one embodiment, a double-stranded RNA having the structure of the aforementioned double-stranded RNA as its basic backbone and additionally having chemical modifications can induce a strong innate immune response despite the processing of double-stranded RNA having a relatively short base length, through an additional signaling mechanism in which MDA5 acts as a key factor.

In one specific embodiment, the double-stranded RNA may be one in which at least one inter-nucleotide bond is modified with phosphorothioate, boranophosphate, or methyl phosphonate. Specifically, the double-stranded RNA may be one in which at least one, for example, 2 to 4, inter-nucleotide bonds adjacent to the 5'-end and/or the 3'-end of the first strand and/or the second strand are modified with phosphorothioate, boranophosphate, or methyl phosphonate.

In one embodiment, a double-stranded RNA having the structure of a double-stranded RNA with the chemical modifications described above as its basic backbone and further modified in the bonds between nucleotides can enable the induction of a strong innate immune response despite such modifications.

Double-stranded RNA with innate immune response induction effect

In the present specification, the double-stranded RNA may be one that induces or enhances an innate immune response. To this end, the double-stranded RNA may satisfy the following conditions: 1) having a length of 25 to 60 base pairs, 2) the innate immune activation motif is located at one terminal region of one strand of the double-stranded RNA, 3) either the first strand or the second strand includes at least 10 nucleotides to which a chemical modification has been introduced.

In one specific embodiment, the double-stranded RNA may additionally have one or both ends of the double-stranded RNA be blunt ends. In one specific embodiment, one end of the double-stranded RNA may be an overhang. In one specific embodiment, the double-stranded RNA may have an innate immune activation motif positioned at the 3' end of the first strand and the 5' end of the second strand complementary thereto, forming a blunt end. For example, in the double-stranded RNA, one end at which the innate immune activation motif is positioned may be a blunt end, and the other end where the innate immune activation motif is not positioned may have an overhang. For example, in the double-stranded RNA, both one end at which the innate immune activation motif is positioned and the other end where the innate immune activation motif is not positioned may be blunt ends.

In one specific example, the double-stranded RNA may be a double-stranded RNA having a length of 25 to 60 base pairs, and may have a property of inducing an innate immune response mediated by RIG-I and MDA-5, and for this purpose, the double-stranded RNA may include an innate immune activation motif, and either the first strand or the second strand may have a structure including at least 10 nucleotides to which a chemical modification has been introduced. The double-stranded RNA may be a double-stranded RNA having a length of 25 to 60 base pairs, and may have a property of inducing RNA interference that inhibits the expression of a specific gene, and for this purpose, the double-stranded RNA may additionally include a region that complementarily binds to a specific gene in a terminal region.

In one specific example, the double-stranded RNA may have an innate immune activation sequence located at the 3' end of the first strand and the 5' end region of the second strand complementary thereto. The innate immune activation sequence may be 10 to 30 nucleotides including the nucleotide sequence of SEQ ID NO: 2, for example, the innate immune activation sequence may refer to a nucleotide consisting of any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 62, or a variant of the nucleotide. The double-stranded RNA may contribute to enhancing an innate immune response of an individual, specifically, a RIG-I-mediated innate immune response and/or an MDA5-mediated innate immune response, by including the above-mentioned innate immune activation motif and the structure of the double-stranded RNA (e.g., chemical modification, terminal form).

According to one embodiment, the double-stranded RNA according to one embodiment comprises a RIG-I-mediated innate immune activation motif located at one end and at least 10 nucleotides into which a chemical modification has been introduced, and the double-stranded RNA having the structure described above can induce a high level of innate immune response, regardless of the sequence of a region outside the innate immune activation motif, the type of strand on which the innate immune activation motif is located, and the region located within the double strand.

Pharmaceutical composition

As used herein, the term "effective ingredient" means an appropriate effective amount of an ingredient that affects a beneficial or desirable clinical or biochemical result. Specifically, it may mean an effective amount of a preparation, an active agent, or a double-stranded RNA. The pharmaceutical composition may optionally include a pharmaceutically acceptable carrier, diluent, excipient, buffer, salt, surfactant, cryoprotectant, etc.

The effective amount may be administered once or more and may be an amount appropriate for preventing a disease, or for alleviating symptoms, reducing the extent of a disease, stabilizing (i.e. not worsening) a disease, delaying or reducing the rate of disease progression, or improving or temporarily alleviating and alleviating (partially or completely) a disease.

The term "prevention" as used herein refers to any action that prevents the occurrence of a disease in advance, suppresses a disease, or delays its progression. For example, it refers to preventing the occurrence of the disease or its characteristic characteristics, interfering with the occurrence, or protecting or protecting against the occurrence of the disease or its characteristic characteristics.

The term "treatment" as used herein refers to both therapeutic treatment and preventive or prophylactic measures. It also refers to any action that improves or beneficially alters the symptoms of a disease. For example, preventing, reducing or ameliorating the disease or its characteristic features, or delaying (attenuating) the progression of the disease or its characteristic features in a subject.

The disease or pathological condition may be an infection caused by a virus or bacteria, a disease associated with immunosuppression, cancer (e.g., a solid tumor such as breast cancer, liver cancer, esophageal cancer, pancreatic cancer, lung cancer, stomach cancer, squamous cell carcinoma of the head and neck, prostate cancer, colon cancer, lymphoma, gallbladder cancer, kidney cancer, multiple myeloma, ovarian cancer, cervical cancer or glioma, or a non-solid tumor such as leukemia).

As used herein, the term "effective amount" refers to its generally accepted meaning in the art. The term can generally mean an amount of a molecule, compound, or composition that will elicit a desired biological response (e.g., a beneficial response) in a cell, tissue, system, animal, or human, as sought by a researcher, veterinarian, physician, or other clinician. Specifically, a "therapeutically effective amount" can mean an amount of a molecule, compound, or composition that will elicit a desired medical response, such as a therapeutically relevant change in a measurable parameter associated with a disease or disorder, such that a particular clinical treatment can be considered efficacious. A therapeutically effective amount of a drug for treating the disease or disorder can be the amount necessary to effect a therapeutically relevant change in the parameter.

The term "pharmaceutically acceptable excipient" as used herein may be one which, when combined/mixed with double-stranded RNA, maintains the activity of the double-stranded RNA. Examples thereof include, but are not limited to, any standard pharmaceutical excipient, such as a buffer system such as phosphate buffered saline, a surfactant, water, emulsions such as oil/water emulsions and various forms of wetting agents, starches, milk, sugars, certain forms of clay, gelatin, stearic acid or its salts, magnesium or calcium stearate, talc, vegetable oils, gums, glycols, or other known excipients.

The method of administration of the above pharmaceutical composition can be determined by a person skilled in the art based on the symptoms of the patient and the severity of the disease. In addition, it can be formulated in various forms such as powder, tablet, capsule, liquid, injection, ointment, syrup, etc., and can be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles.

The pharmaceutical composition of the present invention can be administered orally or parenterally. The route of administration of the composition according to the present invention is not limited thereto, but for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, enteral, sublingual, or topical administration is possible. The dosage of the composition according to the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, method, excretion rate, or disease severity, and can be easily determined by a person skilled in the art. In addition, the composition of the present invention can be formulated into a suitable dosage form using a known technique for clinical administration.

Composition for enhancing innate immunity

As used herein, the term "composition for enhancing innate immunity" refers to a biological agent containing the above-mentioned double-stranded RNA and for inducing or enhancing the innate immune response of an administered subject. The composition for enhancing innate immunity can be used interchangeably with an immune enhancer/adjuvant, an RNA-based immunogenic composition, or an immunostimulation agent. The composition can optionally contain a pharmaceutically acceptable carrier, diluent, excipient, buffer, salt, surfactant, cryoprotectant, etc., and the implementation conditions and implementation embodiments are as described for the pharmaceutical composition.

The above composition for enhancing innate immunity can be utilized as an effective ingredient in a vaccine adjuvant, an immune anticancer agent, or an antiviral agent (e.g., hepatitis B virus), depending on the intended use.

The composition for enhancing innate immunity may, for example, form a complex with one or more lipid components to form liposomes, lipid nanoparticles and/or lipoplexes. The lipid nanoparticles include an ionizable cationic lipid as one component, and may also include other components such as a helper lipid or a stabilizer that encapsulates mRNA or helps with delivery efficiency and stabilization. The lipid nanoparticles may apply components known in the art, and may include cationic lipids, helper lipids, and PEG-conjugated lipids. In addition to the above-mentioned components, techniques known in the art may be utilized without limitation with respect to effective amounts, formulations, administration methods, combination preparations, etc.

Another aspect provides a method of treating a disease or ameliorating a pathological condition comprising administering to a subject the double-stranded RNA.

Another aspect provides a method of enhancing an innate immune response comprising administering to a subject the pharmaceutical composition.

Another aspect provides the use of said double-stranded RNA for the manufacture of a medicament for treating or ameliorating a disease or pathological condition.

Another aspect provides the use of said double-stranded RNA for the manufacture of a medicament for enhancing innate immune response.

Since the above method or use includes or utilizes the above-described double-stranded RNA or pharmaceutical composition as it is, the description of common contents between them is omitted to avoid excessive complexity of this specification. In addition, the disease or pathological condition is as described above.

The term "subject" as used herein means a subject in need of treatment for a disease, specifically a condition, and more specifically, may include any mammal, such as a human or non-human primate, mouse, dog, cat, horse, cow, sheep, pig, goat, camel, or antelope.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

Example 1. Production of double-stranded RNA containing an innate immune activation motif

In this example, double-stranded RNAs of various lengths capable of activating innate immunity were produced. As illustrated in Fig. 1, the double-stranded RNA is composed of 38 to 60 base pairs, which are composed of a first strand and a second strand complementarily binding to the first strand, and includes an innate immune activation motif located at the 3' terminal region of the first strand and the 5' terminal region of the second strand, wherein the innate immune activation sequence of SEQ ID NO: 6 (19 nt) or SEQ ID NO: 8 (20 nt) is located at the 5' terminal region of the second strand. In addition, in order to compare the efficacy according to the structure of the double-stranded RNA, the terminal region of the double-stranded RNA was modified based on the above-described basic structure to produce double-stranded RNAs having a smooth end, one protrusion, or two protrusions, respectively. Specifically, the double-stranded RNA was purchased from Bioneer Co. Ltd (Republic of Korea), and Dharmacon, Inc (UK), and the double-stranded RNA comprising the innate immune activation sequence of SEQ ID NO: 6 is as shown in Table 1, and the double-stranded RNA comprising the innate immune activation sequence of SEQ ID NO: 8 is as shown in Table 2. In Tables 1 and 2, the innate immune activation sequence is underlined, and the protrusion formed in the terminal region is shaded.

[Table 1]

[Table 2]

Example 2. Confirmation of the effect of introducing an innate immune activation motif

2-1. Knockdown effect on target gene

In this example, the knockdown effect of the double-stranded RNA according to Example 1 on the target gene was confirmed. The knockdown effect of the 19, 38, 50, or 60 bp double-stranded RNA (Table 1) containing the innate immune activation motif according to the above Example 1 on the Luciferase gene was confirmed in HeLa (CCL-2, ATCC) cell line treated with Luciferase plasmid (E1741, Promega). Specifically, the HeLa (CCL-2, ATCC) cell line was plated in a 24-well plate at 30% confluency in complete medium without antibiotics. Then, the cell line was transfected with double-stranded RNA using Lipofectamine RNAiMAX (13778150; Invitrogen). After 24 hours from the time of transfection, total RNA was extracted using Tri-RNA Reagent (Favorgen), and 0.5 μg of total RNA was used for cDNA synthesis. Primers for the target gene were mixed with SYBR Green PCR Master Mix and quantified by relative standard curve. The target gene mRNA levels were measured using the relative standard curve quantitation method. Then, IC ₅₀ values were calculated based on the dose-response curve using GraphPad Prism 8.

As a result, as shown in Table 3, all double-stranded RNAs showed IC ₅₀ values of less than 0.5 nM for the target gene, regardless of length or terminal structure. The results indicate that the introduction of the innate immune activation motif into the double-stranded RNA does not affect the target gene knockdown effect, i.e., it can act independently without affecting the original function of the double-stranded RNA.

[Table 3]

2-2. Effect of inducing innate immune response

In this example, we attempted to determine the effect of the innate immune activation motif introduced at the end of double-stranded RNA on the innate immune response. The expression of cytokines related to the innate immune response was analyzed for the 38, 50, or 60 bp double-stranded RNAs comprising the innate immune activation sequence of SEQ ID NO: 6 according to Example 1 (Table 1) and the 40 bp double-stranded RNAs comprising the innate immune activation sequence of SEQ ID NO: 8 (Table 2). Specifically, T98G (CRL-1690, ATCC) cell line was plated in a 24-well plate at 30% confluency in complete medium without antibiotics. Thereafter, the double-stranded RNA was transfected into the cell line using Lipofectamine RNAiMAX (13778150; Invitrogen), and the transfected cells were harvested after 12 or 24 hours, respectively. Thereafter, the levels of IFIT1, ISG15, and CXCL10 were analyzed using qRT-PCR according to the method of Example 2-1. Meanwhile, in this example, the positive control group was administered Poly I:C, and the control group was used as a group to which only the transfection reagent was added (mock) and a group not treated with double-stranded RNA (NT).

As a result, as shown in FIGS. 2 and 3, the double-stranded RNA including the innate immune activation sequence of SEQ ID NO: 6 induced high expression of IFIT1, CXCL10, ISG15, TNF-α, and IL-1β related to the innate immune response. In particular, this effect was more excellent when the terminal structure of the double-stranded RNA was a blunt end, and was also very remarkable compared to the effect of the double-stranded RNA having the same structure as above but without the motif introduced at the terminal of the double-stranded RNA. These results indicate that the induction of the innate immune response is affected by the innate immune activation motif located at the terminal region of the double-stranded RNA.

Also, as shown in FIG. 4, the double-stranded RNA including the innate immune activation sequence of SEQ ID NO: 8 also induced high expression of IFIT1, CXCL10, and ISG15 related to the innate immune response. On the other hand, the group treated with the 40 bp double-stranded RNA (Lamin A/G-GAPDH, Lamin A/C-Luciferase, GAPDH-Luciferase) having the same structure as above but without the motif introduced at the end of the double-stranded RNA, and the group treated by mixing each of the short-length target binding sequence and motif sequence (mixture of 19 bp) did not induce an effective level of innate immune response. Therefore, it is shown that the above-mentioned innate immune response induction effect is derived from the innate immune activation motif according to one embodiment introduced into the double-stranded RNA.

2-3. Effect of RIG-I mediated innate immune response induction

In this example, we attempted to confirm the relationship with RIG-I as a factor mediating the innate immune response. The activity of IRF for the RAW-Lucia ISG cell line (WT) and RAW-Lucia ISG-KO-RIG-I cell line (RIG-I KO) was measured for the 38, 50, or 60 bp double-stranded RNA (Table 1) including the innate immune activation sequence of SEQ ID NO: 6 according to Example 1 and the 40 bp double-stranded RNA (Table 2) including the innate immune activation sequence of SEQ ID NO: 8. Specifically, the RAW-Lucia ISG cell line and the RAW-Lucia ISG-KO-RIG-I cell line were plated at 1 × 10 ⁵ cells per well in a 96-well plate. Thereafter, the double-stranded RNA (10 nM) was transfected into the cell line using Lipofectamine RNAiMAX (13778150; Invitrogen), and the transfected cells were each cultured for 24 hours to obtain the supernatant. Thereafter, the interferon regulatory factor (IRF) activity level was detected in the supernatant using a plate reader (VICTORX2; PerkinElmer). Meanwhile, in this example, the positive control group was a group administered with Poly I:C and/or LPS, and the control group was a group added with only the transfection reagent (mock) and a group not treated with double-stranded RNA (NT).

As a result, as shown in FIGS. 5 and 6, a high level of innate immune response was confirmed in the RAW-Lucia ISG cell line treated with the double-stranded RNA including the innate immune activation sequence of SEQ ID NO: 6 or SEQ ID NO: 8. In particular, it was found that the innate immune response described above was induced when, among the double-stranded RNAs, both ends were blunt-ended or the end adjacent to the innate immune activation motif was blunt-ended. On the other hand, in the ISG-KO-RIG-I cell line lacking RIG-I, the innate immune response was lost or significantly reduced. In particular, as shown in FIG. 6, in the double-stranded RNA, a similar effect was shown not only when both ends formed blunt-ended, but also when one of the terminal regions adjacent to the innate immune activation motif had a blunt-ended end. The above results indicate that the innate immune response derived from motifs located in the terminal region of double-stranded RNA is mediated by RIG-I.

Example 3. Confirmation of the innate immune response-inducing effect of double-stranded RNA containing a modified innate immune activation motif

3-1. Confirmation of the efficacy of double-stranded RNA with a length of 40 bp

In this example, we aimed to evaluate the level of innate immune activation by double-stranded RNA including a modified innate immune activation motif. To this end, a double-stranded RNA was produced in which the nucleotide sequence of SEQ ID NO: 2 (10 nt) or the nucleotide sequence of SEQ ID NO: 4 (15 nt) is located at the 5' end of the second strand, and its specific structure is as shown in Fig. 1. In addition, in order to compare the difference in effect depending on the region in which the innate immune activation motif is located, a double-stranded RNA in which the nucleotide sequence of SEQ ID NO: 8 was introduced into the extra-terminal region was additionally produced. Specifically, the double-stranded RNA was obtained from Bioneer Co. Ltd (Republic of Korea), and Dharmacon, Inc (UK), and the double-stranded RNA including the innate immune activation sequence of SEQ ID NO: 2 or SEQ ID NO: 4 is as shown in Table 4. In Table 4, the innate immune activation sequence is underlined.

[Table 4]

Thereafter, in the same manner as in Example 2-3, the activity of IRF for RAW-Lucia ISG cell line (WT) and RAW-Lucia ISG-KO-RIG-I cell line (RIG-I KO) was measured. Meanwhile, in this example, the positive control group was a group administered with Poly I:C and/or LPS, and the control group was a group added with only the transfection reagent (mock) and a group not treated with double-stranded RNA (NT).

As a result, as shown in FIG. 7, the double-stranded RNA including the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 4 at the terminal was also able to enhance the RIG-I-mediated innate immune response, similarly to Example 2-3. However, the double-stranded RNA (Motif-20-Shifted) in which the nucleotide sequence of SEQ ID NO: 8 was shifted to the center of the entire sequence showed a significant decrease in this effect. From the above results, it was found that the motif of SEQ ID NO: 2 consisting of 10 nt can be applied as a unit or core sequence for inducing the RIG-I-mediated innate immune response, and that the sequence can exhibit effective efficacy when introduced or positioned at the terminal in the double-stranded RNA.

3-2. Confirmation of the efficacy of double-stranded RNA with a length of 30 bp

In this example, based on the experimental results of Example 2 and Example 3-1, a 30 bp double-stranded RNA was prepared in which the nucleotide sequence (10 nt) of SEQ ID NO: 2 is located at the 5' end of the second strand or the 3' end of the first strand in the double-stranded RNA and both ends are blunt ends. In order to confirm the effect of the position of the innate immune activation motif in the double-stranded RNA on the innate immune response, a double-stranded RNA (LaminA/C-Motif-10) in which the innate immune activation sequence is located at the 5' end of the second strand and a double-stranded RNA (LaminA/C-inv. Motif-10) in which the innate immune activation sequence is located at the 3' end of the first strand were prepared, respectively. Specifically, the double-stranded RNA was obtained from Bioneer Co. Ltd (Republic of Korea), and Dharmacon, Inc (UK), and the above-mentioned double-stranded RNAs are as shown in Table 5. In Table 5, the innate immune activation sequences are underlined.

[Table 5]

Thereafter, the knockdown effect of the target gene, Lamin A/C, upon treatment with 10 nM of double-stranded RNA was confirmed using the Huh7 cell line in the same manner as in Example 2-1. As a result, as shown in Fig. 8, the double-stranded RNA having a length of 30 bp also showed a knockdown effect on the target gene, similar to Example 2-1.

In addition, in the same manner as in Example 2-3, the activity of IRF for RAW-Lucia ISG cell line (WT) and RAW-Lucia ISG-KO-RIG-I cell line (RIG-I KO) according to treatment with 10 nM of double-stranded RNA was measured. As a result, as shown in Fig. 9, it was found that the innate immune response can be enhanced not only when the innate immune activation sequence is located at the 5' end of the second strand but also when it is located at the 3' end of the first strand, and that this innate immune response is also mediated by RIG-I.

Example 4. Confirmation of the innate immune response induction effect of double-stranded RNA with chemical modifications

In this example, a double-stranded RNA containing an innate immune activation motif was prepared by introducing chemical modifications into a double-stranded RNA, and the innate immune response induction effect of the double-stranded RNA with the chemical modifications was evaluated.

4-1. Confirmation of the effect according to the strand into which chemical modification was introduced

In this example, a 30-bp double-stranded RNA having the innate immune activation sequence of sequence number 2 inserted at the 5' end of the second strand was prepared as a basic backbone, and a double-stranded RNA was prepared in which a chemical modification was introduced in which the -OH group at the 2' carbon position of the nucleotide was substituted with -OCH ₃ (-O-methyl). In order to confirm the effect of introducing a chemical modification on the innate immune response induction effect, Double-stranded RNA having a chemical modification introduced into either one of the first strand or the second strand, and double-stranded RNA having chemical modifications introduced into both the first strand and the second strand were respectively prepared. Specifically, double-stranded RNA having a chemical modification introduced into either one of the first strand or the second strand are as shown in Table 6, and double-stranded RNA having chemical modifications introduced into both the first strand and the second strand are as shown in Table 7. In Tables 6 and 7, the nomenclature of double-stranded RNA is expressed as [strand having chemical modification introduced]-[terminus having chemical modification introduced and first nucleotide position having chemical modification introduced]-[number of nucleotides having chemical modification introduced]. In addition, the innate immune activation motif and the innate immune activation sequence are respectively shaded and/or bolded, and the nucleotides having 2'-OCH ₃ (-O-methyl) chemical modification introduced are underlined.

[Table 6]

[Table 7]

Thereafter, in the same manner as in Example 2-3, the activity of IRF for RAW-Lucia ISG cell line (WT) and RAW-Lucia ISG-KO-RIG-I cell line (RIG-I KO) was measured. Meanwhile, in this example, the positive control group was a group administered with Poly I:C and/or LPS, and the control group was a group added with only the transfection reagent (mock) and a group not treated with double-stranded RNA (NT).

As a result, as shown in Fig. 10, in the double-stranded RNA having chemical modifications introduced into either the first strand or the second strand, the double-stranded RNA containing eight nucleotides to which chemical modifications were introduced (#1 5'-1-8, #2 3'-2-8, #1 5'-2-8, and #2 3'-1-8) completely lost the effect derived from the innate immune response motif, i.e., the innate immune response induction effect. In contrast, the double-stranded RNA having ten nucleotides to which chemical modifications were introduced into either the first strand or the second strand (#1 5'-1-10, #2 3'-2-10, and #1 5'-2-10, #2 3'-1-10) exhibited a high level of innate immune induction effect, and this effect was significantly reduced in the ISG-KO-RIG-I cell line lacking RIG-I. In addition, as shown in Fig. 11, double-stranded RNA with chemical modifications introduced to both the first and second strands completely lost the innate immune response induction effect.

4-2. Confirmation of the effect according to the position of the nucleotide where chemical modification is introduced

This study was conducted to determine the effect of the position of the chemically modified nucleotides on the innate immune response induction effect. To this end, using the double-stranded RNA of Example 4-1 as the basic framework, 10 nucleotides with chemical modifications were positioned at the 5' end or 3' end of the first strand (#1 5'-1-10, #1 3'-1-10), or the position of the first nucleotide with chemical modifications was changed (#1 5'-1-10, #1 5'-2-10). Additionally, a double-stranded RNA with chemical modifications was prepared by changing the sequence of a region other than the innate immune activation motif in the double-stranded RNA (GAPDH-Motif). Specifically, the double-stranded RNA with the chemical modifications introduced as described above is as shown in Table 8. In Table 8 above, innate immune activation motifs and innate immune activation sequences are shaded and/or bold, respectively, and nucleotides with 2'-OCH ₃ (-O-methyl) chemical modifications are underlined.

[Table 8]

Thereafter, in the same manner as in Example 2-3, the activity of IRF for RAW-Lucia ISG cell line (WT) and RAW-Lucia ISG-KO-RIG-I cell line (RIG-I KO) was measured. Meanwhile, in this example, the positive control group was a group administered with Poly I:C and/or LPS, and the control group was a group added with only the transfection reagent (mock) and a group not treated with double-stranded RNA (NT).

As a result, as shown in Fig. 12, the double-stranded RNA without the innate immune activation motif did not induce an effective level of innate immune response regardless of the introduction of chemical modifications (Lamin A/C-GAPDH, Lamin A/C-GAPDH #1 5'-2-10), whereas the double-stranded RNA containing the innate immune activation motif exhibited a high level of innate immune induction effect when 10 chemical modifications were introduced, regardless of the sequence of the region outside the innate immune activation motif in the double-stranded RNA and the location of the innate immune activation motif in the single strand.

4-3. Confirmation of the effect according to the number of nucleotides to which chemical modifications have been introduced

This study was conducted to determine the effect of the number of chemically modified nucleotides on the innate immune response induction effect. To this end, using the double-stranded RNA of Example 4-1 as the basic framework, 8, 10, 12, or 15 chemically modified nucleotides were positioned at the 5' end of the first strand (AS 5'-2-8, AS 5'-2-10, AS 5'-2-12, and AS 5'-2-15). Additionally, double-stranded RNAs in which chemical modifications were introduced to both the first strand and the second strand were prepared along with the chemical modifications described above, and their innate immune response induction effects were compared. Specifically, the double-stranded RNAs in which the chemical modifications described above were introduced are as shown in Table 9. In Table 9 above, innate immune activation motifs and innate immune activation sequences are shaded and/or bolded, respectively, and nucleotides with 2'-OCH ₃ (-O-methyl) chemical modifications are underlined.

[Table 9]

Thereafter, in the same manner as in Example 2-3, the activity of IRF for RAW-Lucia ISG cell line (WT) and RAW-Lucia ISG-KO-RIG-I cell line (RIG-I KO) was measured. Meanwhile, in this example, the positive control group was a group administered with Poly I:C and/or LPS, and the control group was a group added with only the transfection reagent (mock) and a group not treated with double-stranded RNA (NT).

As a result, as shown in Fig. 13, in the double-stranded RNA including the innate immune activation motif, when at least 10 chemical modifications (10, 12, 15) were introduced, a high level of innate immunity induction effect was exhibited. In addition, this effect was significantly reduced in the ISG-KO-RIG-I cell line lacking RIG-I. In addition, similar to the results of the examples described above, the double-stranded RNA including 8 nucleotides into which chemical modifications were introduced, and the double-stranded RNA into which chemical modifications were introduced into both the first strand and the second strand, all innate immune response induction effects derived from the innate immune response motif were lost.

In summary of the above results, it was found that when at least 10 chemical modifications are introduced into either the first strand or the second strand in a double-stranded RNA including an innate immune activation motif according to one embodiment, the effect of enhancing the RIG-I-mediated innate immune response is achieved regardless of the sequence of the region outside the innate immune activation motif in the double-stranded RNA and the location of the innate immune activation motif in the single strand.

Example 5. Confirmation of the mechanism of induction of innate immune response by double-stranded RNA with chemical modifications

The double-stranded RNA with the chemical modification of Example 4 above showed an enhanced innate immune response induction effect compared to the double-stranded RNA according to Examples 1 to 3 without the chemical modification. In addition, the double-stranded RNA with the chemical modification introduced, unlike the double-stranded RNA without the chemical modification introduced, showed a certain level of IRF activity without completely disappearing or significantly reducing the innate immune response in the ISG-KO-RIG-I cell line lacking RIG-I (see FIGS. 10, 12, and 13).

Therefore, in this example, under the premise that the innate immune response induction effect by introduction of chemical modifications can be achieved by a different mechanism from RIG-I, the activity of IRF was measured for RAW-Lucia ISG cell line (WT), RAW-Lucia ISG-KO-RIG-I cell line (RIG-I KO), and RAW-Lucia ISG-KO-MDA5-I cell line (MDA5 KO) in the same manner as in Example 2-3. Specifically, the double-stranded RNAs into which chemical modifications were introduced used in this example are as shown in Table 10. In Table 10, the innate immune activation motifs and innate immune activation sequences are indicated in shaded and/or bold, respectively, and the nucleotides into which 2'-OCH ₃ (-O-methyl) chemical modifications were introduced are indicated in underline.

[Table 10]

As a result, as shown in Fig. 14, similar to the experimental results of Example 4, double-stranded RNA not containing an innate immune activation motif did not induce an effective level of innate immune response, whereas in double-stranded RNA containing an innate immune activation motif, when at least 10 or more chemical modifications were introduced to either the first strand or the second strand, a high level of innate immune induction effect was exhibited. In particular, it was confirmed that this effect tended to be somewhat reduced in the ISG-KO-RIG-I cell line lacking RIG-I, similar to the experimental results of the previous examples, whereas it was lost or significantly reduced in the ISG-KO-MDA5 cell line lacking MDA5.

From the above results, it was found that the innate immune response to double-stranded RNA with chemical modification introduced according to one embodiment is mediated by MDA5 as a major factor in addition to RIG-I.

Example 6. Verification of the effect of inducing innate immune response using human monocyte cell line

In this example, the effect of inducing an innate immune response according to the treatment of the double-stranded RNA with the chemical modification of Example 4 was verified again, using the human monocyte cell line THP1-Dual (thpd-nfis) cell line. In this example, a 30-bp double-stranded RNA in which an innate immune activation sequence consisting of nucleotides of SEQ ID NO: 2 is inserted at the 5' end of the second strand as a basic framework was prepared to introduce a chemical modification in which the -OH group at the 2' carbon position of the nucleotide is substituted with -OCH ₃ (-O-methyl). Specifically, information on the double-stranded RNA with the chemical modification introduced used in this example is as shown in Tables 6, 7, and 9 above.

In the same manner as in the above Example 2-3, double-stranded RNA (10 nM) was transfected into the THP1-Dual (thpd-nfis) cell line using Lipofectamine RNAiMAX (13778150; Invitrogen), and the transfected cells were cultured for 24 or 48 hours, respectively, and then the supernatant was obtained. Thereafter, the level of interferon regulatory factor activity was detected in the supernatant using a plate reader (VICTORX2; PerkinElmer). Meanwhile, in this example, the positive control group was a group administered with Poly I:C, and the control group was a group added with only the transfection reagent (mock) and a group not treated with double-stranded RNA (NT).

As a result, as shown in FIG. 15, it was found that in a double-stranded RNA including an innate immune activation motif according to one embodiment, when at least 10 chemical modifications were introduced to either the first strand or the second strand, the effect of enhancing the innate immune response was achieved.

Example 7. Confirmation of the effect of inducing innate immune response according to nucleotide length

In this example, the 30 bp double-stranded RNA (#1 5'-2-15), which showed a high level of innate immunity induction effect in all of the above Examples 4-3, 5, and 6, was used to evaluate the innate immune response induction effect according to the modification of the entire nucleotide length. To this end, as in the 30 bp double-stranded RNA of Examples 4-3, 5, and 6 (#1 5'-2-15), the innate immune activation sequence of SEQ ID NO: 2 was inserted at the 5' end of the second strand, and a double-stranded RNA in which 50% of the chemical modifications for the entire nucleotide length were introduced into the first strand was used as the basic backbone, and the length of the nucleotides was changed to 20 to 40 bp so as to include at least 10 or more chemical modifications (#1 5'-2-10, #1 5'-2-11, #1 5'-2-12, #1 5'-2-13, #1 5'-2-15, #1 5'-2-20). Specifically, the 20 to 40 bp double-stranded RNAs into which the above-mentioned chemical modifications were introduced are as shown in Table 11. In Table 11, innate immune activation motifs and innate immune activation sequences are shaded and/or bold, respectively, and nucleotides with 2'-OCH ₃ (-O-methyl) chemical modifications are underlined.

[Table 11]

Thereafter, the activity of IRF for RAW-Lucia ISG cell line was measured in the same manner as in Example 2-3. Meanwhile, in this example, the positive control group was administered Poly I:C, and the control group was used as the group added only with the transfection reagent (mock) and the group not treated with double-stranded RNA (NT). The results are shown in Fig. 16. The result using 30 bp Lamin A/C-motif without 2'-OCH3(-O-methyl) chemical modification in Table 11 is indicated as Lamin A/C-motif in Fig. 16; The result using 20 bp Lamin A/C-motif with 2'-OCH3(-O-methyl) chemical modification #1 5'-2-10 introduced in Table 11 is indicated as 20 bp 2'-OMe Lamin A/C-motif in Fig. 16; The result using the 22 bp Lamin A/C-motif into which the 2'-OCH3(-O-methyl) chemical modification #1 5'-2-11 was introduced in Table 11 above is indicated as 22 bp 2'-OMe Lamin A/C-motif in FIG. 16; The result using the 24 bp Lamin A/C-motif into which the 2'-OCH3(-O-methyl) chemical modification #1 5'-2-12 was introduced in Table 11 above is indicated as 24 bp 2'-OMe Lamin A/C-motif in FIG. 16; The result using the 26 bp Lamin A/C-motif into which the 2'-OCH3(-O-methyl) chemical modification #1 5'-2-13 was introduced in Table 11 above is indicated as 26 bp 2'-OMe Lamin A/C-motif in FIG. 16; The results using the 30 bp Lamin A/C-motif introduced with the 2'-OCH3(-O-methyl) chemical modification #1 5'-2-15 in Table 11 above are indicated as 30 bp 2'-OMe Lamin A/C-motif in Figure 16; The results using the 40 bp Lamin A/C-motif introduced with the 2'-OCH3(-O-methyl) chemical modification #1 5'-2-20 in Table 11 above are indicated as 40 bp 2'-OMe Lamin A/C-motif in Figure 16.

As a result, as shown in FIG. 16, the 20-24 bp double-stranded RNA containing the innate immune activation motif and having at least 10 2'-OCH3(-O-methyl) chemical modifications introduced into either the first strand or the second strand completely lost the effect derived from the innate immune response motif, i.e., the innate immune response induction effect. In contrast, the 26-40 bp double-stranded RNA containing the innate immune activation motif and having at least 10 2'-OCH3(-O-methyl) chemical modifications introduced into either the first strand or the second strand exhibited a high level of innate immune induction effect.

From the above results, it was found that the double-stranded RNA containing an innate immune activation motif according to one embodiment and having at least 10 chemical modifications introduced into either the first strand or the second strand exhibited efficacy due to the innate immune activation motif and chemical modifications in the range of a total nucleotide length of 26 bp to 40 bp.

Example 8. Verification of the effect of inducing innate immune response by innate immune activation motif

8-1. Evaluation of efficacy according to presence or absence of innate immune activation motif

In this example, the 30 bp double-stranded RNA that showed a high level of innate immunity induction effect in Example 7 was used as the target for the human monocyte cell line, THP1-Dual (thpd-nfis), to evaluate the innate immune response induction effect according to the presence or absence of the innate immune activation motif. In this example, using the 30 bp double-stranded RNA (#1 5'-2-15) of the above Example 7 as a basic framework, a double-stranded RNA with or without an innate immune activation motif was produced. Specifically, the 30 bp double-stranded RNAs with or without the above-mentioned innate immune activation motif are as shown in Table 12. In Table 12, the innate immune activation motif and the innate immune activation sequence are indicated in shaded and/or bold, respectively, and the nucleotides introduced with a 2'-OCH ₃ (-O-methyl) chemical modification are indicated in underline.

[Table 12]

Thereafter, in the same manner as in Example 6, the activity of IRF for the THP1-Dual (thpd-nfis) cell line was measured. Meanwhile, in this example, the positive control group was administered Poly I:C, and the control group was used as the group added only with the transfection reagent (mock) and the group not treated with double-stranded RNA (NT). The results are shown in Fig. 17. The result using 30 bp Lamin A/C-GAPDH without 2'-OCH3(-O-methyl) chemical modification in Table 12 is denoted as Lamin A/C-GAPDH in Fig. 17; The result using 30 bp Lamin A/C-GAPDH with 2'-OCH3(-O-methyl) chemical modification #1 5'-2-15 introduced in Table 12 is denoted as 2'-OMe Lamin A/C-GAPDH in Fig. 17; The results using the 30 bp Lamin A/C-Motif without the 2'-OCH3(-O-methyl) chemical modification introduced in Table 12 above are denoted as Lamin A/C-Motif in Figure 17; the results using the 30 bp Lamin A/C-Motif with the 2'-OCH3(-O-methyl) chemical modification #1 5'-2-15 introduced in Table 12 above are denoted as 2'-OMe Lamin A/C-Motif in Figure 17.

As a result, as shown in Fig. 17, the 30 bp double-stranded RNA containing the innate immune activation motif and having at least 10 2'-OCH3(-O-methyl) chemical modifications introduced into either the first or second strand exhibited a high level of innate immune induction effect in human monocyte cell lines, similar to the experimental result of Example 7. On the other hand, the group treated with the 30 bp double-stranded RNA (Lamin A/G-GAPDH) having the same structure as above but without the motif introduced into the end of the double-stranded RNA did not induce a significant level of innate immune response. Therefore, it was confirmed once again that the introduction of the innate immune activation motif is an essential element for the innate immune induction effect.

8-2. Efficacy evaluation according to application of modified innate immune activation motif

In this example, we attempted to confirm the effect of modifying the length of the innate immune activation motif on the innate immune response induction effect based on the 30 bp double-stranded RNA that showed a high level of innate immune induction effect in the above Example 7. To this end, using the 30 bp double-stranded RNA (#1 5'-2-15) of the above Example 7 as the basic framework, we additionally produced a double-stranded RNA in which the innate immune activation sequence of SEQ ID NO: 8 (20 nt) or SEQ ID NO: 62 (30 nt) was introduced instead of the innate immune activation sequence of SEQ ID NO: 2. Specifically, the innate immune activation sequences of SEQ ID NO: 8 (20 nt) and SEQ ID NO: 62 (30 nt) are shown in Table 13, and the 30 bp double-stranded RNA (Lamin A/C- Motif2, Motif3) containing the innate immune activation sequence of SEQ ID NO: 8 (20 nt) or SEQ ID NO: 62 (30 nt) in the 5'-terminal region of the second strand are as shown in Table 14. In Tables 13 and 14, the innate immune activation motif and the innate immune activation sequence are shaded and/or bold, respectively, and the nucleotides introduced with the 2'-OCH ₃ (-O-methyl) chemical modification are underlined.

[Table 13]

[Table 14]

Thereafter, the activity of IRF for RAW-Lucia ISG cell line was measured in the same manner as in Example 2-3. Meanwhile, in this example, the positive control group was administered Poly I:C, and the control group was used as the group added only with the transfection reagent (mock) and the group not treated with double-stranded RNA (NT). The results are shown in Fig. 18. The result using 30 bp Lamin A/C-Motif into which 2'-OCH3(-O-methyl) chemical modification #1 5'-2-15 was introduced in Table 14 is indicated as 2'-OMe Lamin A/C-Motif in Fig. 18; The result using 30 bp Lamin A/C-Motif2 into which 2'-OCH3(-O-methyl) chemical modification #1 5'-2-15 was introduced in Table 14 is indicated as 2'-OMe Lamin A/C-Motif2 in Fig. 18; The results using 30 bp Motif3 with 2'-OCH3(-O-methyl) chemical modification #1 5'-2-15 introduced in Table 14 above are indicated as 2'-OMe Motif3 in Figure 18.

As a result, as shown in Fig. 18, the double-stranded RNA including the nucleotide sequence of sequence number 8 at the end also exhibited a high level of innate immunity induction effect, similar to Example 7, and it was confirmed that the nucleotide sequence of sequence number 62 also exhibited an excellent innate immunity induction effect.

Therefore, it was confirmed that the double-stranded RNA with chemical modification introduced according to one embodiment can exhibit effective innate immune induction efficacy even in the modified innate immune activation motif.

Example 9. Confirmation of the effect of inducing innate immune response by the form of introduced chemical modification

In this example, we attempted to confirm the influence of the type of chemical modification introduced based on the 30 bp double-stranded RNA showing a high level of innate immune induction effect in the above Example 7 on the innate immune response induction effect. To this end, using the 30 bp double-stranded RNA (#1 5'-2-15) of the above Example 7 as a basic framework, a double-stranded RNA was additionally produced in which a chemical modification was introduced in which the -OH group at the 2' carbon position of the nucleotide was substituted with -F instead of -OCH3(-O-methyl). Specifically, the 30 bp double-stranded RNA into which the above-mentioned 2'-F chemical modification was introduced is as shown in Table 15. In Table 15, the innate immune activation motif and the innate immune activation sequence are indicated in shaded and/or bold, respectively, and the nucleotides into which the 2'- _OCH3 (-O-methyl) or 2'-F chemical modification was introduced are indicated in underline.

[Table 15]

Thereafter, the activity of IRF for the RAW-Lucia ISG cell line was measured in the same manner as in the above Example 2-3. In addition, the activity of IRF for the THP1-Dual (thpd-nfis) cell line was measured in the same manner as in the above Example 6. Meanwhile, in this example, the positive control group was administered Poly I:C, and the control group was used as the group added only with the transfection reagent (mock) and the group not treated with double-stranded RNA (NT). The results for the RAW-Lucia ISG cell line are shown in Fig. 19, and the results for the THP1-Dual (thpd-nfis) cell line are shown in Fig. 20. The results using the 30 bp Lamin A/C-motif without chemical modification of 2'-OCH3(-O-methyl) or 2'-F in Table 15 are indicated as Lamin A/C-motif in Figs. 19 and 20; The results using the 30 bp Lamin A/C-Motif in which the 2'-OCH3(-O-methyl) chemical modification was introduced at position #1 5'-2-15 in the above Table 15 are denoted as 2'-OMe Lamin A/C-Motif in FIGS. 19 and 20 ; the results using the 30 bp Motif in which the 2'-F chemical modification #1 5'-2-15 was introduced in the above Table 15 are denoted as 2'-F Lamin A/C-Motif in FIGS. 19 and 20 .

As a result, as shown in Fig. 19, the double-stranded RNA into which 2'-F or 2'-OCH ₃ (-O-methyl) chemical modifications were introduced exhibited an effective innate immune response induction effect, and in particular, it was confirmed that the double-stranded RNA into which 2'-OCH ₃ (-O-methyl) chemical modifications were introduced exhibited a much higher level of innate immune induction effect. In addition, it was confirmed that this tendency was similarly shown in Fig. 20 using a human monocyte cell line.

Example 10. Evaluation of efficacy according to modification of nucleotide bond

In this example, we attempted to verify whether the innate immunity induction effect could be maintained even when the nucleotide bond was modified to improve RNA stability. To this end, using the 30 bp double-stranded RNA (#1 5'-2-15) that showed a high level of innate immunity induction effect in Example 7 as a basic framework, a double-stranded RNA was additionally produced in which two nucleotide bonds adjacent to each other from at least one strand, i.e., the bond between the 1st nucleotide and the 2nd nucleotide and the bond between the 2nd nucleotide and the 3rd nucleotide, were modified from phosphodiester bond to phosphorothioate bond. Specifically, the 30 bp double-stranded RNA in which the nucleotide bond described above was modified is as shown in Table 16. In Table 16, innate immune activation motifs and innate immune activation sequences are shaded and/or bold, respectively, nucleotides with 2'-OCH3(-O-methyl) chemical modifications are underlined, and forms in which nucleotide bonds are substituted with phosphorothioate (PS) bonds are marked with "*".

[Table 16]

Thereafter, the activity of IRF for RAW-Lucia ISG cell line was measured in the same manner as in Example 2-3. Meanwhile, in this example, the positive control group was administered Poly I:C, and the control group was used as the group added only with the transfection reagent (mock) and the group not treated with double-stranded RNA (NT). The results are shown in Fig. 21. The result using the 30 bp Lamin A/C-Motif introduced with the 2'-OCH3(-O-methyl) chemical modification #1 5'-2-15 in Table 16 is indicated as 2'-OMe Lamin A/C-Motif in Fig. 21; The results using 30 bp Lamin A/C-Motif (AS 2PS) in which 2'-OCH3(-O-methyl) chemical modification #1 5'-2-15 was introduced in Table 16 above and two PS bonds were introduced at both ends in the first strand were denoted as AS 2PS in FIG. 21; The results using 30 bp Lamin A/C-Motif (SS 2PS) in which 2'-OCH3(-O-methyl) chemical modification #1 5'-2-15 was introduced in Table 16 above and two PS bonds were introduced at both ends in the second strand were denoted as SS 2PS in FIG. 21; The results using 30 bp Lamin A/C-Motif (AS 2PS SS 2PS) with 2'-OCH3(-O-methyl) chemical modification #1 5'-2-15 introduced in Table 16 above and two PS bonds introduced at both ends of both the first and second strands are denoted as AS 2PS SS 2PS in Figure 21.

As a result, as shown in Fig. 21, it was confirmed that the innate immune response induction effect was significantly maintained despite phosphorothioate (PS) modification, and it was confirmed that a high level of innate immune response induction effect was exhibited in all cases, regardless of whether the PS bond was introduced only to the first strand, only to the second strand, or both to the first and second strands.

Example 11. Confirmation of the effect of inducing innate immune response in various cell lines

In this example, the 30 bp double-stranded RNA, which showed a high level of innate immunity induction effect in the above Example 7, was treated to various cell lines to re-verify the innate immune response induction effect. In the case of mouse cell lines, RAW-Lucia ISG, EMT-6, or CT-26 cell lines were used to measure the protein expression levels of RIG-I and MDA5 by Western blot, and the expression levels of RIG-I, MDA5, and IFIT1 mRNA by RT-qPCR. In addition, in the case of human cell lines, THP1-Dual (thpd-nfis), THP, THP-PMA, PBMS, HCC-1143, SK-BR-3, HCC-70, HCC-38, BT-549, SK BR3, Hep3 B, SN12C, or A498 cell lines were used to measure the mRNA expression levels of RIG-I and MDA5 by RT-qPCR. The results are shown in Table 17 below.

[Table 17]

As a result, as shown in Table 17 above, when the 30 bp double-stranded RNA of Example 7 was applied, it was confirmed that MDA and RIG-I-mediated innate immune responses could be effectively induced in human immune cells such as THP, THP differentiated by PMA treatment (THP-PMA), and PBMC, as well as breast, liver, and renal cancer cell lines.

While the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereby. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (23)

A double-stranded RNA of 25 to 60 base pairs (bp) comprising a first strand; and a second strand complementarily binding to the first strand, One strand of the above double-stranded RNA contains at least 10 nucleotides to which chemical modification has been introduced, The above double-stranded RNA is a double-stranded RNA that enhances the innate immune response mediated by MDA5 (Melanoma differentiation-associated protein 5). A double-stranded RNA according to claim 1, wherein the double-stranded RNA comprises an innate immune activation motif of 10 to 30 base pairs. In claim 2, the innate immune activation motif is a double-stranded RNA that enhances a RIG-I (Retinoic acid-inducible gene I)-mediated innate immune response. A double-stranded RNA according to claim 2, wherein the innate immune activation motif is located at one terminal region of the double-stranded RNA. A double-stranded RNA according to claim 2, wherein the innate immune activation motif comprises a sequence having at least 80% homology to the nucleotide sequence of SEQ ID NO: 2. A double-stranded RNA according to claim 2, wherein the innate immune activation motif comprises a nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 62, and a nucleotide sequence complementarily binding to the nucleotide sequence. A double-stranded RNA according to claim 1, wherein one or both ends of the double-stranded RNA are blunt ends. A double-stranded RNA according to claim 1, wherein one strand of the double-stranded RNA comprises 10 to 60 nucleotides into which a chemical modification has been introduced. A double-stranded RNA according to claim 8, wherein the nucleotide into which the chemical modification is introduced is a double-stranded RNA in which the -OH group at the 2' carbon position is substituted with -H, -CH ₃ (methyl), -OCH ₃ (-O-methyl), -NH ₂ , -F, -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl. A double-stranded RNA according to claim 9, wherein the nucleotide into which the chemical modification is introduced has the -OH group at the 2' carbon position replaced with -OCH ₃ (-O-methyl). A double-stranded RNA according to claim 8, wherein the nucleotides to which the chemical modification has been introduced are arranged alternately with nucleotides to which the chemical modification has not been introduced. A double-stranded RNA according to claim 11, wherein nucleotides having a chemical modification in which an -OH group at the 2' carbon position is substituted with -OCH ₃ (-O-methyl) and nucleotides having no chemical modification are arranged alternately. A double-stranded RNA according to claim 8, wherein at least one internucleotide bond is modified with phosphorothioate, boranophosphate, or methyl phosphonate. A double-stranded RNA according to claim 13, wherein at least one internucleotide bond is modified with phosphorothioate. A double-stranded RNA according to claim 1, wherein one end of the double-stranded RNA is an overhang. A composition for enhancing innate immune response comprising a double-stranded RNA according to any one of claims 1 to 15. A composition for enhancing an innate immune response according to claim 16, wherein the composition simultaneously induces a RIG-I mediated innate immune response and an MDA5 mediated innate immune response. A composition for enhancing innate immune response, wherein the composition according to claim 16 is a vaccine adjuvant. A composition for enhancing innate immune response, wherein the composition according to claim 16 is an immuno-oncology agent. A composition for enhancing innate immune response according to claim 16, wherein the composition is an antiviral agent. A pharmaceutical composition comprising a double-stranded RNA according to any one of claims 1 to 15. A pharmaceutical composition according to claim 21, wherein the pharmaceutical composition further comprises at least one therapeutic substance. A method for enhancing an innate immune response, comprising administering to a subject a pharmaceutical composition according to claim 21.

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