Double-stranded RNA for innate immune response-based anticancer therapy, and use thereof

Abstract

The present invention relates to a double-stranded RNA having an innate immune response-inducing effect, and a 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 comprising the double-stranded RNA for enhancing innate immunity; and a pharmaceutical composition comprising the double-stranded RNA.

Description

Double-stranded RNA and its use for innate immune response-based anticancer therapy

[0001] The present invention relates to double-stranded RNA and its use for innate immune response-based anticancer therapy, and more particularly, to double-stranded RNA comprising an innate immune activation motif and a specific chemical modification pattern.

[0002] Induction of the innate and adaptive immune responses plays a crucial role in the treatment of various diseases. Among these, the innate immune response, a nonspecific immune response, recognizes external factors and serves as the body's first line of defense. Since it was discovered that enhancing or activating the innate immune response can improve difficult-to-treat pathological conditions such as cancer, pathogenic infections, and genetic diseases, research into therapeutics utilizing the innate immune response has been actively conducted. To this end, research has focused on the mechanisms of action that can activate or regulate the innate immune response. For example, it has been reported that activating the innate immune response as an anticancer treatment can activate the recruitment of macrophages or dendritic cells to cancer cells through chemokine production and inflammation promotion, and increase the production of cytokines such as interferon to induce a normal state.

[0003] Additionally, 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 that recognize pattern recognition molecules and mediate innate immune responses. Pattern recognition receptors are distributed in dendritic cells, macrophages, monocytes, neutrophils, epithelial cells, and other cells, 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 absent-in-melanoma-like receptors (ALR). Each receptor recognizes a different type of ligand and contributes to the enhancement of the immune system in various ways.

[0004] Meanwhile, technologies utilizing RNA interference (RNAi) have been developed as RNA-based therapies. RNA interference is a method of suppressing target gene expression by inducing cleavage of target mRNA. Specifically, small interfering RNA (sRNA) can be used as an RNAi agent. In this case, the induction of an innate immune response by administration of an exogenous substance, siRNA, is recognized as a non-specific response and can even hinder the efficacy of the RNAi agent. Therefore, the introduction of modified RNA nucleotides, such as chemically modified nucleotides, has been proposed as a technique to reduce the innate immune response induced by siRNA administration. However, RNA-based therapeutics can be applied not only as RNAi agents but also in passive and active immunotherapy, vaccine formulations, and genetic engineering in general. Furthermore, RNA molecules can also be used as therapeutic agents for replacement therapies, such as protein replacement therapy to replace missing or mutated proteins.

[0005] Despite the recent ongoing interest in anti-tumor immunotherapy leveraging the activation of RIG-I-like receptors, such as RIG-I and MDA5, research on double-stranded RNAs capable of effectively inducing RIG-I and / or MDA5-mediated innate immune responses remains limited. Against this backdrop, the inventors of the present invention have identified the innate immune response-enhancing and tumor growth-inhibiting effects resulting from the chemical modification of specific motifs and nucleotides, and have subsequently developed the present invention.

[0006] [Prior Art Literature]

[0007] [Non-patent literature]

[0008] Jiang J Exp Med. 2019 Dec 2;216(12):2854-2868. (2019 Oct 10)

[0009] The purpose of the present invention is to provide double-stranded RNA capable of activating an innate immune response.

[0010] 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, a method for enhancing an innate immune response comprising a step of administering the pharmaceutical composition to a subject, or a method for treating or improving cancer by inducing an innate immune response comprising a step of administering the pharmaceutical composition to a subject.

[0011] One aspect provides a double-stranded RNA having a first strand and a second strand forming a complementary bond to the first strand, the double-stranded RNA having blunt ends and having 25 to 50 base pairs (bp), wherein the first strand comprises at least five 2'-OMe modified nucleotides at positions 10 to 27 from the 5' end.

[0012] Another aspect provides a composition for enhancing an innate immune response comprising the double-stranded RNA.

[0013] Another aspect provides a pharmaceutical composition comprising the double-stranded RNA.

[0014] Another aspect provides a method of enhancing an innate immune response comprising administering to a subject the double-stranded RNA or the pharmaceutical composition.

[0015] Another aspect provides a method for treating or ameliorating cancer by inducing an innate immune response comprising administering to a subject the double-stranded RNA or the pharmaceutical composition.

[0016] Another aspect provides a method for treating or ameliorating an infection caused by a virus or bacteria, comprising administering to a subject the double-stranded RNA or the pharmaceutical composition.

[0017] Another aspect provides a method of treating or ameliorating a disease associated with immunosuppression, comprising administering to a subject the double-stranded RNA or the pharmaceutical composition.

[0018] Another aspect provides a medicinal use of the double-stranded RNA to enhance innate immune responses.

[0019] Another aspect provides a medicinal use of the double-stranded RNA for treating or ameliorating cancer by inducing an innate immune response.

[0020] Another aspect provides a medicinal use of the double-stranded RNA for treating or ameliorating infections caused by viruses or bacteria.

[0021] Another aspect provides a medicinal use of the double-stranded RNA for treating or ameliorating diseases associated with immunosuppression.

[0022] Double-stranded RNA according to one aspect contains an innate immune response activating motif and can induce a high level of innate immune response by having a specific chemical modification pattern.

[0023] Additionally, double-stranded RNA according to one aspect may enhance the efficacy of cancer therapy by enhancing MDA5 and / or RIG-I-mediated innate immune responses.

[0024] Additionally, double-stranded RNA according to one aspect may enhance the efficacy of vaccine formulations or immunotherapeutic agents by enhancing MDA5 and / or RIG-I mediated innate immune responses.

[0025] Figure 1 is a schematic diagram showing the structure of a double-stranded RNA containing an innate immune activation motif according to one aspect.

[0026] Figure 2 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting double-stranded RNAs having various fusion sequences and 2'-OMe chemical modification patterns capable of activating innate immune responses into RAW-Lucia ISG cell line (WT), RAW-Lucia ISG-KO-RIG-I cell line (RIG-I KO), or RAW-Lucia ISG-KO-MDA5-I cell line (MDA5 KO).

[0027] Figure 3 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting double-stranded RNA having different numbers of nucleotides with 2'-OMe chemical modification introduced on both strands into RAW-Lucia ISG cell line (WT) or RAW-Lucia ISG-KO-RIG-I cell line (RIG-I KO).

[0028] Figure 4 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting a human monocyte cell line (THP-1 Dual) with double-stranded RNA having various 2'-F or 2'-OMe chemical modification patterns on the second strand.

[0029] Figure 5 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting a human monocyte cell line (THP-1 Dual) with double-stranded RNA having various 2'-F and 2'-OMe chemical modification patterns on the first strand and various 2'-F and / or 2'-OMe chemical modification patterns on the second strand.

[0030] Figure 6 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting each candidate double-stranded RNA into a human monocyte cell line (THP-1 Dual) to evaluate the low-concentration efficacy of double-stranded RNA according to the daily aspect.

[0031] Figure 7 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting double-stranded RNA according to one aspect into a human monocyte cell line (THP-1 Dual) or a THP1-Dual KO-MDA5 cell line (MDA5 KO) to evaluate the effect of double-stranded RNA according to one aspect on inducing an MDA5-mediated innate immune response.

[0032] Figure 8 shows the results of a serum stability evaluation performed under 50% FBS conditions to evaluate the stability of double-stranded RNA according to various aspects.

[0033] Figure 9a shows the results of confirming the amount of RIG-I mRNA expression after forming a complex with double-stranded RNA according to one aspect and treating human PBMCs at various concentrations.

[0034] Figure 9b shows the results of confirming the expression level of MDA5 mRNA after forming a complex with LNP and treating human PBMCs at various concentrations according to the daily aspect.

[0035] Figure 10a shows the results of confirming the spheroid survival rate after forming a complex with double-stranded RNA according to one aspect and treating cancer cells (LN229) cultured in the form of spheroids.

[0036] Figure 10b shows the results of confirming the spheroid survival rate after forming a complex with double-stranded RNA according to one aspect and treating cancer cells (HCC70) cultured in the form of spheroids.

[0037] Figure 11a shows the results of confirming the tumor growth inhibitory effect after forming a complex with invivofectamin according to one aspect and injecting it into the tumor of an EMT6 mouse tumor model; Figure 11b shows the results of confirming the mouse survival rate.

[0038] Figure 12 shows the results of confirming the mRNA expression levels of RIG-I, MDA5, CXCL10, and IFIT-1 by isolating tumor tissue after forming a complex with double-stranded RNA according to one aspect and invivofectamin and injecting it into the tumor of an EMT6 mouse tumor model.

[0039] Figure 13a shows the results of confirming the tumor growth inhibitory effect after forming a complex with invivofectamin according to one aspect and injecting it into a B16F10 mouse tumor model; Figure 13b shows the results of confirming the mRNA expression levels of RIG-I, MDA5, CXCL10, IFIT-1, and TNF-α by isolating tumor tissue.

[0040] Figure 14a shows the results of confirming the tumor growth inhibitory effect after forming a complex with invivofectamin according to one aspect and injecting it into a B16F10 mouse tumor model; Figure 14b shows the results of confirming the mouse survival rate; Figure 14c shows the results of confirming the mRNA expression levels of RIG-I, MDA5, CXCL10, IFIT-1, TNF-α, and IFN-α by isolating tumor tissue.

[0041] Figure 15 shows the results of examining the serum IFN-α concentration after intramuscular injection of double-stranded RNA complexed with LNP according to the daily pattern into a B16F10 mouse tumor model.

[0042] Figure 16 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 RNAs having various shifts in the position of the 2'-OMe chemical modification pattern of the first strand.

[0043] Figure 17 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting double-stranded RNA with various shifts in the position of the 2'-OMe chemical modification pattern of the first strand into a human monocyte cell line (THP-1 Dual) or a THP1-Dual KO-MDA5 cell line (MDA5 KO).

[0044] Figure 18 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting double-stranded RNAs with various shifts in the position of the 2'-OMe chemical modification pattern of the first strand for various nucleotide sequences into a human monocyte cell line (THP-1 Dual), a THP1-Dual KO-RIG-I cell line (RIG-I KO), or a THP1-Dual KO-MDA5 cell line (MDA5 KO), respectively.

[0045] Figure 19 shows the results of confirming the activity level of interferon regulatory factor (IRF) after transfecting double-stranded RNA introducing an innate activation motif according to one aspect into a human monocyte cell line (THP-1 Dual) or a THP1-Dual KO-MDA5 cell line (MDA5 KO).

[0046] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0047]

[0048] One aspect provides a double-stranded RNA having a first strand and a second strand forming a complementary bond to the first strand, the double-stranded RNA having blunt ends and having 25 to 50 base pairs (bp), wherein the first strand comprises at least five 2'-OMe modified nucleotides at positions 10 to 27 from the 5' end.

[0049]

[0050] double-stranded RNA

[0051] As used herein, the term “double-stranded RNA” means RNA with two strands (a first strand and a second strand) that bind complementarily to each other.

[0052] As used herein, the terms “first strand” or “second strand” may refer to the antisense strand or sense strand, respectively, when the double-stranded RNA comprises a region for RNA interference.

[0053] The double-stranded RNA may have a length of 25 to 50 base pairs, for example, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 27 to 50, 27 to 45, 27 to 40, 27 to 35, 27 to 30, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 27, 30, 34, 36, 38, or 40 base pairs. Preferably, the double-stranded RNA may have a length of 30 base pairs. Both ends of the double-stranded RNA may form blunt ends.

[0054] In one specific example, the first strand may be composed of the nucleotide sequence of SEQ ID NO: 9. The first strand may include a variant of the nucleotide sequence having at least 80%, for example, 85%, 90%, 95%, 97%, or 98% sequence identity with the nucleotide sequence of SEQ ID NO: 9. The double-stranded RNA may be composed of a first strand consisting of the nucleotide sequence of SEQ ID NO: 9 and a second strand consisting of a nucleotide sequence that complementarily binds to the nucleotide sequence. The second strand may be composed of the nucleotide sequence of SEQ ID NO: 10. The second strand may include a variant of the nucleotide sequence having at least 80%, for example, 85%, 90%, 95%, 97%, or 98% sequence identity with the nucleotide sequence of SEQ ID NO: 10.

[0055] 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 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-base-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 its corresponding target nucleic acid molecule.

[0056]

[0057] Innate immune activation motif

[0058] As used herein, the term "Innate Immune Response Activating Motif" refers to a region within a double-stranded RNA for inducing or enhancing an innate immune response in a subject to which the double-stranded RNA is administered, and refers to a double-stranded structure consisting of a first-strand nucleotide sequence of the innate immune activating motif and a second-strand nucleotide sequence of the innate immune activating motif that forms a complementary bond with the first-strand nucleotide sequence.

[0059] As used herein, the term "innate immune response" may refer to an innate defense immune system in vivo derived from double-stranded RNA, which may contribute to enhancing 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).

[0060] The innate immune response induced by double-stranded RNA is known to depend on the terminal structure of the double-stranded RNA. Therefore, approaches that introduce 5'-triphosphate or modified terminal structures as technical means to enhance the innate immune response have been reported. Furthermore, MDA5-mediated immune responses have been reported to be induced by long-length double-stranded RNA. Meanwhile, the present inventors have discovered that, despite the relatively short length of double-stranded RNA according to one embodiment, an innate immune response can be induced or enhanced through innate immune activation motifs and specific chemical modification patterns.

[0061] As used herein, the term "first strand nucleotide sequence of an innate immune activation motif" refers to a nucleotide sequence located on the first strand in a double-stranded RNA that activates an innate immune response. For example, the first strand nucleotide sequence of the innate immune activation motif may comprise a structure capable of activating an innate immune response mediated by RIG-I or MDA5. Specifically, the first strand nucleotide sequence of the innate immune activation motif may be the nucleotide sequence of SEQ ID NO: 15 or SEQ ID NO: 17. In one embodiment, the first strand nucleotide sequence of the innate immune activation motif may comprise a variant of the nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 15. In one specific example, the first strand nucleotide sequence of the innate immune activation motif may comprise a variant of the nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 17.

[0062] In one specific example, the innate immune activation motif may be comprised of a nucleotide sequence of SEQ ID NO: 15 and a nucleotide sequence that complementarily binds to said nucleotide sequence.

[0063] In one specific example, the innate immune activation motif may be comprised of a nucleotide sequence of SEQ ID NO: 17 and a nucleotide sequence that complementarily binds to said nucleotide sequence.

[0064] The first strand of the double-stranded RNA according to one aspect may have an innate immune activation motif at positions 10 to 27 from the 5' end, for example, at positions 14 to 24, 16 to 24, 12 to 22, 16 to 26, 10 to 20, 14 to 22, 16 to 26, or 12 to 20.

[0065] In one specific example, the nucleotide sequence at positions 10 to 27 from the 5' end of the first strand of the double-stranded RNA may include the nucleotide sequence of SEQ ID NO: 15 or SEQ ID NO: 17. For example, the nucleotide sequence at positions 14 to 24, 16 to 24, 12 to 22, 16 to 26, 10 to 20, 14 to 22, 16 to 26, or 12 to 20 from the 5' end of the first strand of the double-stranded RNA may consist of or include the nucleotide sequence of SEQ ID NO: 15 or SEQ ID NO: 17. In one specific example, the first strand of the double-stranded RNA may be such that the nucleotide sequence at positions 16 to 24 from the 5' end is composed of the nucleotide sequence of SEQ ID NO: 15. In one specific example, the first strand of the double-stranded RNA may be such that the nucleotide sequence at positions 14 to 24 from the 5' end is composed of the nucleotide sequence of SEQ ID NO: 17.

[0066] The first strand nucleotide sequence of the innate immune activation motif may comprise at least five, for example, five to nine, five, six, seven, eight, or nine 2'-OMe modified nucleotides. In one embodiment, the first strand nucleotide sequence of the innate immune activation motif may be an alternating sequence of five 2'-OMe modified nucleotides.

[0067] In one embodiment, the first strand of the double-stranded RNA can comprise at least 5, for example, 5 to 9, 5, 6, 7, 8, or 9 2'-OMe modified nucleotides at positions 10 to 27 from the 5' end. For example, the first strand of the double-stranded RNA can comprise at least 5, for example, 5 to 9, 5, 6, 7, 8, or 9 2'-OMe modified nucleotides at positions 14 to 24, 16 to 24, 12 to 22, 16 to 26, 10 to 20, 14 to 22, 16 to 26, or 12 to 20 from the 5' end of the first strand of the double-stranded RNA.

[0068] In one specific example, the first strand of the double-stranded RNA may have 5 to 9, for example, 5, 6, 7, 8, or 9, 2'-OMe modified nucleotides alternately arranged at positions 10 to 27 from the 5' end.

[0069] In one specific embodiment, the first strand of the double-stranded RNA may comprise at least five, for example, five to nine, five, six, seven, eight, or nine 2'-OMe modified nucleotides at positions 16 to 24 from the 5' end, specifically, five 2'-OMe modified nucleotides may be arranged alternately at positions 16 to 24 from the 5' end.

[0070] In one specific embodiment, the first strand of the double-stranded RNA may comprise at least six, for example, six to nine, six, seven, eight, or nine 2'-OMe modified nucleotides at positions 14 to 24 from the 5' end, specifically, six 2'-OMe modified nucleotides may be arranged alternately at positions 14 to 24 from the 5' end.

[0071] 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 chemical moieties (e.g., carbohydrates, lipids, etc.) covalently linked to the polynucleotide backbone. Such variants exhibit an overall sequence identity with the reference polynucleotide of at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0072] 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 a mathematical algorithm. As is well known to those skilled in the art, nucleotide sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN.

[0073] In one embodiment, the innate immune activation motif can induce an innate immune response when introduced at positions 16 to 24 from the 5'-end of the first strand of a double-stranded RNA that does not induce an innate immune response.

[0074]

[0075] Double-stranded RNA with chemical modifications

[0076] In the double-stranded RNA of the present specification, the first strand and / or the second strand may include nucleotides into which chemical modifications have been introduced.

[0077] The term "modified nucleotide" may be used interchangeably with the terms "chemically modified nucleotide" and "nucleotide to which a chemical modification has been introduced".

[0078] The term "2'-OMe modified nucleotide" refers to a nucleotide in which the -OH group at the 2' carbon position of the sugar structure within the nucleotide is replaced with -OCH3 (-O-methyl).

[0079] The term "2'-F modified nucleotide" refers to a nucleotide in which the -OH group at the 2' carbon position of the sugar structure within the nucleotide is replaced with -F(-fluoro).

[0080] In a double-stranded RNA according to one aspect, the 2'-OMe modified nucleotides may be included in either or both of the first and second strands.

[0081] In one embodiment, the first strand may comprise 10 to 15, for example, 12 to 15, 10 to 12, 10, 11, 12, 13, 14, or 15 2'-OMe modified nucleotides. The first strand may have 10 to 12, for example, 10 or 12, 2'-OMe modified nucleotides arranged alternately from the 3'-end or the 5'-end. Preferably, the first strand may have 15 2'-OMe modified nucleotides arranged alternately, specifically, 15 2'-OMe modified nucleotides arranged alternately starting from the first nucleotide or the second nucleotide from the 5'-end of the first strand.

[0082] In one embodiment, the second strand may comprise 2 to 6, for example, 2 to 4, 2, 3, 4, or 6 2'-OMe modified nucleotides. The second strand may have 2 to 4 2'-OMe modified nucleotides arranged alternately from one or both ends. Preferably, the second strand may have 2 to 4, for example, 2, 3, or 4 2'-OMe modified nucleotides arranged alternately from the 5' end, and specifically, 2 to 4 2'-OMe modified nucleotides may be arranged alternately from the first nucleotide or the second nucleotide from the 5' end of the second strand.

[0083] In a double-stranded RNA according to one aspect, the 2'-F modified nucleotide may be included in either or both of the first strand and the second strand.

[0084] In one embodiment, the second strand may comprise 3 to 30, for example, 3 to 15, 10 to 30, 20 to 30, 24 to 30, 26 to 30, 27 to 30, or 28 to 30 2'-F modified nucleotides. The second strand may have 2 to 10, for example, 2 to 3, 2 to 4, 3 to 5, 5 to 10, 2, 3, 5, or 10 2'-F modified nucleotides arranged alternately from one or both ends. The second strand may have 15 2'-F modified nucleotides arranged alternately. Preferably, the second strand may have 20 to 30, for example, 24 to 30, 25 to 30, or 27 to 30 2'-F modified nucleotides arranged in sequence. Preferably, the second strand may have 30 2'-F modified nucleotides arranged in sequence, or 25 to 27 2'-F modified nucleotides arranged in sequence from the 3' end. When 25 to 27 2'-F modified nucleotides are arranged in sequence from the 3' end of the second strand, 2 or 3 2'-OMe modified nucleotides may be arranged alternately with 2'-F modified nucleotides from the 5' end. Specifically, in a structure in which 30 2'-F modified nucleotides are sequentially arranged on the second strand, two or three 2'-F modified nucleotides at one end or both ends may be substituted with 2'-OMe modified nucleotides so that two or three 2'-OMe modified nucleotides are alternately arranged at one end or both ends.

[0085] In one embodiment, the first strand can comprise 5 to 11, for example, 5 to 6, 9 to 11, 5, 6, 7, 8, 9, 10, or 11 2'-F modified nucleotides. The first strand can have 3 to 10, for example, 3 to 5, 3 to 6, or 5 to 6 2'-F modified nucleotides arranged alternately from one or both ends. The first strand may have 6 to 7, for example, 6 or 7, 2'-F modified nucleotides arranged alternately from the 5'-end, and 3 to 5, for example, 3, 4, or 5, 2'-F modified nucleotides arranged alternately from the 3'-end. Preferably, the first strand may have 6 2'-F modified nucleotides arranged alternately from the 5'-end, or the first strand may have 6 2'-F modified nucleotides arranged alternately from the 5'-end, and 5 2'-F modified nucleotides arranged alternately from the 3'-end. Specifically, six 2'-F modified nucleotides may be arranged alternately from the first nucleotide or the second nucleotide from the 5'-end of the first strand, or six 2'-F modified nucleotides may be arranged alternately from the first nucleotide or the second nucleotide from the 5'-end of the first strand, and five 2'-F modified nucleotides may be arranged alternately from the first nucleotide or the second nucleotide from the 3'-end.

[0086] In one specific example, the nucleotide at position 19 from the 5'-end of the first strand may not include a chemical modification. In other words, the nucleotide at position 19 from the 5'-end of the first strand may be a naked nucleotide to which no chemical modification has been introduced.

[0087] The term "alternating sequence" refers to a repetitive arrangement of modified nucleotides, starting from the first nucleotide of one end or a specified region, alternately with unmodified nucleotides or nucleotides modified with different chemical modifications, i.e., in an AB or BA alternating pattern. For example, the alternating sequence may be a discontinuous sequence, being arranged at odd positions a specified number of times counted from the first nucleotide of one end or a specified region, or being arranged at even positions.

[0088] The term "contiguous sequence" refers to a repetitive arrangement of modified nucleotides in an AA or BB contiguous pattern, starting from the first nucleotide of one end or a specified region. For example, the contiguous sequence may be arranged at both odd and even positions a specified number of times, counting from the first nucleotide of one end or a specified region.

[0089] The above 2'-OMe modified nucleotide or 2'-F modified nucleotide may be located in a region within the innate immune activation motif and / or a region outside the innate immune activation motif.

[0090] In one embodiment, no more than 50%, for example, 50%, 45%, 40%, 35%, or 30% of the total nucleotides of the first strand may be the 2'-OMe modified nucleotides.

[0091] In one embodiment, no more than 40%, for example, 40%, 35%, 30%, 25%, 20%, or 10% of the total nucleotides of the first strand may be the 2'-F modified nucleotides.

[0092] In one embodiment, from 60% to 100%, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the total nucleotides of the second strand may be the 2'-F modified nucleotides.

[0093] In one embodiment, no more than 20%, for example, 20%, 15%, 10%, or 5% of the total nucleotides of the second strand may be the 2'-F modified nucleotides.

[0094] Double-stranded RNAs having the aforementioned chemical modification patterns, together with the innate immune activation motif, can induce or enhance innate immune responses. Specifically, double-stranded RNAs containing the innate immune activation motif and having the aforementioned chemical modification patterns introduced can induce or enhance MDA5-mediated and / or RIG-I-mediated innate immune responses.

[0095] 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, sharing the structure of N-tandem amino-terminal caspase activation and recruitment domains (CARDs) 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). Both MDA5 and RIG-I bind to foreign RNA, i.e., double-stranded RNA, and initiate signal transduction. However, unlike RIG-I, which prefers binding to short dsRNA, MDA5 is known to initiate signal transduction by relatively long dsRNA.

[0096] In one embodiment, double-stranded RNA comprising an innate immune activation motif applied with the specific chemical pattern described above can enable the induction of a strong innate immune response despite the processing of double-stranded RNA having a relatively short base pair length, through an additional signaling mechanism in which MDA5 acts as a key factor.

[0097] In one specific embodiment, the double-stranded RNA may have at least one nucleotide bond modified with phosphorothioate, boranophosphate, or methyl phosphonate. Specifically, the double-stranded RNA may have two to four nucleotide bonds adjacent from both ends or one end of the first strand and / or the second strand modified with phosphorothioate, boranophosphate, or methyl phosphonate.

[0098]

[0099] pharmaceutical composition

[0100] As used herein, the term "effective ingredient" refers to an appropriate effective amount of a component that affects a beneficial or desirable clinical or biochemical outcome. Specifically, it may refer to an effective amount of a formulation, 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.

[0101] 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 the disease, stabilizing the disease (i.e., not worsening), delaying or reducing the rate of disease progression, or improving or temporarily alleviating and alleviating (partially or completely) the disease.

[0102] As used herein, the term "prevention" refers to any action that prevents the onset of a disease, suppresses the disease, or delays its progression. For example, it refers to preventing the onset of the disease or its characteristic features, interfering with its development, or protecting against or protecting against the onset of the disease or its characteristic features.

[0103] As used herein, the term "treatment" refers to both therapeutic treatment and preventative or prophylactic measures. It also refers to any action that improves or beneficially alters the symptoms of a disease. For example, it prevents, reduces, or improves the disease or its characteristic features, or delays (attenuates) the progression of the disease or its characteristic features in a subject.

[0104] The pharmaceutical composition may be for preventing, treating, or ameliorating an infection caused by a virus or bacteria, a disease associated with immunosuppression, or a cancer (e.g., a solid tumor such as breast cancer, glioblastoma, liver cancer, esophageal cancer, pancreatic cancer, lung cancer, stomach cancer, head and neck squamous cell carcinoma, 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).

[0105] As used herein, the term "effective amount" refers to its generally accepted meaning in the art. The term may generally refer to the 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" may refer to an amount of a molecule, compound, or composition that elicits 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 may be the amount necessary to effect a therapeutically relevant change in the parameter.

[0106] As used herein, the term "pharmaceutically acceptable excipient" may be one that, 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, starch, milk, sugar, certain forms of clay, gelatin, stearic acid or its salts, magnesium or calcium stearate, talc, vegetable oils, gums, glycols, or other known excipients.

[0107] The method of administration of the above pharmaceutical composition can be determined by a person skilled in the art based on the typical symptoms and severity of the patient's disease. Furthermore, the composition can be formulated in various forms, such as powders, tablets, capsules, liquids, injections, ointments, and syrups, and can be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles.

[0108] 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 to these, 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 known techniques for clinical administration.

[0109] The pharmaceutical composition may be administered in combination with one or more drugs, for example, an anticancer agent, and may be administered simultaneously or sequentially with the anticancer agent.

[0110]

[0111] Composition for enhancing innate immunity

[0112] 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 an innate immune response of an administered subject. The composition for enhancing innate immunity may be used interchangeably with an immune enhancer / adjuvant, an RNA-based immunogenic composition, or an immunostimulating agent. The composition may optionally contain a pharmaceutically acceptable carrier, diluent, excipient, buffer, salt, surfactant, cryoprotectant, etc., and the implementation conditions and implementation modes are as described in the pharmaceutical composition.

[0113] The above composition for enhancing innate immunity can be used as an active ingredient in a vaccine adjuvant, an immuno-oncology agent, or an antiviral agent (e.g., hepatitis B virus), depending on the intended use, and specifically, can be an immuno-oncology agent.

[0114] The term "immuno-oncology agent" refers to a drug or substance that helps treat or improve cancer by activating or enhancing the patient's immune system, and includes not only an anti-cancer agent that exhibits anti-cancer effects alone, but also an anti-cancer adjuvant agent for use in combination with other anti-cancer agents. In one embodiment, the double-stranded RNA of the present invention can be useful as an immuno-oncology agent because it has been confirmed to be capable of inducing a strong MDA5 and / or RIG-I-mediated innate immune response, significantly inhibiting tumor growth, and enhancing the expression of cytokines associated with the innate immune response in tumor tissue. This MDA5 and / or RIG-I activation may induce tumor cell death by promoting type I interferon production and increase the infiltration of immune cells (dendritic cells, natural killer cells, T cells, etc.), thereby exhibiting an anti-cancer effect.

[0115] 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 utilize 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.

[0116]

[0117] Another aspect provides a method of enhancing an innate immune response comprising administering to a subject the pharmaceutical composition.

[0118] Another aspect provides a method of treating or ameliorating cancer by inducing an innate immune response comprising administering to a subject the double-stranded RNA.

[0119] As used herein, the term "subject" 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.

[0120]

[0121] Another aspect provides the use of the double-stranded RNA for the manufacture of a medicament for treating or ameliorating cancer by inducing an innate immune response.

[0122] Another aspect provides the use of said double-stranded RNA for the manufacture of a medicament for enhancing innate immune response.

[0123] Since the above method or use includes or utilizes the double-stranded RNA or pharmaceutical composition described above, the common content between them is omitted to avoid excessive complexity of the present specification.

[0124]

[0125] The present invention will be described in more detail below 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.

[0126]

[0127] Example 1. Confirmation of the innate immune response-inducing effect of double-stranded RNA with 2'-OMe chemical modification.

[0128] 1.1. Production of double-stranded RNA with 2'-OMe chemical modification

[0129] In this example, double-stranded RNAs having various fusion sequences and 2'-OMe chemical modification patterns capable of activating an innate immune response were produced. The double-stranded RNAs were each produced as a double-stranded RNA consisting of a first strand composed of a fusion sequence of 5'-SEQ ID NO: 1 (20 nt)-SEQ ID NO: 3 (10 nt)-3' (SEQ ID NO: 9), a fusion sequence of 5'-SEQ ID NO: 1 (20 nt)-SEQ ID NO: 5 (10 nt)-3' (SEQ ID NO: 11), or a fusion sequence of 5'-SEQ ID NO: 7 (20 nt)-SEQ ID NO: 3 (10 nt)-3' (SEQ ID NO: 13) and a second strand complementarily binding to the first strand, each of which is 30 base pairs, and in which both ends of the first strand and the second strand form blunt ends. Also, in order to compare the efficacy of introducing 2'-OMe chemical modifications in which the -OH group at the 2' carbon position of the nucleotide is replaced with -OCH3(-O-methyl), double-stranded RNAs with various 2'-OMe chemical modification patterns were produced by alternately introducing 2'-OMe chemical modifications based on the basic structure described above, but varying the positions and numbers of the introduced strands, introduced terminals, and introduced nucleotides. The double-stranded RNAs were obtained from Bioneer Co. Ltd (Republic of Korea) and Dharmacon, Inc (UK), and the produced double-stranded RNAs are shown in Tables 1 and 2. In Table 2, the nomenclature of the double-stranded RNAs was expressed as [strand with introduced chemical modification]-[terminus with introduced chemical modification and first nucleotide position with introduced chemical modification]-[number of nucleotides with introduced chemical modification]. Additionally, nucleotides with a 2'-OCH3 (2'-O-methyl; 2'-OMe) chemical modification are underlined.

[0130] [Table 1]

[0131]

[0132] [Table 2]

[0133]

[0134]

[0135]

[0136] 1.2. Effect of inducing RIG-I or MDA5-mediated innate immune response

[0137] For the double-stranded RNA produced in Example 1.1, we aimed to identify a double-stranded RNA capable of inducing a strong innate immune response by introducing a 2'-OMe chemical modification. Furthermore, we aimed to determine whether such an innate immune response was mediated by RIG-I (Retinoic acid-inducible gene Ⅰ) or MDA5 (Melanoma differentiation-associated gene 5).

[0138] To this end, the activity of IRF was measured in 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) for the double-stranded RNA of Table 2 above. Specifically, RAW-Lucia ISG cell line, RAW-Lucia ISG-KO-RIG-I cell line, and RAW-Lucia ISG-KO-MDA5-I cell line were seeded at 1 × 10 per well in a 96-well plate. 5Each cell line was plated. Afterwards, the double-stranded RNA (10 nM) was transfected into the cell line using Lipofectamine RNAiMAX (13778150; Invitrogen), and the transfected cells were cultured for 24 hours each, and the supernatant was obtained. Thereafter, the level of interferon regulatory factor (IRF) 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 / 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). The results are shown in Fig. 2.

[0139] As a result, as shown in Fig. 2, double-stranded RNAs that did not contain a GFP sequence did not induce an effective level of innate immune response regardless of the introduction of chemical modifications (OLX013A-001-1 (Lamin A / C-GAPDH), OLX013A-001-3 (Lamin A / C-GAPDH #1 5'-2-10)), and among double-stranded RNAs containing a GFP sequence, when not containing a Lamin A / C sequence, a high level of innate immune response was not induced even when a 2'-OMe chemical modification was introduced (OLX013A-004-1 (GAPDH-GFP), OLX013A-004-2 (GAPDH-GFP #1 5'-2-10)). On the other hand, it was confirmed that double-stranded RNAs containing both a Lamin A / C sequence and a GFP sequence were capable of inducing a high level of innate immune response upon the introduction of a 2'-OMe chemical modification. These effects were significantly reduced in the ISG-KO-MDA5 cell line lacking MDA5, confirming that this innate immune response is mediated by MDA5.In particular, in the case of double-stranded RNAs in which the same number of 2'-OMe modifications were introduced into both the first and second strands, no significant level of innate immune response was induced (OLX013A-002-19 (Lamin A / C-GFP #1 5'-2-8 #2 3'-1-8), OLX013A-002-22 (Lamin A / C-GFP #1 5'-2-10 #2 3'-1-10), OLX013A-002-36 (Lamin A / C-GFP #1 5'-2-12 #2 3'-1-12), OLX013A-002-38 (Lamin A / C-GFP #1 5'-2-15 #2 3'-1-15)), whereas induction of an innate immune response was possible when 2'-OMe modifications were introduced into only one strand, among which the first Double-stranded RNA (OLX013A-002-2 (Lamin A / C-GFP #1 5'-2-15)) with alternating 2'-OMe modifications introduced throughout the strands was found to exhibit the highest level of innate immune induction.

[0140]

[0141] 1.3. Confirming the possibility of introducing 2'-OMe into both strands

[0142] In the above 1.2, it was shown that when the same number of 2'-OMe chemical modifications were introduced into both the first and second strands, a significant level of innate immune response was not induced. Therefore, the number of nucleotides into which 2'-OMe chemical modifications were introduced that are permissible in both strands was sought to be determined. To this end, double-stranded RNAs into which 2'-OMe chemical modifications were introduced were additionally produced using OLX013A-002-1 (Lamin A / C-GFP naked) as the basic backbone, as shown in Table 3 below. Specifically, the number of nucleotides into which 2'-OMe chemical modifications were introduced was fixed in one strand, and the number of nucleotides into which 2'-OMe chemical modifications were introduced in the other strand was gradually increased. In Table 3, the nomenclature of double-stranded RNA is indicated as [strand with chemical modification]-[terminus with chemical modification and first nucleotide position with chemical modification]-[number of nucleotides with chemical modification]. In addition, nucleotides with 2'-OCH3 (2'-O-methyl; 2'-OMe) chemical modifications are underlined.

[0143] [Table 3]

[0144]

[0145] Thereafter, for the double-stranded RNA of Table 3, the activity of IRF was measured in the RAW-Lucia ISG cell line (WT) and the RAW-Lucia ISG-KO-RIG-I cell line (RIG-I KO) using the same method as in Example 1.2. 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). The results are shown in Fig. 3.

[0146] As a result, as shown in Fig. 3, it was confirmed that introduction of 2'-OMe modifications to both strands is also allowed when a relatively small number of 2'-OMe modifications, such as 2 to 4, is introduced to one strand instead of the same number to both strands (OLX013A-002-23 (#1 5'-2-10 #2 3'-1-2). OLX013A-002-27 (#1 5'-2-2 #2 3'-1-10), OLX013A-002-28 (#1 5'-2-4 #2 3'-1-10)).

[0147]

[0148] Example 2. Confirmation of the innate immune response-inducing effect of double-stranded RNA with 2'-OMe, 2'-F, and PS linkage modifications.

[0149] 2.1. Confirmation of the effect of introducing 2'-F chemical modification to the second strand

[0150] In the above Example 1.2, the double-stranded RNA (OLX013A-002-2 (Lamin A / C-GFP #1 5'-2-15)) in which 2'-OMe chemical modifications were alternately introduced only to the first strand induced the strongest innate immune response. Therefore, we attempted to determine whether the innate immune response induction effect could be further enhanced by introducing a 2'-F chemical modification in which the -OH group at the 2' carbon position of the nucleotide is substituted with -F(-fluoro) to the second strand, or by introducing a small number of 2'-OMe chemical modifications. On the other hand, since modifying the nucleotide bond to a phosphorothioate (PS) bond may be necessary to improve the stability of the double-stranded RNA, we also introduced a modification to a PS bond to identify a combination of chemical modifications capable of inducing a strong innate immune response.

[0151] To this end, based on a double-stranded RNA (OLX013A-002-2 (Lamin A / C-GFP #1 5'-2-15)) in which 2'-OMe chemical modifications were alternately introduced throughout the first strand, a double-stranded RNA in which 2'-F chemical modifications were alternately introduced throughout the second strand, sequentially introduced, or alternately introduced in a fixed number of 5'-terminal or 3'-terminal regions, or in which a small number of 2'-OMe chemical modifications were alternately introduced in the 5'-terminal or 3'-terminal regions, or in which a fixed number of PS bonds were introduced in the first strand and / or the second strand was additionally prepared as shown in Table 4 below. In Table 4, nucleotides with 2'-OCH3 (2'-O-methyl; 2'-OMe) chemical modification are underlined, nucleotides with 2'-F (2'-fluoro; 2'-F) chemical modification are indicated in parentheses, and those in which the nucleotide linkage is replaced by a phosphorothioate (PS) linkage are indicated with "*". 2'-OMe or The naming of double-stranded RNA with 2'-F chemical modification was written as [strand with chemical modification]-[terminus with chemical modification and first nucleotide position with chemical modification]-[number of nucleotides with chemical modification], and the naming of double-stranded RNA with nucleotide bonds replaced by PS bonds was written as [strand with PS bonds]-[2PS (two adjacent nucleotide bonds from both ends of the strand replaced by PS bonds)].

[0152] [Table 4]

[0153]

[0154]

[0155]

[0156] Afterwards, the activity of IRF was measured in the human monocyte cell line THP1-Dual (thpd-nfis) for the double-stranded RNA of Table 4 above. Specifically, the THP1-Dual (thpd-nfis) cell line was seeded at 1 × 10 per well in a 96-well plate. 5 Each cell line was plated. Afterwards, the double-stranded RNA (10 nM) was transfected into the cell line using Lipofectamine RNAiMAX (13778150; Invitrogen), and the transfected cells were cultured for 24 hours each, and the supernatant was obtained. Thereafter, the interferon regulatory factor (IRF) activity level was detected in the supernatant using a plate reader (INNO-M, LTek). 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). The results are shown in Fig. 4.

[0157] As a result, as shown in Fig. 4, the highest level of innate immune response was induced in the case of double-stranded RNA in which 2'-OMe chemical modifications were alternately introduced throughout the first strand and 2'-F chemical modifications were sequentially introduced throughout the second strand (OLX013A-002-77 (2'-OMe #1 5'-2-15 / 2'-F #2 3'-1-30 / SS 2PS), OLX013A-002-88 (2'-OMe #1 5'-2-15 / 2'-F #2 3'-1-30 / AS 2PS SS 2PS), which was found to have the best innate immune response induction effect in Example 1.2, compared to double-stranded RNA in which 2'-OMe chemical modifications were alternately introduced throughout only the first strand (OLX013A-002-2 (2'-OMe #1 5'-2-15). The immune induction effect was significantly enhanced.

[0158]

[0159] 2.2. Confirmation of the effect of introducing 2'-F chemical modification to the first strand

[0160] In the above Example 2.1, the strongest innate immune response was induced in the case of double-stranded RNA (OLX013A-002-77 (2'-OMe #1 5'-2-15 / 2'-F #2 3'-1-30 / SS 2PS), OLX013A-002-88 (2'-OMe #1 5'-2-15 / 2'-F #2 3'-1-30 / AS 2PS SS 2PS)) in which 2'-OMe chemical modifications were alternately introduced throughout the first strand and 2'-F chemical modifications were continuously introduced into the second strand. Therefore, it was attempted to determine whether a strong innate immune response could be induced even when a 2'-F chemical modification was additionally introduced into the first strand or a small number of 2'-OMe modifications were introduced into the second strand. As in the above Example 2.1, a modification to a PS bond was also introduced and evaluated.

[0161] To this end, based on double-stranded RNA (OLX013A-002-88 (2'-OMe #1 5'-2-15 / 2'-F #2 3'-1-30 / AS 2PS SS 2PS) in which 2'-OMe chemical modifications were alternately introduced throughout the first strand and 2'-F chemical modifications were sequentially introduced into the second strand, double-stranded RNAs in which a constant number of 2'-F chemical modifications were alternately introduced into one or both terminal regions of the first strand, a small number of 2'-OMe chemical modifications were alternately introduced into one or both terminal regions of the second strand, or a constant number of PS bonds were introduced into the first strand and / or the second strand were additionally prepared as shown in Table 5 below. In Table 5, nucleotides in which 2'-OCH3 (2'-O-methyl; 2'-OMe) chemical modifications were introduced are underlined, and 2'-F (2'-fluoro; 2'-F) Nucleotides with chemical modifications are indicated in parentheses, and those in which nucleotide bonds are replaced with phosphorothioate (PS) bonds are indicated with "*". The naming of double-stranded RNAs with 2'-OMe or 2'-F chemical modifications is written as [strand with chemical modification]-[terminus with chemical modification and position of the first nucleotide with chemical modification]-[number of nucleotides with chemical modification], and the naming of double-stranded RNAs in which nucleotide bonds are replaced with PS bonds is written as [strand with PS bond introduced]-[2PS (two adjacent nucleotide bonds from both ends of the strand are replaced with PS bonds) or fPS (all nucleotide bonds of the strand are replaced with PS bonds)].

[0162] [Table 5]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171] Thereafter, for the double-stranded RNA of Table 5, the activity of IRF was measured in the human monocyte cell line THP1-Dual (thpd-nfis) using the same method as in Example 2.1. Meanwhile, in this example, the positive control group was a group administered with Poly I:C, and the control group was a group treated with only the transfection reagent (mock) and a group not treated with double-stranded RNA (NT). The results are shown in Fig. 5.

[0172] As a result, as shown in Fig. 5, when 2'-F chemical modifications were continuously introduced into the second strand, it was confirmed that in some cases where 2'-OMe chemical modifications were alternately introduced into the first strand as a whole and 2'-F chemical modifications were alternately introduced partially into one or both terminal regions, it was possible to induce an innate immune response at a significant level (OLX013A-002-5 (2'-OMe #1 5'-2-15 / 2'-F #1 5'-1-6 #2 3'-1-30 / AS 2PS SS 2PS), OLX013A-002-148 (2'-OMe #1 5'-2-15 / 2'-F #1 5'-1-6 #1 3'-2-5 #2 3'-1-30 / AS 2PS SS 2PS), OLX013A-002-154 (2'-OMe #1 5'-2-15 / 2'-F #1 5'-1-6 #1 3'-2-4 #2 3'-1-30 / AS 2PS SS 2PS), OLX013A-002-161 (2'-OMe #1 5'-2-15 / 2'-F #1 5'-1-6 #1 3'-2-3 #2 3'-1-30 / AS 2PS SS 2PS), OLX013A-002-168 (2'-OMe #1 5'-2-15 / 2'-F #1 5'-1-7 #1 3'-2-3 #2 3'-1-30 / AS 2PS SS 2PS)).However, when a 2'-F chemical modification is introduced at position 19 from the 5' end of the first strand (OLX013A-002-101 (2'-OMe #1 5'-2-15 / 2'-F ##1 5'-1-10 #2 3'-1-30 / AS 2PS SS 2PS), OLX013A-002-141 (2'-OMe #1 5'-2-15 / 2'-F #1 5'-1-5 #1 3'-2-6 #2 3'-1-30 / AS 2PS SS 2PS)), when the position is naked (OLX013A-002-5 (2'-OMe #1 5'-2-15 / 2'-F #1 5'-1-6 #2 3'-1-30 / AS 2PS SS 2PS), OLX013A-002-148 (2'-OMe #1 5'-2-15 / 2'-F #1 5'-1-6 #1 3'-2-5 #2 3'-1-30 / AS 2PS SS 2PS), OLX013A-002-154 (2'-OMe #1 5'-2-15 / 2'-F #1 5'-1-6 #1 3'-2-4 #2 3'-1-30 / AS 2PS SS 2PS), OLX013A-002-161 (2'-OMe #1 5'-2-15 / 2'-F #1 5'-1-6 #1 3'-2-3 #2 3'-1-30 / AS 2PS SS 2PS), OLX013A-002-168 (2'-OMe #1 Unlike the 5'-2-15 / 2'-F #1 5'-1-7 #1 3'-2-3 #2 3'-1-30 / AS 2PS SS 2PS)), the innate immune response was found to be significantly reduced, so it was predicted that position 19 may be the site recognized by MDA5. In addition, in particular, when 2'-F chemical modifications were introduced continuously to the second strand, it was confirmed that the case in which 2'-OMe chemical modifications were introduced alternately throughout the first strand and six 2'-F chemical modifications were introduced alternately partially to the 5'-terminal region (OLX013A-002-5 (2'-OMe #1 5'-2-15 / 2'-F #1 5'-1-6 #2 3'-1-30 / AS 2PS SS 2PS)) induced the highest level of innate immune response.

[0173] In addition, in a structure in which 2'-F chemical modifications were successively introduced into the second strand, in a case in which two to three 2'-OMe chemical modifications were alternately introduced into one or both terminal regions, in a case in which two to three 2'-OMe chemical modifications were alternately introduced into only the 3' terminal region, an innate immune response was not significantly induced (OLX013A-002-137, OLX013A-002-144, OLX013A-002-150, OLX013A-002-157, OLX013A-002-164, OLX013A-002-171, OLX013A-002-140, OLX013A-002-147, OLX013A-002-153, When two to three 2'-OMe chemical modifications were alternately introduced only in the 5'-terminal region (OLX013A-002-160, OLX013A-002-167, OLX013A-002-174), or when two to three 2'-OMe chemical modifications were alternately introduced in both terminal regions, it was confirmed that an innate immune response was significantly induced. In particular, it was confirmed that the strongest innate immune response was induced when three 2'-OMe chemical modifications were alternately introduced only in the 5'-terminal region (OLX013A-002-136, OLX013A-002-143, OLX013A-002-6, OLX013A-002-156, OLX013A-002-163, OLX013A-002-170).In addition, in a structure in which 2'-F chemical modifications were successively introduced into the second strand, when two to three 2'-OMe chemical modifications were alternately introduced into one or both terminal regions, it was confirmed that a high level of innate immune response was induced when 2'-OMe chemical modifications were alternately introduced into the entire first strand and 4 to 6 2'-F chemical modifications were alternately introduced into both terminal regions at the same time (OLX013A-002-6 (2'-OMe #1 5'-2-15 #2 5'-1-3 / 2'-F #1 5'-1-6 #1 3'-2-5 #2 3'-1-27 / AS 2PS SS 2PS), OLX013A-002-156 (2'-OMe #1 5'-2-15 #2 5'-1-3 / 2'-F #1 5'-1-6 #1 3'-2-4 #2 3'-1-27 / AS 2PS SS 2PS)). Among them, it was confirmed that the case in which six 2'-F chemical modifications were introduced alternately at the 5' end and five 2'-F chemical modifications were introduced alternately at the 3' end region induced the highest level of innate immune response (OLX013A-002-6 (2'-OMe #1 5'-2-15 #2 5'-1-3 / 2'-F #1 5'-1-6 #1 3'-2-5 #2 3'-1-27 / AS 2PS SS 2PS)). In addition, similar to the previously confirmed case, when a 2'-F chemical modification was introduced at position 19 from the 5' end of the first strand (OLX013A-002-143 (2'-OMe #1 5'-2-15 #2 5'-1-3 / 2'-F #1 5'-1-5 #1 3'-2-6 #2 3'-1-27 / AS 2PS SS 2PS)), the innate immune response was found to be significantly reduced, unlike when the position was naked (OLX013A-002-6 (2'-OMe #1 5'-2-15 #2 5'-1-3 / 2'-F #1 5'-1-6 #1 3'-2-5 #2 3'-1-27 / AS 2PS SS 2PS)).

[0174]

[0175] In summary of the results of the above Examples 1 and 2, a double-stranded RNA in which 2'-OMe modifications are alternately introduced throughout the first strand (OLX013A-002-3 (2'-OMe #1 5'-2-15 / SS 2PS)), a double-stranded RNA in which 2'-OMe modifications are alternately introduced throughout the first strand and six 2'-F chemical modifications are alternately introduced into the 5'-terminal region, and 2'-F chemical modifications are continuously introduced into the second strand (OLX013A-002-05 (2'-OMe #1 5'-2-15 / 2'-F #1 5'-1-6 #2 3'-1-30 / AS 2PS SS 2PS)), and a double-stranded RNA in which 2'-OMe modifications are alternately introduced throughout the first strand and six 2'-F chemical modifications are alternately introduced into the 5'-terminal region, and six 2'-F chemical modifications are alternately introduced into the 3'-terminal region Three types of double-stranded RNA (OLX013A-002-6 (2'-OMe #1 5'-2-15 #2 5'-1-3 / 2'-F #1 5'-1-6 #1 3'-2-5 #2 3'-1-27 / AS 2PS SS 2PS)) in which five 2'-F chemical modifications were alternately introduced and 2'-F chemical modifications were sequentially introduced to the second strand were selected as final candidates for inducing innate immune responses, and subsequent experiments were conducted.

[0176]

[0177]

[0178] Example 3. Efficacy evaluation of candidate double-stranded RNA

[0179] 3.1. Low-concentration efficacy evaluation

[0180] The three double-stranded RNAs (OLX013A-002-3, OLX013A-002-5, and OLX013A-002-6) selected as final candidates were compared with naked double-stranded RNA (OLX013A-002-1) without chemical modification to see if they could induce significant innate immune responses even at low concentrations. The four double-stranded RNAs are shown in Table 6 below. In Table 6, nucleotides with 2'-OCH3 (2'-O-methyl; 2'-OMe) chemical modifications are underlined, nucleotides with 2'-F (2'-fluoro; 2'-F) chemical modifications are indicated in parentheses, and nucleotides in which nucleotide linkages are substituted with phosphorothioate (PS) linkages are indicated with “*”. The naming of double-stranded RNA with 2'-OMe or 2'-F chemical modifications was written as [strand with chemical modification]-[terminus with chemical modification and first nucleotide position with chemical modification]-[number of nucleotides with chemical modification], and the naming of double-stranded RNA with nucleotide bonds replaced by PS bonds was written as [strand with PS bonds]-[2PS (two adjacent nucleotide bonds from both ends of the strand replaced by PS bonds) or fPS (all nucleotide bonds of the strand replaced by PS bonds)].

[0181] [Table 6]

[0182]

[0183] Thereafter, for the double-stranded RNA of Table 6, the activity of IRF was measured in the human monocyte cell line THP1-Dual (thpd-nfis) cell line using the same method as in Example 2.1. Meanwhile, in this example, the positive control group was a group administered with Poly I:C, and the control group was a group treated with only the transfection reagent (mock) and a group not treated with double-stranded RNA (NT). The results are shown in Fig. 6.

[0184] As a result, as shown in Fig. 6, OLX013―002-5 and OLX013―002-6 induced higher innate immune responses than OLX013A-002-3 at all treatment concentrations, and in particular, it was confirmed that OLX013―002-6 could induce a sufficient innate immune response even at a very low concentration of 0.625 nM. This means that, compared to double-stranded RNA in which the 2'-OMe modification is introduced only in the first strand, double-stranded RNA in which the 2'-OMe modification and the 2'-F modification are combined and introduced in the first strand, and the 2'-F modification or the 2'-F modification and the 2'-OMe modification are combined and introduced in the second strand can induce a more potent innate immune response.

[0185]

[0186] 3.2. Evaluation of MDA5-mediated innate immune responses according to scrambled base sequences

[0187] The three double-stranded RNAs (OLX013A-002-3, OLX013A-002-5, and OLX013A-002-6) selected as final candidates were comparatively evaluated for their effects on inducing MDA5-dependent innate immune responses. In this example, in order to confirm the effect of the Lamin A / C-GFP sequence on innate immune response induction, double-stranded RNAs in which 2'-OMe modifications were introduced alternately throughout the first strand of a scrambled sequence (Lamin A / C-GFP scramble) in which nucleotides included in the Lamin A / C-GFP sequence were randomly shuffled and rearranged were additionally produced. The four double-stranded RNAs are shown in Table 7 below. In Table 7, nucleotides with a 2'-OCH3 (2'-O-methyl; 2'-OMe) chemical modification are underlined, nucleotides with a 2'-F (2'-fluoro; 2'-F) chemical modification are indicated in parentheses, and those in which the nucleotide bond is replaced with a phosphorothioate (PS) bond are indicated with "*". The naming of double-stranded RNA with 2'-OMe or 2'-F chemical modifications was written as [strand with chemical modification]-[terminus with chemical modification and first nucleotide position with chemical modification]-[number of nucleotides with chemical modification], and the naming of double-stranded RNA with nucleotide bonds replaced by PS bonds was written as [strand with PS bonds]-[2PS (two adjacent nucleotide bonds from both ends of the strand replaced by PS bonds) or fPS (all nucleotide bonds of the strand replaced by PS bonds)].

[0188] [Table 7]

[0189]

[0190] Thereafter, for the double-stranded RNA of Table 7, the activity of IRF was measured for the THP1-Dual (thpd-nfis) cell line (WT) and the THP1-Dual KO-MDA5 cell line (MDA5 KO) in the same manner as in Example 2.1. Meanwhile, in this example, the positive control group was the group administered with Poly I:C and / or LPS, and the control group was the group added with only the transfection reagent (mock) and the group not treated with double-stranded RNA (NT). The results are shown in Fig. 7.

[0191] As a result, as shown in Fig. 7, all of the OLX013A-002-3, OLX013A-002-5, and OLX013A-002-6 treatment groups induced an MDA5-dependent innate immune response, and in particular, the strongest innate immune response was induced in OLX013A-002-6. In addition, OLX013A-scr-3, in which the Lamin A / C-GFP sequences were randomly mixed and 2'-OMe modifications were alternately introduced throughout the first strand, induced IRF activity that was 75%p lower than OLX013A-002-3 with similar chemical modifications. This implies that the base sequence of Lamin A / C-GFP is involved in inducing an MDA5-dependent innate immune response.

[0192]

[0193] 3.3. Stability assessment according to chemical transformation

[0194] To evaluate the stability of double-stranded RNA according to chemical modification, serum stability evaluation was performed on the three double-stranded RNAs (OLX013A-002-3, OLX013A-002-5, and OLX013A-002-6) selected as final candidates under 50% FBS conditions.

[0195] Specifically, 20 μL of 10 μM double-stranded RNA and 20 μL of FBS were mixed and then dispensed into PCR tubes (4 μL each) according to the time points to be analyzed. The Day 0 group was immediately stored at -80℃, and the remaining groups were incubated at 37℃. After that, each group was taken out according to the time points to be analyzed and immediately stored at -80℃. RNA was purified from all groups through protein removal and ethanol precipitation using Tissue & Cell Lysis Solution (MTC096H, Biosearch Technologies) and Proteinase K (MPRK092, Biosearch Technologies). 130 ng of the purified RNA from each group was mixed with Blue / Orange Loading Dye, 6X (G1881, Promega) and electrophoresed on a 10% non-denaturing agarose gel to confirm the results. The results are shown in Figure 8.

[0196] As a result, as shown in Fig. 8, OLX013A-002-3 showed that the double-stranded RNA was degraded after only one day, but in the case of OLX013A-002-5, about 60% of the double-stranded RNA was maintained without degradation until the 8th day, and in the case of OLX013A-002-6, about 86% of the double-stranded RNA was maintained without degradation until the 8th day. This means that the more nucleotides with 2'-F or 2'-OMe modifications are introduced, the greater the improvement in dsRNA stability.

[0197]

[0198] 3.4. Evaluation of RIG-I and MDA5 mRNA expression levels in human PBMCs

[0199] The two double-stranded RNAs (OLX013A-002-3, OLX013A-002-5) selected as final candidates were compared with the negative control OLX013A-002-4 to see whether they could increase the mRNA expression levels of RIG-I and MDA5 by innate immune response in human PBMC cells. OLX013A-002-4 is a double-stranded RNA produced by introducing PS bonds to both ends of the second strand of OLX013A-002-38 (#1 5'-2-15 #2 3'-1-15), which was confirmed in Example 1.2 to not induce a significant level of innate immune response when the same number of 2'-OMe modifications were introduced to both the first and second strands. The three double-stranded RNAs are shown in Table 8 below. In Table 8, nucleotides with a 2'-OCH3 (2'-O-methyl; 2'-OMe) chemical modification are underlined, nucleotides with a 2'-F (2'-fluoro; 2'-F) chemical modification are indicated in parentheses, and those in which the nucleotide bond is replaced with a phosphorothioate (PS) bond are indicated with "*". The naming of double-stranded RNA with 2'-OMe or 2'-F chemical modifications was written as [strand with chemical modification]-[terminus with chemical modification and first nucleotide position with chemical modification]-[number of nucleotides with chemical modification], and the naming of double-stranded RNA with nucleotide bonds replaced by PS bonds was written as [strand with PS bonds]-[2PS (two adjacent nucleotide bonds from both ends of the strand replaced by PS bonds) or fPS (all nucleotide bonds of the strand replaced by PS bonds)].

[0200] [Table 8]

[0201]

[0202]

[0203] Specifically, on the day of the experiment, 4X10 human PBMCs (Stemcell Technologies) 5 Cells / well were seeded in a 96-well plate. Complete RPMI media (10% FBS) was used as the medium. The double-stranded RNA in Table 8 was complexed using LNP, a delivery vehicle. The double-stranded RNA-LNP complexes were treated at concentrations of 5 nM, 10 nM, and 50 nM. Each substance was treated in triplicate in three wells. Treated cells were cultured overnight at 37°C / 5% CO2. After culture, the cells obtained were washed with 1X PBS, and then cell lysis and cDNA synthesis were performed using Toyobo SuperPrep II Cell Lysis & RT Kit. mRNA expression was compared through qPCR using Thunderbird Taqman qPCR Mix. The results are shown in Figures 9a and 9b.

[0204] As a result, as shown in Figures 9a and 9b, compared to the negative control group OLX013A-002-4, both the OLX013A-002-3 and OLX013A-002-5 treatment groups were found to increase the expression levels of RIG-I and MDA5 mRNA in a concentration-dependent manner, and in particular, it was confirmed that the expression levels increased further in OLX013A-002-5.

[0205]

[0206] 3.5. Confirmation of in vitro cytotoxicity against cancer cells

[0207] The cytotoxic effects of the three double-stranded RNAs (OLX013A-002-3, OLX013A-002-5, and OLX013A-002-6) selected as final candidates were compared with those of the negative control, OLX013A-002-4, in cancer cells cultured in a three-dimensional spherical shape to simulate microtumor conditions. The four double-stranded RNAs are shown in Table 8 above.

[0208] Specifically, for spheroid culture in the form of spheres, human glioblastoma cell line LN229 and human breast cancer cell line HCC70 were cultured in 96-well ultra low attachment plates at 5,000 cells / well each for 3 days (37°C, 5% CO2). On the third day of culture, to differentiate human PBMC (Stemcell Technologies) into T cells, PMBC were added to the spheroids in a ratio of 1:5 or 1:10 relative to the number of each cancer cell line in complete RPMI media supplemented with IL-2 (100 IU / mL). The double-stranded RNA in Table 8 was complexed using LNP, a delivery vehicle. Double-stranded RNA-LNP complexes were added to each well at a concentration of 10 nM immediately after the addition of PBMC and cultured for 4 days. On the fourth day of culture, both spheroids and PBMCs were transferred to a white flat-bottom 96-well plate, and an equal amount of CellTiter-Glo 3D was added to each well. Luminescence was measured using a Promega GloMax Discover instrument, and spheroid viability was determined. The results are shown in Figures 10a and 10b.

[0209] As a result, as shown in Figures 10a and 10b, compared to the negative control group OLX013A-002-4, both the OLX013A-002-3 and OLX013A-002-5 treatment groups showed a decrease in cancer cell survival rate, confirming that they had excellent anticancer effects.

[0210]

[0211] 3.6. Confirmation of in vivo tumor growth inhibition effect in the EMT6 mouse tumor model

[0212] After injecting OLX013A-002-3 into the EMT6 mouse tumor model, the tumor growth inhibitory effect and mouse survival rate were evaluated. In addition, tumor tissues were isolated from the EMT6 mouse tumor model and analyzed for the mRNA expression levels of RIG-I and MDA5, as well as the mRNA expression levels of CXCL10 and IFIT-1, cytokines associated with the innate immune response. OLX013A-002-4 was used as a negative control. The two types of double-stranded RNA are shown in Table 8.

[0213] Specifically, EMT6 cells (2x 10 5 ) was injected subcutaneously into the mammary fat pad to create an EMT6 mouse tumor model. The tumor was approximately 80 mm 3 When the size was reached, each group was injected intratumorally 4 times at 3-day intervals starting on the 17th day after transplantation with vehicle, OLX013A-002-4 (control) 25 μg, OLX013A-002-3 25 μg, or OLX013A-002-3 50 μg. Control and OLX013A-002-3 used in the experiment were complexed using invivofectamine, a delivery vehicle, and injected. The tumor size of 8 mice in each group was measured to confirm the tumor growth inhibition effect (long axis (mm) x short axis 2 (mm 2) / 2), and the mouse survival rate was confirmed. The results are shown in Figures 11a and 11b. The degree of significance for each control group was indicated by the number of symbols, and according to the p value, 1 was indicated as p<0.05, 2 as p<0.01, and 3 as p<0.001.

[0214] Additionally, the tumors were removed the day after the final injection, RNA was extracted using the RNeasy plus mini kit (Qiagen, 74136), and cDNA was synthesized using the High-capacity cDNA reverse transcription kit (Applied Biosystems, 4368813). The mRNA expression of RIG-I, MDA5, CXCL10, and IFIT-1 in each group was compared using qPCR. The results are shown in Figure 12.

[0215] As a result, as shown in Fig. 11a and Fig. 11b, 3 days after the last substance injection, the OLX013A-002-3 50 μg administration group showed a significant tumor growth inhibition effect compared to the non-administered group (*), the vehicle administration group (†), and the OLX013A-002-4 (control) 25 μg administration group (§). The OLX013A-002-3 25 μg administration group was also confirmed to have a significant tumor inhibition effect compared to the non-administered group, the vehicle administration group, and the OLX013A-002-4 (control) 25 μg administration group. In addition, it was confirmed that the mouse survival rate was superior in the OLX013A-002-3 administration group compared to the OLX013A-002-4 administration group.

[0216] In addition, as shown in Fig. 12, the OLX013A-002-3 25 μg administration group showed higher expression of mRNA of RIG-I, MDA5, CXCL10, and IFIT-1 compared to the non-administered group, the vehicle administration group, and the OLX013A-002-4 (control) 25 μg administration group. This means that the OLX013A-002-3 double-stranded RNA was well delivered to cancer cells, and an innate immune response occurred within the cancer cells.

[0217]

[0218] 3.7. Confirmation of in vivo tumor growth inhibition effect in the B16F10 mouse tumor model

[0219] The tumor growth inhibitory effect of OLX013A-002-5 was evaluated after injection into a B16F10 mouse tumor model. Furthermore, tumor tissues were isolated from the B16F10 mouse tumor model and analyzed for the expression levels of RIG-I and MDA5 mRNA, as well as CXCL10, IFIT-1, and TNF-α mRNA, cytokines associated with the innate immune response. The double-stranded RNAs of this type are shown in Table 8.

[0220] Specifically, a B16F10 mouse tumor model was created by subcutaneously injecting B16F10 cells into the left flank of the mouse. The tumor was approximately 230 mm 3 When the size reached, the mice were divided into a non-administered group and a group administered 25 μg of OLX013A-002-5, and intratumoral injections were performed four times at three-day intervals starting on the 14th day after transplantation. OLX013A-002-5 used in the experiment was injected as a complex using invivofectamine, a delivery vehicle. Five mice were used for each group to measure the tumor size and confirm the tumor growth inhibition effect (long axis (mm) x short axis 2 (mm 2 ) / 2). The results are shown in Fig. 13a.

[0221] Additionally, the day after the last substance injection, tumors were removed, RNA was extracted using the RNeasy plus mini kit (Qiagen, 74136), and cDNA was synthesized using the High-capacity cDNA reverse transcription kit (Applied Biosystems, 4368813). The mRNA expression of RIG-I, MDA5, CXCL10, IFIT-1, and TNF-α in each group was compared using qPCR. The results are shown in Figure 13b.

[0222] As a result, as shown in Figure 13a, in the non-administered group, 3 out of 5 animals died during the observation period or the tumor size continued to increase. On the other hand, in the OLX013A-002-5 25 μg administered group, no animals died, confirming the tumor growth inhibitory effect compared to the non-administered group.

[0223] In addition, as shown in Fig. 13b, it was confirmed that the OLX013A-002-5 administration group had higher mRNA expression of RIG-I, MDA5, CXCL10, and IFIT-1 compared to the non-administered group. This means that the OLX013A-002-5 double-stranded RNA was well delivered to cancer cells and an innate immune response was induced within the cancer cells.

[0224] Similarly, the tumor growth inhibitory effect and mouse survival rate were evaluated after injection of OLX013A-002-3 or OLX013A-002-6 into a B16F10 mouse tumor model. In addition, tumor tissues were isolated from the B16F10 mouse tumor model and the expression levels of RIG-I and MDA5 mRNA, as well as CXCL10, IFIT-1, TNF-α, and IFN-α mRNA, which are cytokines associated with the innate immune response, were analyzed. The two types of double-stranded RNAs are shown in Table 8 above.

[0225] Specifically, B16F10 tumor cells (4X10 5) were transplanted to create a B16F10 mouse tumor model. They were divided into a non-administered group and a group administered OLX013A-002-3 25 μg, OLX013A-002-3 10 μg, OLX013A-002-6 25 μg, and OLX013A-002-6 10 μg. The tumors were injected intratumorally four times at 3-day intervals starting from the 7th day after transplantation, and the tumor sizes were measured (long axis (mm) x short axis 2 (mm 2 ) / 2) to confirm the tumor growth inhibition effect and mouse survival rate. OLX013A-002-3 and OLX013A-002-6 used in the experiment were complexed using invivofectamine, a delivery vehicle, and injected. The results are shown in Figures 14a and 14b.

[0226] Additionally, tumor tissues were isolated 24 hours after the third administration, and the mRNA expression of various genes was compared. Tumors were excised and stored in RNAlater™ Stabilization Solution (Invitrogen, AM7021). RNA was extracted from the tumors using the RNeasy plus mini kit (Qiagen, 74136), and cDNA was synthesized using the High-capacity cDNA reverse transcription kit (Applied Biosystems, 4368813). Then, the mRNA expression of RIG-I, MDA5, CXCL10, IFIT-1, TNF-α, and IFN-α was measured using primers or Taqman probes for each gene. The expression of each gene was corrected with RPL32. The results are shown in Figure 14c.

[0227] As a result, as shown in FIGS. 14a and 14b, OLX013A-002-3 and OLX013A-002-6 showed a significant tumor growth inhibitory effect even in the 10 μg administration group, and it was confirmed that the mouse survival rate was better in the OLX013A-002-6 administration group than in the OLX013A-002-3 administration group.

[0228] In addition, as shown in Fig. 14c, it was confirmed that the OLX013A-002-3 and OLX013A-002-6 administration groups had higher expression of mRNA of RIG-I, MDA5, CXCL10, IFIT-1, TNF-α, and IFN-α compared to the non-administered group, and in particular, it was confirmed that the expression increased in the OLX013A-002-6 administration group. This means that the OLX013A-002-3 and OLX013A-002-6 double-stranded RNAs were well delivered to cancer cells, and an innate immune response was induced within the cancer cells.

[0229]

[0230] 3.8 Evaluation of serum IFN-α concentrations after intramuscular administration in the B16F10 mouse tumor model

[0231] B16F10 tumor cells (4X10) were injected into the flanks of 6-week-old C57BL / 6 mice 5 ) One week after transplantation, three types of LNP-encapsulated double-stranded RNA (OLX013A-002-3-LNP, OLX013A-002-5-LNP, OLX013A-002-6-LNP) were administered intramuscularly at each concentration. Five hours after administration, blood was collected, centrifuged at 1400 xg for 10 minutes, and serum was obtained to measure and compare IFN-α concentrations. IFN-α was measured using Mouse IFN-Alpha All Subtype ELISA Kit (PBL assay science, 42115-1). The results are shown in Fig. 15.

[0232] As a result, as shown in Fig. 15, in the OLX013A-002-3 administration group and the OLX013A-002-5 administration group, serum IFN-α was not detected at low concentrations (5 ng), whereas in the OLX013A-002-6 administration group, serum IFN-α was detected even at low concentrations (5 ng).

[0233]

[0234] Example 4. Analysis of the innate immune activation motif recognized by MDA5.

[0235] 4.1. Analysis by position of 2'-OMe chemical modification introduction

[0236] In order to identify the immune activation site recognized by MDA5, based on the double-stranded RNA (OLX013A-002-2 (2'OMe #1 5'-2-15)) in which 2'-OMe modifications were introduced alternately throughout the first strand, which was identified as the basic structure for inducing a strong innate immune response in Example 2.1, double-stranded RNAs in which six or five 2'-OMe modifications were introduced alternately at one end or the middle region of the first strand were additionally prepared as shown in Table 9 below, and it was evaluated whether the innate immune response was maintained in OLX013A-002-2 in which 2'-OMe modifications were introduced alternately throughout. Table 10 shows the first-strand nucleotide sequence and the second-strand nucleotide sequence of the innate immune activation motif in OLX013A-002-51 and OLX013A-002-176 of Table 9. In Tables 9 and 10, nucleotides with 2'-OCH3 (2'-O-methyl; 2'-OMe) chemical modifications are underlined, and the nomenclature of double-stranded RNAs with 2'-OMe chemical modifications is written as [strand with chemical modification]-[terminus with chemical modification and first nucleotide position with chemical modification]-[number of nucleotides with chemical modification]. Innate immune activation motifs are shaded, and the first-strand nucleotide sequences of innate immune activation motifs are bold.

[0237] [Table 9]

[0238]

[0239] [Table 10]

[0240]

[0241] Thereafter, for the double-stranded RNA of Table 9, the activity of IRF was measured for the THP1-Dual (thpd-nfis) cell line (WT) and the THP1-Dual KO-MDA5 cell line (MDA5 KO) in the same manner as in Example 2.1. Meanwhile, in this example, the positive control group was the group administered with Poly I:C and / or LPS, and the control group was the group added with only the transfection reagent (mock) and the group not treated with double-stranded RNA (NT). The results are shown in Figures 16 and 17.

[0242] As a result, as shown in FIGS. 16 and 17, OLX013A-002-14 (2'-OMe #1 5'-2-6), in which six 2'-OMes were alternately introduced into the 5'-terminal region, failed to activate the innate immune response, whereas OLX013A-002-51 (2'-OMe #1 5'-14-6) and OLX013A-002-52 (2'-OMe #1 3'-1-6) induced the innate immune response. In particular, OLX013A-002-51 (2'-OMe #1 5'-14-6), in which six 2'-OMe nucleotides were alternately introduced at positions 14-24 from the 5' end of the first strand, showed an innate immune response at a level similar to that of OLX013A-002-2 (2'OMe #1 5'-2-15), in which 2'-OMe modifications were alternately introduced throughout. In addition, OLX013A-002-176 (2'-OMe #1 5'-16-5), in which the number of nucleotides with alternate 2'-OMe was reduced to five, also induced a strong innate immune response and showed an MDA5-dependent immune response. Therefore, the strong MDA5-dependent innate immune response induction effect in OLX013A-002-2 (2'OMe #1 5-2-15), in which 2'-OMe modifications were introduced alternately throughout the first strand, was found to be derived from the introduction of 2'-OMe at positions 14 to 24, preferably 16 to 24, from the 5' end of the first strand, which means that the region may be a core region recognized by MDA5, i.e., a region that acts as an innate immune activation motif.

[0243]

[0244] 4.2. Analysis by base sequence

[0245] To further verify the region that acts as an innate immune activation motif, we compared the cases in which 2'-OMe modifications were introduced alternately throughout the first strand and the cases in which 2'-OMe modifications were introduced alternately partially at positions 2 to 12, 6 to 16, 16 to 26, or 20 to 30 throughout the first strand, targeting different base sequences, to identify the region that can induce an innate immune response like the case in which 2'-OMe modifications were introduced alternately throughout the first strand.

[0246] In this example, a scrambled sequence (scrambled Lamin A / C-GFP (GC content 43%)) in which nucleotides included in the Lamin A / C-GFP sequence are randomly mixed and rearranged, a random sequence (random sequence (GC content 43%)) in which only the GC content is the same as that of the Lamin A / C-GFP sequence at 43%, but which is randomly constructed using nucleotides that are completely different from those included in the Lamin A / C-GFP sequence, and a GAPDH sequence having a different GC content from that of the Lamin A / C-GFP sequence, were additionally produced as shown in Table 11 below. Double-stranded RNAs in which 2'-OMe modifications were alternately introduced throughout the first strand and double-stranded RNAs in which 2'-OMe modifications were alternately introduced partially at one end or the middle region of the first strand were additionally produced. In Table 11, nucleotides with 2'-OCH3 (2'-O-methyl; 2'-OMe) chemical modifications are underlined, and the nomenclature of double-stranded RNAs with 2'-OMe chemical modifications is written as [strand with chemical modification]-[terminus with chemical modification and position of first nucleotide with chemical modification]-[number of nucleotides with chemical modification].

[0247] [Table 11]

[0248]

[0249]

[0250] Thereafter, for the double-stranded RNA of Table 10, the activity of IRF was measured for the THP1-Dual (thpd-nfis) cell line (WT), the THP1-Dual KO-RIG-I cell line (RIG-I KO), and the THP1-Dual KO-MDA5 cell line (MDA5 KO) in the same manner as in Example 2.1. Meanwhile, in this example, the positive control group was the group administered with Poly I:C and / or LPS, and the control group was the group added with only the transfection reagent (mock) and the group not treated with double-stranded RNA (NT). 3p-hpRNA was used as a positive control group for WT and MDA5 KO cells and as a negative control group for RIG-I KO cells. The results are shown in Fig. 18.

[0251] As a result, as shown in Fig. 18, when the random sequence was used as the basic framework, OLX013A-ran-1, in which 2'OMe was introduced alternately throughout, and OLX013A-ran-5 and OLX013A-ran-6, in which 2'OMe was introduced alternately partially, induced similar levels of MDA5-dependent immune responses. When the GAPDH sequence was used as the basic framework, OLX013A-025-8, in which 2'OMe was introduced alternately partially, showed a somewhat reduced immune response compared to OLX013A-025-4, in which 2'-OMe was introduced alternately throughout, but induced an MDA5-dependent immune response. OLX013A-ran-6 and OLX013A-025-8 are cases in which 2'-OMe was introduced alternately at the same position, 16th to 26th base sequence. All regions recognized by MDA5 appear to be located at the same location. However, OLX013A-scr-3~8, which uses a scrambled sequence as its backbone, failed to elicit an innate immune response despite the introduction of a 2'-OMe modification in the first strand. This confirms that MDA5 recognition involves not only the chemical pattern but also the base sequence.

[0252]

[0253] 4.3. MDA5-dependent innate immune activation by introduction of innate immune activation motifs

[0254] Based on the results of the above Examples 4.1 and 4.2, it was assumed that the innate immune activation motif recognized by MDA5 exists at positions 16 to 24 from the 5' end of the first strand, and that the sequence of that region is also involved. It was attempted to confirm whether an innate immune response by MDA5 could be induced by introducing the innate immune activation motif identified in the above Example 4.1 at positions 16 to 24 of a double-stranded RNA that does not induce an innate immune response by MDA5.

[0255] In this example, in addition to the double-stranded RNA (OLX013A-002-2 (2'OMe #1 5'-2-15)) in which 2'-OMe modifications were alternately introduced throughout the first strand used in the analysis of the introduction site of 2'-OMe chemical modification in Example 4.1, and the scramble sequence (OLX013A-scr-3, OLX013A-scr-7) that failed to induce an innate immune response despite the introduction of 2'-OMe modifications in the first strand in Example 4.2, a double-stranded RNA in which the innate immune activation motif of OLX013A-002-2 was introduced into the scramble sequence of OLX013A-scr-7 was additionally produced as shown in Table 13 below. Table 12 shows the first-strand nucleotide sequence and the second-strand nucleotide sequence of the innate immune activation motif. In Tables 12 and 13, nucleotides with 2'-OCH3 (2'-O-methyl; 2'-OMe) chemical modifications are underlined, and the nomenclature of double-stranded RNAs with 2'-OMe chemical modifications is written as [strand with chemical modification]-[terminus with chemical modification and position of first nucleotide with chemical modification]-[number of nucleotides with chemical modification]. Innate immune activation motifs are shaded, and the first-strand nucleotide sequences of innate immune activation motifs are bold.

[0256] [Table 12]

[0257]

[0258] [Table 13]

[0259]

[0260] Thereafter, for the double-stranded RNA of Table 12, the activity of IRF was measured for the THP1-Dual (thpd-nfis) cell line (WT) and the THP1-Dual KO-MDA5 cell line (MDA5 KO) in the same manner as in Example 2.1. Meanwhile, in this example, the positive control group was the group administered with Poly I:C and / or LPS, and the control group was the group added with only the transfection reagent (mock) and the group not treated with double-stranded RNA (NT). The results are shown in Fig. 19.

[0261] As a result, as shown in Fig. 19, OLX013A-scr-9, which introduced the innate immune activation motif of OLX013A-002-2 into OLX013A-scr-7, which failed to induce an immune response by MDA5, was confirmed to induce an MDA5-dependent immune response. This means that the sequence at positions 16 to 24 from the 5' end of the first strand of the Lamin A / C-GFP sequence and the 2'-OMe modification introduced alternately within the corresponding region act together as a core unit or innate activation motif for inducing an MDA5-mediated innate immune response.

Claims

1. A double-stranded RNA of 25 to 50 base pairs (bp) consisting of a first strand and a second strand forming a complementary bond to the first strand, and having blunt ends at both ends, A double-stranded RNA, wherein the first strand comprises at least five 2'-OMe modified nucleotides at positions 10 to 27 from the 5' end.

2. In claim 1, a double-stranded RNA, wherein the double strand composed of the first strand and the second strand has 30 base pairs.

3. A double-stranded RNA according to claim 1, wherein the nucleotide sequence at positions 10 to 27 comprises the nucleotide sequence of SEQ ID NO:

15.

4. A double-stranded RNA according to claim 1, wherein the nucleotide sequence at positions 10 to 27 comprises the nucleotide sequence of SEQ ID NO:

17.

5. A double-stranded RNA according to claim 1, wherein the first strand has 5 to 9 2'-OMe modified nucleotides arranged alternately at positions 10 to 27 from the 5' end.

6. A double-stranded RNA according to claim 1, wherein the first strand comprises 10 to 15 2'-OMe modified nucleotides.

7. A double-stranded RNA according to claim 1, wherein the second strand comprises 2 to 6 2'-OMe modified nucleotides.

8. A double-stranded RNA according to claim 1, wherein the second strand comprises 3 to 30 2'-F modified nucleotides.

9. A double-stranded RNA according to claim 1, wherein the first strand comprises 5 to 11 2'-F modified nucleotides.

10. A double-stranded RNA according to claim 1, wherein the nucleotide at position 19 from the 5'-end of the first strand does not contain a chemical modification.

11. A double-stranded RNA according to claim 1, wherein the first strand has 10 to 12 2'-OMe modified nucleotides arranged alternately from the 5'-terminus.

12. A double-stranded RNA according to claim 1, wherein the first strand comprises 15 2'-OMe modified nucleotides arranged alternately.

13. A double-stranded RNA according to claim 1, wherein the second strand has two to four 2'-OMe modified nucleotides arranged alternately from one end or both ends.

14. A double-stranded RNA according to claim 1, wherein the second strand has 2 to 4 2'-OMe modified nucleotides arranged alternately from the 5' end.

15. A double-stranded RNA according to claim 1, wherein the second strand has 2 to 10 2'-F modified nucleotides arranged alternately from one end or both ends.

16. A double-stranded RNA according to claim 1, wherein the second strand has 15 2'-F modified nucleotides arranged alternately.

17. A double-stranded RNA according to claim 1, wherein the second strand comprises 20 to 30 2'-F modified nucleotides arranged in a continuous manner.

18. A double-stranded RNA according to claim 1, wherein the second strand has 25 to 27 2'-F modified nucleotides arranged consecutively from the 3' end.

19. A double-stranded RNA according to claim 1, wherein the first strand has 3 to 10 2'-F modified nucleotides arranged alternately from one end or both ends.

20. A double-stranded RNA according to claim 1, wherein the first strand has six 2'-F modified nucleotides arranged alternately from the 5'-terminus.

21. A double-stranded RNA according to claim 1, wherein the first strand has 6 to 7 2'-F modified nucleotides arranged alternately from the 5'-terminus and 3 to 5 2'-F modified nucleotides arranged alternately from the 3'-terminus.

22. A double-stranded RNA according to claim 1, wherein the first strand has six 2'-F modified nucleotides arranged alternately from the 5'-terminus and five 2'-F modified nucleotides arranged alternately from the 3'-terminus.

23. A double-stranded RNA according to claim 1, wherein the first strand is composed of a sequence having at least 80% homology with the nucleotide sequence of SEQ ID NO:

9.

24. A double-stranded RNA according to claim 1, wherein two to four nucleotide bonds adjacent to both ends of the first strand are modified with phosphorothioate, boranophosphate, or methyl phosphonate.

25. A double-stranded RNA according to claim 1, wherein two to four nucleotide bonds adjacent to the second strand from both ends are modified with phosphorothioate, boranophosphate, or methyl phosphonate.

26. In claim 1, the double-stranded RNA induces an MDA5 (Melanoma differentiation-associated protein 5)-mediated innate immune response.

27. In claim 1, the double-stranded RNA is a double-stranded RNA that induces a RIG-I (Retinoic acid-inducible gene I)-mediated innate immune response.

28. A composition for enhancing innate immunity comprising a double-stranded RNA according to any one of claims 1 to 27.

29. A composition for enhancing innate immunity according to claim 28, wherein the composition induces a RIG-I mediated innate immune response or an MDA5 mediated innate immune response.

30. A composition for enhancing innate immunity, wherein the composition is an immuno-cancer agent according to claim 28.

31. A composition for enhancing innate immunity, according to claim 28, wherein the composition is a vaccine adjuvant.

32. A pharmaceutical composition comprising a double-stranded RNA according to any one of claims 1 to 27.

33. A pharmaceutical composition according to claim 32, wherein the pharmaceutical composition is for treating or improving cancer by inducing an innate immune response.

34. A pharmaceutical composition according to claim 32, wherein the pharmaceutical composition is administered in combination with at least one anticancer agent.

35. A method for enhancing an innate immune response, comprising administering to a subject a pharmaceutical composition according to claim 32.

36. A method for treating or improving cancer by inducing an innate immune response, comprising administering to a subject a pharmaceutical composition according to claim 32.

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