KR102256001B1 - Novel ribonucleic acid and pharmaceutical composition based on the same - Google Patents

Abstract

The present invention has an arbitrary length and a double-stranded ribonucleic acid (dsRNA) site that acts as a TLR3 ligand having complete complementarity with an arbitrary sequence, is located in the middle, and has an arbitrary length with an arbitrary sequence (preferably, a TLR7 ligand sequence). A single-stranded ribonucleic acid (ssRNA) relates to a hetero structured RNA (hsRNA) linked to both 3'ends of a double-stranded ribonucleic acid. In addition, it relates to a composition for preventing and treating viral or bacterial infections and cancers comprising the same.

Description

Novel ribonucleic acid and pharmaceutical composition based on it {NOVEL RIBONUCLEIC ACID AND PHARMACEUTICAL COMPOSITION BASED ON THE SAME}

The present invention relates to a novel ribonucleic acid overcoming the problems of Poly(I:C). Specifically, a double-stranded ribonucleic acid (dsRNA) site that has complete complementarity with an arbitrary sequence and acts as a TLR3 ligand is located in the middle, and has an arbitrary sequence and an arbitrary length at both 3'ends of the dsRNA. -It relates to a mixed structured ribonucleic acid (hsRNA) linked to a single-stranded ribonucleic acid (ssRNA) acting as a similar ligand, and the length of the resulting mixed structured ribonucleic acid is highly homogeneous and the mixed structure ribonucleic acid has a high congenital profile. It has an active function and has a stable advantage.

In addition, the present invention relates to a composition for preventing and treating viral or bacterial infection, cancer, and treatment comprising the mixed structure ribonucleic acid or double-stranded ribonucleic acid.

A) TLR3 ligand as an innate immune activator

TLR3 is normally expressed in DC (dendritic cells), B cells, monocyte-derived macrophages, and endosome compartments on many tumor tissues and detects dsRNA. dsRNA can be derived from viruses, bacteria or abnormal cells. When the TLR3 receptor recognizes dsRNA, it stimulates the secretion of type I interferons and proinflammatory cytokines. DCs are activated into mature antigen presenting cells (APCs), and antigenic epitopes are loaded onto the MHC-1 molecule and provided to naive T cells. Activation of DC by the TLR3 enzyme not only contributes to the induction of innate and adaptive immune responses to microbial pathogens, but also activates CD8+ T cells and natural killer (NK) cells (Non-Patent Document 1). It is known that the minimum length of dsRNA capable of binding to TLR3 and inducing dimerization is 45 mer. 150 to 540 bp dsRNA is known to be a length that activates DC in vitro (Non-Patent Document 1). However, in vitro dendritic progenitor cells were treated with FLT3 ligand or GM-CSF, and the dsRNA-treated cells could not be considered to have analyzed the function in vivo.

There is a report of applying Poly(I:C), which is a dsRNA analogue, to infections and tumors (Patent Documents 1-14). For example, in mice, Poly(I:C) stimulated DC, NK, and CTL (cytotoxic lymphocyte) to inhibit tumor growth very much (Non-Patent Literature 2), but Poly(I:C) itself has high toxicity. In addition, since it is impossible to produce it in a certain length, there is a problem that its application to the clinical field is very limited. Often, Poly(I:C) exhibits unique cellular responses along its length, with shorter forms activating TLR3 and longer forms activating innate immunity through TLR3 and MDA5 activities, respectively (Non-Patent Document 1).

B) ssRNA that acts as a TLR7/TLR8 ligand in innate immunity

SsRNA containing guanosine (G) and uridine (U) (preferably having a repeating sequence of'GUU') stimulates DC and macrophages to activate NF-λB, secrete cytokines, and MYD88 in innate immunity. And induces an inflammatory response through TRAF6. TLR7 and TLR8 exist on the membrane of endosomes and recognize ssRNA. TLR7 is a dual receptor for ssRNA including guanosine and uridine. The first site of binding is used for binding small ligands and the second site is used for binding ssRNA. That is, the first site detects guanosine preferentially, and the second site specifically binds to the uridine portion of the ssRNA.

C) Extreme length heterogeneity and toxicity of poly (I:C) family dsRNA

There is a need to more accurately define the dsRNA length that activates DC in a TLR3-dependent manner in vitro and in vitro. Although several ribonucleic acid derivatives have been suggested as vaccine adjuvant candidates (Patent Document 1-18), identity and homogeneity cannot be guaranteed. Because of this, the activity is unstable and there is no safety, so it is used for limited (Patent Documents 1-14). The manufacturing process of the poly(I:C)-based material, which is a dsRNA analogue, and its problems are as follows.

Poly(I:C) is a synthetic dsRNA analog consisting of a complementary bond of an inosine homopolymer (inosine homopolymer) strand Poly(I) and a cytidyl homopolymer strand Poly(C). Since each strand is enzymatically synthesized by adding a new base to a base located at the end using polynucleotide phosphorylase (PNPase), the length of the resulting homopolymer is very variable. In addition, when annealing is performed by matching a long homopolymer (e.g., an average of 400 base Poly(C)) to a long homopolymer sequence (e.g., an average of 400 base Poly(I)) and annealing, complementary binding at a random position is achieved. Even after bonding, the strands slide from side to side (slippage), resulting in a portion remaining as a single strand having different lengths at each end of the chain of the two strands, and the complementary strands recombine to this site to expand the chain. ) Is made, and it is made in extremely various lengths over tens to hundreds of kbps. In addition, after chain expansion, phosphate diester bonds do not occur between adjacent strands, so that an unspecified number of nicks exist at unspecified positions. Therefore, when RNase T1, which recognizes the location of the nick and cuts it, is treated, the length is randomly reduced (see FIG. 8C).

Poly(I:C)-L-lysine-methylcellulose (Poly(I:C)-LC) is one of the poly(I:C) derivatives and is less sensitive to degradation by RNase in serum, but the manufacturing method is poly( Since it is essentially the same as I:C), it has the same problem that the length is not constant (Patent Documents 1-14). Poly(I:C)-LC induces IFN-γ secretion and increases CTL response and antigen specific antibodies. Poly(I:C)-LC has somewhat moderate toxicity, but still exhibits side effects in clinical trials. PIKA, which is another Poly(I:C) derivative, also has a form of Poly(I:C) whose length is not constant (Non-Patent Document 3 and Non-Patent Document 4).

Poly(I:C12U) is another derivative of Poly(I:C), and uridine (U) is added for every 12 cytidine (C), which is complementary because it has a partly inconsistent site with the Poly(I) strand. It is sensitive to degradation by RNase T1 because there is no natural binding and there are multiple nicks on a single strand. Therefore, it has a shorter half-life than Poly(I:C) and has a higher selectivity for TRIF other than Myd88, resulting in improved side effects of Poly(I:C). However, it still has not been able to overcome the fact that the length is not constant and has extreme diversity. Poly(I:C12U) was approved for use in humans in Canada and Argentina. In 2000, it was approved by the European Union as an orphan drug for the treatment of chronic fatigue syndrome (CFS), but it was not approved by the US FDA. In addition, there is Poly(I:C) with improved length control, but the fundamental length diversity problem still remains. A method of making Poly(I:C) (100~400) by complementary bonding a short chain (e.g., an average of 108 base Poly(I) 100) to a long chain (e.g., an average of 344 base Poly(C) 400) Was devised. In this way, dsRNA with an average length close to 400 bp was prepared, but even if the average length is close to 400 bp, the entire length range is very wide (100-2000 base), and the length diversity problem of Poly(I:C) is Still not overcome. That is, the short homopolymer sequence (eg, Poly(I) 100) binds complementarily to the long homopolymer (eg, Poly(C) 400), and chain slippage and chain expansion could not be prevented from occurring. In summary, the poly (I:C) family of dsRNA analogues does not have a certain length, and because there are an unspecified number of nicks at an unspecified position inside the dsRNA, the stability against RNase T1 is very low (patent Document 3, Non-Patent Document 2, Non-Patent Document 5).

D) Role of OX40 and PD-1 molecules in the cancer microenvironment

Both the innate immune response and the adaptive immune response are involved in tumor growth and control (Non-Patent Document 7). Various methods for enhancing the activity of T cells in a tumor microenvironment in which immune function is suppressed, that is, a strategy using an antagonist for a T cell inhibitory receptor or a stimulator for a T cell activating receptor has been proposed (Non-Patent Document 8).

OX40, OX40R, CD134, TNFRSF4, TXGP1L, ACT35 and ACT-4 are members of the tumor necrosis factor receptor superfamily OX40, which are not expressed on naive T cells, but are induced to increase after antigen binds to T cell receptor (TCR) ( Non-Patent Document 9). In addition, OX40 is a major active receptor that is expressed in active B cells, active dendritic cells, active eosinophils, natural killer T (NKT) cells, and NK cells to regulate the function of these cells (Non-Patent Document 9). OX40L, an OX40 ligand that binds to OX40, is primarily expressed in antigen presenting cells. OX40 is highly expressed on activated CD4+ T cells, activated CD8+ T cells, memory T cells and regulatory T cells. The signaling process of OX40 provides a co-stimulatory signal to CD4 and CD8 T cells, enhancing cell proliferation, survival, effector function and migration. In addition, the OX40 signal can induce memory T cell development and function enhancement to overcome the immune suppression environment inside and outside cancer tissues. According to the results of in vivo studies, antigen-specific T cells are preferentially expanded through stimulation of OX40L-OX40.

When antigen presenting cells such as dendritic cells mature, co-stimulatory factors including OX40L as well as the B7 family (e.g., CD80 and CD86) increase, which is not only substantial memory cell differentiation, but also the kinetics and scale of T cell immune responses. Help you decide. In addition, B cells, vascular endothelial cells, mast cells, and in some cases activated T cells also induce OX40L (Non-Patent Document 10).

The OX40:OX40L binding linkage (interaction) forms a trivalent receptor (trimer) to induce higher-order clustering and subsequent signaling (Non-Patent Document 11). It has been demonstrated that mouse OX40-stimulated rat-derived antibody (anti-mOX40 rat agonist Ab) can induce tumor rejection in a mouse model (Non-Patent Document 12, Non-Patent Document 13), and human OX40-stimulated mouse-derived antibody (anti-hOX40mouse agonist Ab) has also been shown to enhance cancer immune function in cancer patients (Non-Patent Document 14). In addition, the OX40:OX40L interaction is associated with graft versus host disease (e.g. organ transplant rejection), asthma/atopic, encephalomyelitis, rheumatoid arthritis, colitis/inflammatory bowel disease, diabetes and atherosclerosis, lupus, It is also associated with the immune response in inflammatory and autoimmune diseases and disorders (Non-Patent Document 15).

PD-L1 is overexpressed in many cancers and is often associated with poor prognosis (Non-Patent Document 16, Non-Patent Document 17). Interestingly, the majority of tumor-infiltrating T lymphocytes, in contrast to normal tissue and peripheral blood T lymphocytes, primarily express PD-1, which indicates that this upregulation of PD-1 in tumor-reactive T cells is responsible for the tumor immune response. It shows that it can contribute to damage (Non-Patent Document 18). In addition, it is known that signal transduction due to the interaction between PD-1 on tumor infiltrating T cells and PD-L1 on tumor cells weakens T cells (Non-Patent Document 19, Non-Patent Document 20). Thus, inhibition of the PD-1:PD-L1 (or PD-L2) interaction can help to treat cancer or acute and chronic infections by preventing weakening of CD8+ T cells. However, for optimal treatment, in addition to blocking the interaction between the PD-1 receptor and the ligand, it is necessary to develop additional immune enhancing therapy.

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1) Overcoming the extreme length heterogeneity of ribonucleic acid-based adjuvants

The pharmaceutical composition should have consistent reproducibility and homogeneity. The main reasons for limiting the application of the dsRNA analog, Poly(I:C) and its derivatives in clinical use, are due to the extreme length heterogeneity, lack of reproducibility in production, unpredictable pharmacokinetics, and inconsistent efficacy (Non-Patent Literature 3, non-patent document 6). In addition to these characteristics, the main reason for early termination of the application of Poly(I:C) in most clinical trials is due to severe toxicity that causes symptoms such as joint pain, fever, erythema and endotoxin-like shock (Patent Documents 15 and 17). Therefore, there is an essential need for an alternative form of dsRNA having a more clearly specified length and composition, while having consistent efficacy, reproducible production potential and less toxicity. So far, dsRNA analogues include Poly(I:C), Poly(I:C)-LC, and Poly(I:C12U), but all of them are made by complementary bonds between two-stranded homopolymers (homopolymers). The length of ribonucleic acid varies extremely. That is, it is essentially impossible to prepare a dsRNA analog of a certain length by a homopolymer annealing method prepared based on PNPase.

The present invention is to provide a novel hsRNA capable of solving the problems of poly(I:C) in heterogeneity and toxicity of extreme length and mass production. If necessary, the hsRNA of the present invention may be treated with RNase T1 to remove the ssRNA region and selectively extract only the dsRNA region. In addition, the hsRNA of the present invention has the advantage of specifying the exact overall length, the exact length and position of the ssRNA, and the exact length and position of the dsRNA, and overcomes the problems of the existing Poly(I:C) and its derivatives with these characteristics. It can be done.

2) Reduce the amount of antigen required in vaccines

The present invention is to provide hsRNA and dsRNA capable of inducing a stronger protective immunity while reducing the amount of antigen required in a vaccine. Influenza vaccination is one of the most effective defenses against influenza virus, but the currently used seasonal trivalent or tetravalent vaccine requires at least 15 µg per vaccine antigen for each predicted virus for effective prophylactic immunity. . In this case, a large amount of antigen is required, and there is a potential possibility that the production is delayed and the vaccine is insufficient, so there is a need to reduce the amount of antigen required for the vaccine. Despite the presence of various adjuvants, no adjuvants are included in the influenza vaccine currently approved for people under the age of 65 in the United States. The FLUAD flu vaccine, an MF59 (squalene oil-in-water emulsion) adjuvant vaccine approved in the U.S. in 2015, was approved for people over the age of 65, and there are still examples of vaccines that have been approved to reduce the amount of antigen required and improve efficacy. none.

3) Providing a cancer vaccine composition with enhanced anticancer efficacy

The present invention provides a cancer vaccine composition comprising a complex of hsRNA and OX40 antibody and/or PD-1 antibody that consistently and strongly activates dendritic cells in vivo, so as to prevent or treat not only primary cancer, but also distant metastatic cancer. It is intended to provide a cancer vaccine composition that can be used.

The hsRNA of the present invention promotes the death of cancer cells, promotes the outflow of cancer-associated antigens present in the cancer cells, and activates innate immune cells including dendritic cells and natural killer cells. The OX40 agonist antibody functionally plays a role of OX40L, a ligand of OX40, specifically binds to human OX40 present on the surface of immune T cells, and then increases OX40 activity to dissolve cancer cells. PD-1 antagonist antibodies activate T tumor-specific immune cells by binding to PD-1, an inhibitory receptor on the surface of immune cells.

1) The present invention provides ribonucleic acid having a high degree of homogeneity and stability through bidirectional synthesis.

The present invention relates to hsRNA and dsRNA of a specific length having perfect homogeneity and high stability at room temperature in order to overcome the extreme diversity of lengths and toxicity resulting from Poly(I:C).

The method of preparing hsRNA of the present invention is as follows. A DNA fragment capable of directing transcription of the target RNA to be prepared is inserted into an arbitrary plasmid vector. The DNA fragment is amplified by PCR, purified, and used as a template for transcription into RNA in vitro. At this time, a T7 promoter sequence is added to both 5'ends of the DNA fragment. If necessary, a fragment having a TLR7 ligand sequence or a fragment having an arbitrary length and an arbitrary sequence may be added to the 5'end of the T7 promoter sequence. When the same T7 polymerase is used as a template for in vitro transcription, ssRNAs at the top and bottom with perfect complementarity are produced at almost the same molar ratio, so even if they are left at room temperature, almost no ssRNA remains and almost all of them are complementary. By combining, highly pure hsRNA can be prepared. The hsRNA or dsRNA does not encode a protein, but may optionally encode it. That is, ribonucleic acid having a specific length can be prepared through ribonucleic acid polymerase-mediated transcription in vitro using a DNA template with a promoter attached to both 5'ends of DNA composed of a heteropolymer sequence other than the same repeat sequence. At this time, an additional sequence can be added to the 5'end of the T7 promoter primer site to make a longer and more stable structure. When complementary binding is performed after bidirectional transcription, a dsRNA having a specific sequence and length that is 100% complementary to each other is located in a specific intermediate region, and an hsRNA in which an ssRNA having a specific sequence and length is also located at the 3'end is generated. For example, the ssRNA may be a random sequence or may include a GUU repeat sequence, but is not limited thereto, and ssRNA may be arbitrarily removed by treatment with RNase T1.

Until now, dsRNAs having minimum and optimal lengths for activating DC and immune systems in vivo have not been identified, because it is difficult to prepare dsRNAs that differ by one base pair length. When each ribonucleic acid having a variable length prepared by the production method of the present invention is used, it is possible to find the length or structure of ribonucleic acid that exhibits optimal immune activation in vitro and in vivo. From this, the optimal dsRNA length and sequence required for TLR3 activation can be identified.

2) The present invention provides a vaccine composition for preventing and treating infections comprising ribonucleic acid capable of reducing the required amount of antigen used.

The present invention provides a vaccine composition for preventing or treating viral or bacterial infections comprising the hsRNA or dsRNA as an immunogen and an adjuvant. In addition, the present invention provides a pharmaceutical composition for preventing or treating viral or bacterial infections comprising the hsRNA or dsRNA.

Ribonucleic acid, which is strongly negatively charged, can effectively accept many large antigens, making nanocomposites comparable to virus-like particles (VLPs). The nanocomposite encapsulating the antigen in an appropriate formulation is captured by dendritic cells or macrophages, effectively inducing the presentation of the antigen in B cells and T cells. At the same time, cytokines are secreted, resulting in activation of an efficient adaptive immune response. Infection prevention and treatment vaccine composition of the present invention is a group consisting of pathogen proteins, recombinant proteins, subunits, split protein antigens, glycoproteins, peptides, polysaccharides, lipopolysaccharides, polynucleotides, and combinations thereof. It may be selected from, but is not limited thereto.

3) It provides a cancer vaccine composition comprising the ribonucleic acid adjuvant of the present invention and a pharmaceutical composition for preventing or treating cancer comprising ribonucleic acid and an antibody.

The present invention provides a vaccine composition for preventing or treating cancer comprising the hsRNA or dsRNA as an immunogen and an adjuvant. In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the hsRNA or dsRNA, and a pharmaceutical composition for preventing or treating cancer further comprising an OX40 antibody or a PD-1 antibody.

The hsRNA of the present invention overcomes the extreme diversity of length and composition heterogeneity of Poly(I:C), provides the advantage of being able to specify the exact overall length and sequence, and is completely complementary when the ssRNA site is removed from the hsRNA. It can be prepared with dsRNA that induces an improved immune response, which has a homogeneous sequence and exhibits the correct length.

In addition, when a complex containing a specific antigen is prepared in the hsRNA of the present invention, the antigen complex is delivered into the antigen-presenting cell, so that the antigen can be efficiently presented to T cells and B cells. Furthermore, the hsRNA or dsRNA of the present invention may reduce the amount of antigen required, and may induce a protective immune effect against viral or bacterial infection and cancer.

As a result, the ribonucleic acid adjuvant of the present invention (1) strengthens the adaptive immunity to the antigen, (2) reduces the amount of antigen required for the vaccine, and (3) improves the antigen-specific Th1-polarized cross-protection reaction. And (4) when used alone without an antigen, it exhibits an activity that enhances innate immunity.

In addition, the hsRNA of the present invention alone or a cancer vaccine composition comprising hsRNA and an OX40 antibody or PD-1 antibody exhibits anticancer efficacy against both primary and distant cancers. In addition, the cancer vaccine composition of the present invention converts a refractory cancer that does not respond to a reactive cancer when treated with an immune checkpoint inhibitor such as a PD-1 antibody alone, thereby exhibiting a more excellent anticancer effect.

1 shows a schematic diagram and results of verification of in vivo activity for a ribonucleic acid-based substance that activates DC (Example 1).
Figure 1a shows that each of the three C57BL/6 mice was injected with NA by the intraperitoneal (ip) route, and after 24 hours, spleen cells were isolated, and Lineage (CD3, Thy1.1, B220, Gr1, CD49b, TER -119) This is a picture that defines the negative CD11c+ cell group, that is, Lin-CD11c+, as the DC group cDC. For DC separation, gating on mononuclear cells based on forward and lateral scattering was performed, followed by additional gating on Lineage-CD11c+ cells, defined as cDCs, to CD8α+/CD11c+ and CD8α-/CD11c+. Divided further by cDCs.
Figure 1b is a result of measuring the mean fluorescence intensity (MFI) for the number of positive cells of the co-stimulatory factor (CD40, CD86) and MHCII among CD11c+ DC cells. NA increases the DC markers of CD40, CD86, and MHC-II. Show. NA shows significantly stronger DC activation compared to 100 μg of Poly (I:C).
1C is a graph showing that NA strongly induces the secretion of IL-6, IL-12, and TNF-α compared to Poly (I:C).
1D shows that NA induces Th1 cell marker T-bet and Th1 cell cytokine interferon-gamma (IFN-γ) mRNA, not Th2 cytokine (IL-4) and Th17 cytokine (IL-17A). This is the qRT-PCR result showing. Each data is the average of three independent analyzes.
Figure 2 is a result showing the activity of DC in vivo according to the length of the hsRNA of the present invention (Example 2).
Figure 2a is a result of in vitro synthesis of hsRNA series 1 prepared by artificial sequence (Backbone Group 1), respectively, 140 bases long hsRNA (NA1001), 190 bases length (NA1501), 240 bases length (NA 2001), 340 bases length (NA3001), 440 bases length (NA4001), 540 bases length (NA5001), 640 bases length (NA6001), and 840 bases length (NA8001).
Figure 2b is a result of in vitro synthesis of hsRNA series 2 derived from tomato sequence (Backbone Group 2), respectively, 140 bases length (NA1002), 190 bases length (NA1502), 240 bases length (NA2002), 340 bases length (NA3002) ), 440 bases length (NA4002), 540 bases length (NA5002), 640 bases length (NA6002), 740 bases length (NA7002) and 840 bases length (NA8002).
FIG. 2C shows the MFI of CD40 and CD86 surface expressions that appear when 5 μg of each of the hsRNAs shown in FIG. 2A is injected. Each data is an average of three independent analyzes.
FIG. 2D shows the MFI of CD40 and CD86 surface expressions that appear when 5 μg of each of the hsRNAs shown in FIG. 2B is injected. Each data is an average of three independent analyzes.
Figure 3 shows the innate immune activity index and immune response results of the hsRNA of the present invention (Example 3).
Figure 3a is a picture of the preparation of the hsRNA of the present invention (R1 to R11, VP10), and then electrophoresis on a 1% agarose gel to determine the length.
3B is a value showing the corrected IFN-β promoter-Luciferase activity value after 24 hours elapsed in HEK 293 of the additional sequence.
Figure 3c is a mouse intramuscular injection using hsRNA complexed ovalbumin (OVA) as a model antigen, boosting after 2 weeks, and Th2-mediated Anti-OVA IgG1 and Th1 mediated in serum after 1 week again It is a graph showing the level of Anti-OVA IgG2a. The NAR7 (R7) of the present invention enhanced Th1 (IgG2a) and Th2 (IgG1) reactions than R3, R5, and R10.
4 shows the physicochemical properties of NA (NVT), one of the hsRNAs of the present invention (Example 4).
4A shows that the NA length is uniformly constant after purification as a result of measurement by HPLC. Figure 4a shows the homogeneity of the length of the dsRNA treated with hsRNA and RNase T1, and also shows that the dsRNA site is well preserved.
4B is a result showing that stability is maintained even when NA is stored at 45° C. for 150 days or longer.
4C is an experimental result showing that NA has a short half-life of less than 10 minutes in 100% calf serum (Serum).
4D shows that the antigen complexed with NA has a long shelf life.
Figure 5 shows the aspect of the innate immune activity index after subcutaneous (sc) injection of the hsRNA and dsRNA of the present invention (Example 5).
5A is a comparison of the activity between hsRNA (NVT 4-T1) not treated with RNase T1 in NVT 4 (same as NVT IV, VP20) and dsRNA treated (NVT 4 + T1).
5B is a result of activating DC (CD86 induction) of both hsRNA and dsRNA after 24 hours of subcutaneous injection.
Figure 5c is a comparison of the number of immune cells in the drain lymph node (dLN) after 24 hours and 48 hours, and after 48 hours, dsRNA showed higher immunological activity than hsRNA.
5D is a result of dsRNA inducing higher TNF-α, IFN-β, and IL-6 than hsRNA, suggesting that while dsRNA induces innate immunity more strongly, hsRNA is a material that is advantageous for safety.
6 shows that the Ova vaccine complexed with NA (NVT) induces a stronger Th1 polarized immune response compared to other adjuvants (Example 6).
6A is a whole in which an adjuvant-complexed vaccine in BalB/c mice was primed and boosted intramuscularly over days 0 and 14, respectively, and peripheral blood and splenocytes were extracted on the 21st day, followed by antibody and flow cytometric analysis. Indicate the process.
6b and 6c show that NA (NVT) activates Th2 immune response (IgG1) with other adjuvants, but the Th1 response (IgG2c) activates higher than other adjuvants and secretes IFN-γ from the spleen. The number of CD8 T cells and CD4 T cells also showed the greatest increase.
Fig. 7 shows an experiment for induction of innate immunity and induction of adaptive immunity by NA (NVT) and NVT+SE (squalene emulsion) (NVT II) selected from the hsRNA of the present invention (Example 7).
7A is a method for verifying activity through intramuscular injection of NVT and NVT II.
7B shows that PBS (physiological saline), NVT and NVT II induce DC activity in the inguinal lymph nodes (iLN) 24 hours after injection.
7C shows the result that both NVT and NVT II induce neutralizing antibodies in serum, but NVT II induces more than twice as strong as NVT.
8 is a diagram showing that the structure specificity and length homogeneity of the hsRNA of the present invention is superior to that of the competing drug Poly(I:C) (Example 8).
8A is a schematic diagram of hsRNA according to the present invention. A dsRNA site having complete complementarity with an arbitrary length and an arbitrary sequence is located in the center, and ssRNA that can act as a potential ligand of any length and TLR7/8 is bound to both 3'ends of the dsRNA. The dsRNA may be an artificial or natural sequence, but it does not encode a protein. When dsRNA is treated with RNase III, it is cut into dsRNA fragments of 20 to 25 bp on average. If RNAse T1, which does not act on dsRNA, but only acts on ssRNA, is treated, the ssRNA site is completely degraded and disappeared. In the present invention, dsRNA of a length (>100bp) that is difficult to synthesize effectively by enzymatic synthesis is mass-synthesized in vitro using T7 RNA polymerase, and two types of ssRNA derived from both strands of DNA are synthesized in a similar molar ratio. It offers the advantage of being able to do it.
The present invention has a structural feature in which the 5'terminal P (phosphate group) is removed in order to reduce the intracellular reaction due to the phosphate group. In addition, the ssRNA site is not a simple overhang, but is characterized by consisting of a specific length and sequence (a sequence including a plurality of GU, which is a TLR7 ligand sequence) that can act as a specific TLR7/8 ligand. In addition, the ssRNA site may have a function of preventing the end of dsRNA from being degraded, and due to the nature of the manufacturing process, the single strands at the top and bottom are produced at the same molar ratio. And has the characteristic that unbound single strands do not remain.
Figure 8b shows the result of digestion of hsRNA with RNase A, RNase III and RNAse III + RNAse T1. If RNase A, which can degrade both dsRNA and ssRNA, is treated, all RNA is degraded and no band appears.If RNase III, which cleaves dsRNA to a length of 20 to 25 bp, is treated, the band of a fragment having a length of about 25 bp is the most. A lot appeared. When RNase III treatment, the ssRNA fragments (51, 58 base) at both ends that remain undigested are overlapped around 25 bp, so the band is slightly spread out. When RNase III and RNAse T1 are treated at the same time, the ssRNA fragments (51, 58base) are completely degraded and disappeared, and only the 25 bp band clearly appears.
FIG. 8C is a diagram showing that NA (533 base, dsRNA site 424 bp) was separated from an agarose gel before and after treatment with DNase I and RNase T1, and a single result with a specific length appears. After preparation in vitro, when DNA fragments remaining in the reaction product are removed by treatment with DNase I, only hsRNA remains. When the ssRNA site present in hsRNA is removed with RNase T1, only the dsRNA site remains. If there is more than one nick inside the dsRNA site, RNase T1 will cut the site and appear as two or more dsRNA fragments. Otherwise, if only one fragment of a single size is visible, there is no nick in the dsRNA site and ssRNA is It proves that it is present only at the 3'ends of the hsRNA.
8D shows that Poly(I:C), a competing drug of the other invention, has heterogeneity due to extreme length diversity, and this is a problem that cannot be overcome even after fractionation is completed after production as it occurs from the manufacturing stage (non Patent document 2).
8E shows the basic structure of Poly(I:C), a competing drug of another invention, and may be displayed only as an average length, not a specific length. For example, dsRNA is produced by synthesizing an average of ~ 389 base PolyI and an average of 344 base PolyC complementary thereto and making them complementary to each other. However, since chain slippage occurs in which complementary bonding occurs at unpredictable non-specific regions, nicks having unpredictable gaps eventually occur in an unpredictable number. In addition to these nicks, when complementary bonds are additionally generated at both ends, that is, chain repeats occur, the length is extended to hundreds of kb or more.
The hsRNA of the present invention can be designed to form an overhang of an ssRNA having a predetermined length or sequence only at the 3'end, but in the existing Poly(I:C), the ssRNA site is at both the 5'end and the 3'end. It formed sporadically, and this problem could not be controlled (Non-Patent Document 2), and the above problem still remained in the improved manufacturing method (Non-Patent Document 5). In addition, since PNPase is used in the manufacturing process, a phosphoric acid group (P) is always attached to the 5'end, so the yield is low (Non-Patent Document 2, Non-Patent Document 5).
Fig. 9 shows the immune response after administration of the inactivated influenza whole vaccine (iPR8) complexed to the NA (NVT) of the present invention intranasally (in) (Example 9).
Figure 9a is a schematic diagram of the test method.
Figure 9b shows a pattern in which the total number of cells, the number of alveolar macrophages, the number of neutrophils, and the number of natural killer cells in the alveolar lavage solution increase up to 24 hours after priming, and then decrease from the point when 24 hours have elapsed when NA and iPR8+NA are administered. Is showing.
9C shows a marked increase in IL-6, IL-12, and TNF-α after 24 hours.
9D shows an increase in germinal center B (GCB) cells and follicular helper T cells (T _FH ) in mediastinal lymph nodes (mLN) on day 7.
9E shows that serum Anti-iPR8 IgG increases after 21 days only when iPR8+NVT is administered.
9F shows an increase in IgA in nasal lavage on day 21.
Figure 10 relates to the potentiation and antigen reduction effect of influenza vaccines (4IIV, QIV, Vaxigrip, 2018/2019 seasonal tetravalent influenza vaccine) by NA (NVT), an hsRNA of the present invention (Example 10).
Figure 10a is a schematic diagram of the test method.
Figure 10b shows that compared to the case of using Vaxigrip alone, when using a mixture of NA (NVT) or NA+SE (NVT II), the amount of antigen required to induce IgG is reduced to about 1/5 and less than about 1/25, respectively. And neutralizing antibodies are also increased.
Figure 10c shows that up to 17 weeks after vaccination, the amount of total IgG against Vaxigrip is maintained high.
Figure 10d shows that the HAI titer, which is an indicator of protective immunity against IAV H1N1 and IBV infection, is maintained as high as 40 or higher until the 17th week in proportion to the amount of neutralizing antibodies.
11 shows the induction of a neutralizing antibody IgG enhancement against the meningitis ovarian bacteria vaccine antigen complexed with NVT II of the present invention (Example 11).
Figure 11a is a schematic diagram of the test method.
11B and 11C show the increase of antibodies against the conjugated antigen Menactra vaccine as well as the N. Meningitidis antigen (type A is used in this experiment) when 14 and 21 days have elapsed after priming. Show.
12 shows the antigenic restoration efficacy provided by the NVT II of the present invention (Example 12).
Figure 12a is a schematic diagram of the test method.
12B shows that Vaxigrip alone completely lost IgG-induced immunogenicity, but when NVT II was incorporated into Vaxigrip, which lost immunogenicity, antigenicity was restored.
Figure 12c shows that in the vaccine incorporating NVT II, the HAI titer, an indicator of protective immunity against IAV H1N1 and IBV, is increased to 40 or more at 18 weeks.
13 shows the effect of the ribonucleic acid alone immunity enhancing effect of the present invention on melanoma regression (Example 13).
13A is a test for observing a mass while injecting NA (NVT) intratumorally (it) or intramuscularly on days 6, 8, and 10 after subcutaneous transplantation of B16F10-OVA melanoma cells into 7-week-old female C57BL/6 mice. Indicate the way.
13B shows the state of tumor regression caused by the hsRNA of the present invention.
13C shows the actual survival rate.
14 shows the effect of the ribonucleic acid alone immunity enhancing effect of the present invention on the regression of colon cancer and lung cancer (Example 14).
15 shows the effect of the ribonucleic acid alone immunity enhancing effect of the present invention on triple negative breast cancer (TNBC) regression and metastasis inhibition (Example 15).
Figure 15a is a schematic diagram of the test method. 7-week-old female C57BL/6 mice were transplanted with a 4T1 triple-negative breast cancer (TNBC) cell line subcutaneously with mammary adipose tissue, followed by intratumoral (it) NA (NVT) ribolysis over a total of 9 times once every 2 days from the 8th day. The nucleic acid was administered alone and the mass was measured.
15B shows the results of about 60% regression of primary breast cancer by NA (NVT).
Fig. 15c shows a remarkably reduced metastasis of primary breast cancer to the lung in NA (NVT)-administered mice.
Figure 16 shows the effect of inhibiting melanoma cell metastasis from the spleen to the liver when the hsRNA of the present invention alone or the hsRNA complexed cancer antigen vaccine is administered intraperitoneally (Example 14).
Figure 16a is a schematic diagram of the test method.
16B is a measurement of the size of the spleen and liver on the 18th day.
16C shows the average of spleen and liver weights on day 18.
16D shows the number of tumor nodules that have metastasized to the liver. In the NA alone or NA+ OVA treatment group, metastasis to the liver was inhibited.
17 shows the inhibitory effect of primary cancer and distant cancer by the cancer vaccine composition comprising the hsRNA and OX40 antibody of the present invention (Example 17).
Figure 17a is a schematic diagram of the test method.
Figure 17b shows that the PD-1 antibody and IR cell + NVT + OX40 Ab composition significantly inhibited the growth of primary cancer.
Figure 17c shows that only NVT + OX40 Ab without IR cells sufficiently inhibited primary cancer.
17D shows that only NVT+OX40 Ab inhibits the growth of B16F10/Ova remote cancer and EG7/Ova remote cancer.
18 shows the results of comparing the melanoma inhibitory effect when the PD-1 antibody or the OX40 antibody was mixed with the hsRNA of the present invention (Example 18).
Figure 18a is a schematic diagram of the test method.
FIG. 18B shows the results of the NA+PD-1 composition generally similar to that of the NA+OX40 Ab composition in inhibiting primary cancer tumors.
Fig. 18c shows that the two compositions have similar inhibitory effects on tumors of distant cancer.

Hereinafter, embodiments and examples of the present disclosure will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art may easily implement the present disclosure. However, the present application may be implemented in various forms and is not limited to the embodiments and examples described herein.

In the entire specification of the present application, when a certain part "includes" a certain constituent element, it means that other constituent elements may be further included rather than excluding other constituent elements unless otherwise specified.

The present invention relates to a mixed structure ribonucleic acid (hsRNA) comprising a double-stranded ribonucleic acid (dsRNA) and a single-stranded ribonucleic acid (ssRNA) acting as a TLR3 ligand, wherein the double-stranded ribonucleic acid (dsRNA) has complementarity, and the Single-stranded ribonucleic acid (ssRNA) is located at both 3'-ends of the double-stranded ribonucleic acid (dsRNA), and characterized in that it has an arbitrary length and sequence, it provides a mixed structure ribonucleic acid (hsRNA).

The single-stranded ribonucleic acid (ssRNA) may act as a TLR7 ligand or a TLR8 ligand, and may have a G/U sequence that acts as a TLR7 ligand, preferably a GUU repeat, and a length of 10 bases or more, preferably 15 to It may have a length of 80 base, more preferably a length of 17 to 75 base.

The mixed structure ribonucleic acid (hsRNA) of the present invention can be uniformly prepared to a desired length and has high stability.

Specifically, the mixed structure of the ribonucleic acid of the present invention does not cause chain slippage or chain expansion during manufacture, unlike conventional ribonucleic acid, so it can be uniformly manufactured with a certain length. "Chain slip" means that after a long homopolymer ribonucleic acid strand is complementarily bound at an arbitrary position, the binding is not fixed due to the same sequence around the binding site and the binding site moves back and forth, and "chain extension" is complementary It means that the ends of the two strands bonded together do not exactly match, so that the other strand is complementarily bonded to the protruding site as a single strand, and the length of the entire length is extended.

In addition, the present invention provides a method for preparing a mixed structure ribonucleic acid comprising the following steps:

1) inserting a template DNA fragment with a promoter sequence added at both 5'ends into an arbitrary plasmid vector;

2) transcribing RNA from the template DNA in both directions using an RNA polymerase that recognizes a promoter sequence;

3) obtaining two types of ssRNAs that are complementary to each other and have the same length derived from both strands of the template DNA; And

4) Complementary binding of the ssRNA.

At this time, an additional sequence may be added to the 5'end of the promoter sequence, and the step 1) may be used after amplifying the template DNA fragment by PCR and then purifying it. In addition, the promoter of step 2) may be a T7 promoter, and the RNA polymerase may be a T7 polymerase.

In addition, the present invention provides a method for preparing a mixed structure ribonucleic acid comprising the following steps:

1) inserting a template DNA fragment to which a T7 promoter sequence is added to both 5'ends into an arbitrary plasmid vector;

2) adding an additional sequence to the 5'end of the promoter sequence;

3) bidirectional transcription of RNA from template DNA using T7 polymerase;

4) obtaining two types of ssRNAs that are complementary to each other and have the same length derived from both strands of the template DNA; And

5) Complementary binding of the ssRNA.

The hsRNA of the present invention may act as a TLR3 ligand, may have a base length of 140 to 1682 base, preferably 200 to 1500 base, more preferably 300 to 1000 base, most preferably 400 to 900 base. It can be, but is not limited thereto.

The effect of hsRNA tends to show a higher level of dendritic cell activation as the length of the base increases. However, as the length of the base increases, the manufacturing cost increases and mass production may be difficult.

Likewise, the base length of the dsRNA portion of the present invention may be 106 to 1648 bp, preferably 200 to 1500 bp, more preferably 300 to 1000 bp, and most preferably 400 to 900 bp, but is not limited thereto. Does not.

The base length of the hsRNA or dsRNA may be increased or decreased within the range of 20%.

The present invention provides a dsRNA characterized in that it contains any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 63 to 93 and acts as a TLR3 ligand. The base length of the dsRNA may be 106, 156, 206, 306, 319, 397, 406, 424,466, 506, 588, 606, 664, 706, 733, 806, 822, 885, 1032, 1153 or 1648 bp.

The names and basic information of ribonucleic acid used in the present invention are shown in Tables 1 and 2 below. As shown in Table 2, the present invention has the same length and sequence, but has been described by several names. NA, NVT (abbreviation for Next Adjuvant), VP1, VP10, and VP11 are examples. However, VP11 is a dsRNA treated with VP10 hsRNA with RNase T1. In addition, NVT IV (NVT 4) and VP20 refer to the same ribonucleic acid.

The hsRNA or dsRNA may be composed of the nucleotide sequence of the sequence number shown in Table 1 below. Table 1 below shows the correspondence between each hsRNA or dsRNA and sequence described in the examples of the present invention.

designation Sequence number constituting hsRNA Sequence number of dsRNA part NA1001 Binding of SEQ ID NO: 1 and SEQ ID NO: 32 SEQ ID NO: 63 NA1501 Binding of SEQ ID NO: 2 and SEQ ID NO: 33 SEQ ID NO: 64 NA2001 Binding of SEQ ID NO: 3 and SEQ ID NO: 34 SEQ ID NO: 65 NA3001 Binding of SEQ ID NO: 4 and SEQ ID NO: 35 SEQ ID NO: 66 NA4001 Binding of SEQ ID NO: 5 and SEQ ID NO: 36 SEQ ID NO: 67 NA5001 Binding of SEQ ID NO: 6 and SEQ ID NO: 37 SEQ ID NO: 68 NA6001 Conjugation of SEQ ID NO: 7 and SEQ ID NO: 38 SEQ ID NO: 69 NA7001 Binding of SEQ ID NO: 8 and SEQ ID NO: 39 SEQ ID NO: 70 NA8001 Conjugation of SEQ ID NO: 9 and SEQ ID NO: 40 SEQ ID NO: 71 NA1002 Binding of SEQ ID NO: 10 and SEQ ID NO: 41 SEQ ID NO: 72 NA1502 Binding of SEQ ID NO: 11 and SEQ ID NO: 42 SEQ ID NO: 73 NA2002 Conjugation of SEQ ID NO: 12 and SEQ ID NO: 43 SEQ ID NO: 74 NA3002 Conjugation of SEQ ID NO: 13 and SEQ ID NO: 44 SEQ ID NO: 75 NA4002 Conjugation of SEQ ID NO: 14 and SEQ ID NO: 45 SEQ ID NO: 76 NA5002 Combination of SEQ ID NO: 15 and SEQ ID NO: 46 SEQ ID NO: 77 NA6002 Conjugation of SEQ ID NO: 16 and SEQ ID NO: 47 SEQ ID NO: 78 NA7002 Conjugation of SEQ ID NO: 17 and SEQ ID NO: 48 SEQ ID NO: 79 NA8002 Conjugation of SEQ ID NO: 18 and SEQ ID NO: 49 SEQ ID NO: 80 R1 Conjugation of SEQ ID NO: 19 and SEQ ID NO: 50 SEQ ID NO: 81 R2 Conjugation of SEQ ID NO: 20 and SEQ ID NO: 51 SEQ ID NO: 82 R3 Combination of SEQ ID NO: 21 and SEQ ID NO: 52 SEQ ID NO: 83 R4 Conjugation of SEQ ID NO: 22 and SEQ ID NO: 53 SEQ ID NO: 84 R5 Conjugation of SEQ ID NO: 23 and SEQ ID NO: 54 SEQ ID NO: 85 R6 Conjugation of SEQ ID NO: 24 and SEQ ID NO: 55 SEQ ID NO: 86 R7 Conjugation of SEQ ID NO: 25 and SEQ ID NO: 56 SEQ ID NO: 87 R8 Combination of SEQ ID NO: 26 and SEQ ID NO: 57 SEQ ID NO: 88 R9 Conjugation of SEQ ID NO: 27 and SEQ ID NO: 58 SEQ ID NO: 89 R10 Conjugation of SEQ ID NO: 28 and SEQ ID NO: 59 SEQ ID NO: 90 R11 Conjugation of SEQ ID NO: 29 and SEQ ID NO: 60 SEQ ID NO: 91 NA,
NVT,
VP1,
VP10 Conjugation of SEQ ID NO: 30 and SEQ ID NO: 61 SEQ ID NO: 92 NVT IV,
NVT 4,
VP20 Conjugation of SEQ ID NO: 31 and SEQ ID NO: 62 SEQ ID NO: 93

The hsRNA or dsRNA may activate immune cells including dendritic cells, neutrophils, B cells, macrophages, T cells, mast cells, and natural killer cells.

The present invention provides a vaccine composition for preventing or treating viral or bacterial infection, comprising the hsRNA or dsRNA, and an immunogen. The vaccine composition for preventing or treating viral or bacterial infection may be a vaccine composition for preventing or treating influenza virus or meningococcal infection.

The immunogen may be a trivalent seasonal influenza vaccine, a tetravalent seasonal influenza vaccine, an inactivated influenza vaccine, or a Neisseria meningitidis groups A, C, Y and W-135 vaccine, and protection against viruses of allogeneic or heterogeneous subtypes It can have an effect.

The vaccine composition for preventing or treating viral or bacterial infection may reduce the required amount of immunogen compared to the vaccine composition without the hsRNA or dsRNA. The required usage may be reduced, for example, by 2 times or more, 3 times or more, 5 times or more, 10 times or more, 25 times or more, 50 times or more, or 100 times or more.

The vaccine composition for preventing or treating viral or bacterial infection may enhance, maintain or restore the antigenicity of an immunogen.

The vaccine composition for preventing or treating viral or bacterial infection may include an additional adjuvant. The additional adjuvant may be one or more selected from the group consisting of oil-in-water emulsion adjuvant, aluminum salt, Freund adjuvant, and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), The oil-in-water emulsion adjuvant may be a squalene oil-in-water emulsion.

In addition, the present invention provides a pharmaceutical composition for preventing or treating viral or bacterial infections comprising the hsRNA or dsRNA.

The pharmaceutical composition for preventing or treating viral or bacterial infection may include an additional adjuvant. The additional adjuvant may be one or more selected from the group consisting of oil-in-water emulsion adjuvant, aluminum salt, Freund adjuvant, and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), The oil-in-water emulsion adjuvant may be a squalene oil-in-water emulsion.

The present invention provides a vaccine composition for preventing or treating cancer comprising the hsRNA or dsRNA, and an immunogen.

The immunogen is a peptide, antigen, inactivated or living attenuated organism, a cell-derived, inactivated cancer tissue or cancer tissue isolated from a human being irradiated to inactivate cancer tissue, and cancer cells cultured in vitro are irradiated with radiation. It may be selected from the group consisting of inactivated cancer cells, cancer cytoplasm obtained by dissolving cancer cells cultured in vitro in a solvent, cancer-associated antigen proteins or immunodeterminants (epitopes) in cancer-associated antigens, and DNA or mRNA encoding the protein. , Is not limited thereto.

The vaccine composition for preventing or treating cancer may reduce the required dose of the immunogen compared to the vaccine composition without the hsRNA or dsRNA. The required usage may be reduced, for example, by 2 times or more, 3 times or more, 5 times or more, 10 times or more, 25 times or more, 50 times or more, or 100 times or more.

The vaccine composition for preventing or treating cancer may enhance, maintain or restore the antigenicity of an immunogen.

The vaccine composition for preventing or treating cancer may include an additional adjuvant. The additional adjuvant may be one or more selected from the group consisting of oil-in-water emulsion adjuvant, aluminum salt, Freund adjuvant, and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), The oil-in-water emulsion adjuvant may be a squalene oil-in-water emulsion.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the hsRNA or dsRNA.

The cancer includes, but is not limited to, carcinoma, lymphoma, leukemia, blastoma and sarcoma. More specific examples of cancer include squamous cell carcinoma, myeloma, lung cancer, small cell lung cancer, non-small cell lung cancer, colon cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myelogenous leukemia (AML), multiple myeloma, gastrointestinal cancer. , Kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma polymorphic, cervical cancer , Brain cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon carcinoma, head and neck cancer, or metastatic cancer thereof, but are not limited thereto.

The vaccine composition for preventing or treating cancer may exhibit a cancer metastasis inhibitory effect.

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising the hsRNA or dsRNA, and OX40 antibody or PD-1 antibody.

The OX40 antibody may be an OX40 agonist antibody, and the PD-1 antibody may be a PD-1 antagonist antibody. In addition, the OX40 antibody may be a monoclonal antibody or antigen-binding fragment thereof that specifically binds to OX40, and the PD-1 antibody is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to PD-1. Can be Specifically, the OX40 antibody or PD-1 antibody may be a human antibody, a humanized antibody, a chimeric antibody, or a mouse antibody, and may include a human constant region. The human constant region is selected from the group consisting of an IgG1, IgG2, IgG3 and IgG4 constant region, preferably an IgG1 or IgG4 constant region. In one embodiment, the antigen binding fragment is selected from the group consisting of Fab, Fab'-SH, F(ab')2, scFv and Fv fragments. Examples of specific human PD-1 monoclonal antibodies that can be used as the PD-1 antagonist antibody include pembrolizumab, nivolumab, avelumab, and pidilizumab. However, it is not limited thereto.

Table 2 lists hsRNAs derived from four different backbone vectors used in the examples of the present invention. In the following examples, each hsRNA is referred to by its name.

designation hsRNA (base) dsRNA (bp) Upper ssRNA (base) Lower ssRNA (base) hsRNA-20% (base) hsRNA+ 20% (base) dsRNA-20% (base) dsRNA+ 20% (base) Backbone Group NA1001 140 106 17 17 112 168 85 127 One NA1501 190 156 17 17 152 228 125 187 One NA2001 240 206 17 17 192 288 165 247 One NA3001 340 306 17 17 272 408 245 367 One NA4001 440 406 17 17 352 528 325 487 One NA5001 540 506 17 17 432 648 405 607 One NA6001 640 606 17 17 512 768 485 727 One NA7001 740 706 17 17 592 888 565 847 One NA8001 840 806 17 17 672 1008 645 967 One NA1002 140 106 17 17 112 168 85 127 2 NA1502 190 156 17 17 152 228 125 187 2 NA2002 240 206 17 17 192 288 165 247 2 NA3002 340 306 17 17 272 408 245 367 2 NA4002 440 406 17 17 352 528 325 487 2 NA5002 540 506 17 17 432 648 405 607 2 NA6002 640 606 17 17 512 768 485 727 2 NA7002 740 706 17 17 592 888 565 847 2 NA8002 840 806 17 17 672 1008 645 967 2 R1 353 319 17 17 282 424 255 383 3 R2 431 397 17 17 345 517 318 476 3 R3 500 466 17 17 400 600 373 559 3 R4 622 588 17 17 498 746 470 706 3 R5 698 664 17 17 558 838 531 797 3 R6 767 733 17 17 614 920 586 880 3 R7 856 822 17 17 685 1027 658 986 3 R8 919 885 17 17 735 1103 708 1062 3 R9 1066 1032 17 17 853 1279 826 1238 3 R10 1187 1153 17 17 950 1424 922 1384 3 R11 1682 1648 17 17 1346 2018 1318 1978 3 NA, NVT, VP1, VP10 533 424 51 58 426 640 339 509 4 NVT IV,
NVT 4,
VP20 568 424 75 69 454 682 339 509 4 * VP11 is Rnase T1-treated VP10 (VP1)

The present invention is to be described in more detail through the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

In vivo dendritic cell (DC) activation test of ribonucleic acid (RNA) adjuvant

In this example, after injection of NA (same as NVT or VP10) into the intraperitoneal cavity of C57BL/6 mice, the innate immunity activity of ribonucleic acid NA was confirmed in vivo (FIG. 1A ).

As a result, the NA of the present invention induced the differentiation of dendritic cell markers (CD40, CD86, MHC-II) equal or higher than that of 100 μg of Poly (I:C) (Fig. 1b), and naive CD4 T cells were Th1 It was confirmed that the induction of type 1 interferon beta (IFN-β) and the secretion of IL-6 and IL-12 were further increased toward CD4 T cells (FIG. 1C). In addition, T-bet, a Th1 cell marker, and IFN-γ, a secretion marker, were increased. However, there was little effect on the Th2 cytokine (IL-4) and the Th17 cytokine (IL-17A) (Fig. 1D).

As a result, it can be seen that the NA of the present invention strongly activates DC compared to the Poly (I:C) positive control. In addition, it was confirmed that the activity increased as the length of ribonucleic acid increased from about 100 base to 400 base, and excellent activity was observed when the length of the ribonucleic acid was longer than 400 base.

DC activation test in vivo according to the length of hsRNA

HsRNAs derived from two types of vectors were prepared in various lengths (FIGS. 2a and 2b), and they were injected into the intraperitoneal cavity of mice to analyze splenic DC activity caused in vivo.

As a result of examining the ability to induce CD40 and CD86 expression, the level of DC activation in the spleen increased as the length of ribonucleic acid increased, and it was confirmed that the activity was high in 400-900 bases (FIGS. 2c and 2d).

In addition, it was confirmed that all hsRNAs of the present invention exhibited more excellent activity than the positive control Poly (I:C) TLR3 ligand.

In addition, the level of DC activation in the spleen showed a tendency to increase as the length of the dsRNA site increased, and it was confirmed that the level of the dsRNA site exhibited high activity when the length of the dsRNA site was about 406 to 806 bp.

As a result of retesting 540 hsRNA (506bp dsRNA) showing high activity at various concentrations (0.5 ~ 20 μg), it showed maximum activity at 10 μg. In vivo, it was 10 to 20 times higher than that of Poly (I:C). Showed activity.

IFN-β promoter activation and antigen-specific antibody induction test according to hsRNA length

A wider range of ribonucleic acid (353 ~ 1682 base) derived from a vector of another backbone was prepared and mixed with innate immune activity indicators and antigens to investigate the immune response.

The length of the additionally prepared hsRNA was confirmed (Fig. 3a), and activation of the IFN-β promoter in HEK 293 due to the additional sequence was confirmed (Fig. 3b). Several of these (R3, R5, R7, R10) were mixed with OVA antigen and injected intramuscularly, and the levels of Anti-OVA IgG1 and Th1-mediated Anti-OVA IgG2 were quantified. R5 (dsRNA length 664 bp) It showed a relatively high activity in the vicinity (Fig. 3c).

From the above results, the degree of DC activation is not dependent on the specific sequence of ribonucleic acid, but is proportional to the length of hsRNA and dsRNA, and it is confirmed that DC exhibits high activity, especially when the length of the dsRNA site is 406 bp to 806 bp. I did.

Through this, in addition to the specific length of each RNA presented in the present invention, it can be seen that hsRNA having a base length between 140 and 1682 and dsRNA having a base length between 106 and 1648 bp exhibit excellent effects.

Accordingly, the present invention includes ribonucleic acid, which is characterized by having a shorter or longer hsRNA length (112 to 2018 base) within a 20% range in addition to each hsRNA tested in the present invention. In addition, in addition to each dsRNA selected from SEQ ID NOs: 63 to 93, it includes a ribonucleic acid characterized in that the dsRNA length (85 to 1977 bp) is shorter or longer within the range of 20% thereof.

Physicochemical characterization of hsRNA

hsRNA was analyzed using HPLC, and length separation was performed on an agarose gel. As a result, it was found that the hsRNA was uniform in a single length (Fig. 4a). This is in contrast to the fact that Poly (I:C) produced by template-independent enzyme synthesis is essentially bound to have a high degree of heterogeneity (Fig. 8c).

The dsRNA of the present invention was not degraded even when left at 45° C. for 150 days or more (FIG. 4B), but had a very short half-life in the serum medium in which a lot of various RNAse were present (FIG. 4C). The antigen complex complexed to NA could be stored for a long time at room temperature (Fig. 4d).

Difference between hsRNA and dsRNA induction of immune cells and cytokines

The degree of DC activation induced by NVT 4 (same as NVT IV and VP20) in two cases configured as follows was compared in vivo after 24 and 48 hours elapsed.

(i) hsRNA not treated with RNase T1 (NVT 4-T1)

(ii) dsRNA remaining after all single-stranded parts were degraded by treatment with RNaseT1 (NVT 4 +T1)

As a result, both hsRNA and dsRNA induced DC activation, but dsRNA significantly increased the number of immune cells (especially B cells and neutrophils) compared to hsRNA, and TNF-α, IFN-β, and IL-6 were also proportionally higher. Induced (Fig. 5).

These results show that dsRNA strongly induces innate immunity, but hsRNA has a low number of neutrophils and levels of IL-6 and TNF-α, which is advantageous for safety.

Comparison of the ability of NA and other adjuvants to induce Th1 polarization immune response

Model antigen OVA was complexed to NA, Alum, squalene (SE), IFA, and Poly(I:C) of the present invention, and then administered intramuscularly (Fig. 6A) to compare the ability to induce immune responses.

As a result, the activity of the Th2 immune response (IgG1) was similar, but the Th1 response (IgG2c) was activated with a higher NA than that of other adjuvants (FIG. 6B ). The number of CD8 T cells and CD4 T cells secreting IFN-γ in the spleen also increased (Fig. 6c). Even when administered intranasally, NA in body fluids (tears, nasal and drainage) and serum induced mucosal IgA and mucosal IgG more strongly than other adjuvants.

Comparison of immune-inducing ability of compositions comprising hsRNA and additional adjuvants

The degree of DC activation induced by intramuscular administration of NVT (NVT or NVTII) in two cases configured as follows was compared in vivo (FIG. 7A ).

(i) NVT; NVT (NA) alone

(ii) NVTII; NVT (NA)+SE (Squalene Emulsion)

As a result, DC activity was induced in the inguinal lymph node (iLN), and the activity of DC was higher in the case of NVTII than in NVT (Fig. 7b).

In addition, antibody response induction was compared when configured as follows.

(i) OVA alone

(ii) OVA+NVT

(iii) OVA+NVTII

Anti-OVA IgG1 and IgG2a were measured after injection into the mouse muscle. As a result, when OVA+NVT was injected, about 100 times stronger antibody response was induced than when OVA alone was injected. In the case of OVA+NVTII, the antibody response was more than twice as strong as in the case of OVA+NVT (Fig. 7c).

When NVT was administered intranasally, DC was activated in mediastinal lymph nodes (mLN), and IL-6, IL-12 and TNF-α were induced in alveolar lavage fluid (BALF).

Homogeneity of structure and length of hsRNA

The hsRNA of the present invention has a 3'-overhang structure in which both 3'ends of the dsRNA protrude. In the present invention, the ribonucleic acid having the above structure is described as hsRNA.

The dsRNA portion that has any length and any sequence and is completely complementary may be an artificial sequence or a natural sequence, but does not encode a protein. Single-stranded ssRNA having an arbitrary length and arbitrary sequence is bound to the 3'end of the dsRNA by a phosphate diester bond and corresponds to a TLR7/8 potential ligand (FIG. 8A). It can be confirmed through the following experiment that the dsRNA site of the present invention has complete complementarity regardless of sequence or length, and that there is no nick inside and has a uniform length.

When RNase T1 is treated, ssRNA is completely degraded. When RNase III is treated for a short time, dsRNA is cut into fragments of an average of 20 to 25 bp, and when treated for a longer time, fragments having a length of 12 bp or less are cut. It is possible to experimentally confirm the results exactly consistent with the mode of action of these various RNases. That is, when RNAse III is treated on hsRNA having 51 base and 58 base ssRNA protrusions at both 3'ends, 20 to 25 bp dsRNA fragments and about 50 base ssRNA fragments can be identified through the band. When the hsRNA is treated with RNAse III and RNAse T1 at the same time, it can be confirmed through the band that ssRNA is completely degraded and only a 20 to 25 bp dsRNA fragment remains. When RNase A is treated, all RNAs including ssRNA and dsRNA are completely degraded, so no band appears (Fig. 8b).

In addition, when the hsRNA is treated with RNase T1, the complementarity and stability of the dsRNA site is demonstrated through the fact that only one band appears in the agarose gel before and after the treatment (Fig. 8c).

It has been confirmed several times in the above examples that the length of a specific hsRNA has a high degree of homogeneity (FIGS. 2A, 3A, and 4A).

As another specific example, any single strand (≥17 base) containing a polyuridine (U) base can be added to both sides of the dsRNA. In addition, a single strand can be designed to have stability by adding a specific sequence to form an internal dsRNA such as a hairpin structure or a palindromic ring structure.

However, in contrast, the competing drug, Poly(I:C), has extreme length diversity, cannot be prepared with a constant length, and ssRNA stretch sites are practically absent. This is because the length diversity of Poly(I:C) arises from slippage and chain extension in the chain. As a result, it can be seen that nicks for which the position and number cannot be specified exist in the dsRNA site or at the ends, so that when RNase T1 is treated, it is cut into various lengths (FIG. 8C). Poly(I:C) cannot specify the number of chains each forming the upper and lower strands due to chain expansion, etc., and nicks are not supplemented because phosphate diester bonds are not made between adjacent strands. Easy to digest

In contrast to the hsRNA and dsRAN of the present invention, as a result of treatment with RNase III on the existing Poly(I:C)-L (long species) and Poly(I:C)-S (short species), it is shorter than the original but constant Many unnicked dsRNAs were generated (FIG. 8D). This is because Poly(I:C) has an unspecified nick at an unspecified position inside the dsRNA, and the site digested by RNase III is not uniform (Non-Patent Document 2). There were cases where this problem was improved by inducing complementary binding by inducing complementary binding in a way that corresponds to long PolyI (P400 with an average of 400 base) and short PolyC (P100 with an average of 100 base), but the length was still varied from 100 bp to 100 kbp. Patent Document 5).

The ribonucleic acid of the present invention has an essential difference from the existing invention in the following matters.

Compared with Poly(I:C), the ribonucleic acid of the present invention 1) has a high degree of homogeneity in length due to the essential difference in the manufacturing process, is a non-homopolymer of sequence, and does not have nicks in dsRNA with complete complementarity. , It does not have a phosphoric acid group at the 5'end, and 2) can act as a TLR3 ligand and a TLR7-like ligand.

Compared to siRNA, the ribonucleic acid of the present invention 1) has an essential difference in the manufacturing process compared to the synthesized siRNA, 2) does not have a function of targeting a specific gene of siRNA, and 3) the length of siRNA is 21- As short as 25 bp, TLR3 cannot be stimulated, whereas the length of the ribonucleic acid of the present invention is 45 bp or more, so it can act as a TLR3 ligand that exhibits sufficient activity (Non-Patent Document 1), and 4) a very short 3'at the end of siRNA. -The protrusion corresponds to a simple residual sequence without a function, but the ssRNA of the present invention has a specific sequence of a specific length or more (eg, 17 bases or more) and serves as a TLR7 and TLR7-like ligand. In fact, hsRNA to which the above sequence was added showed a low toxicity index in vivo compared to dsRNA to which the sequence was not added (Example 5).

Immune response after nasal administration of inactivated influenza whole vaccine (iPR8) complexed with dsRNA and antigen-independent immunity enhancing effect of hsRNA

When NVT was administered with iPR8 to BalB/c mice, the number of mDC and rDC cells in the mediastinal lymph node increased after 24 hours, and DC (CD80, CD86) was activated and then decreased to the basal level at 96 hours. In addition, when NVT was treated with iPR8, total cells, alveolar macrophages, neutrophils and natural killer cells increased in the alveolar lavage solution, which was proportional to the increase of IL-6, IL-12 and TNF-α. This increase in innate immunity indicators was accompanied by an increase in IgG and IgA in germinal center B (GCB) cells, follicular helper T cells (T _FH ), and serum and nasal lavage (Fig. 9d, e).

As a result of observing the antigen-independent immunity enhancing effect of the NA selected as one of the hsRNAs of the present invention, it was confirmed that the NA adjuvant of the present invention alone has a protective effect against deadly live virus infection in the absence of the antigen. Multiple administrations of NA into the nasal cavity of the mouse resulted in an average life span of more than 4 days, and the mortality associated with viral infection was reduced by about 20%. That is, even with the NA adjuvant of the present invention that did not form a complex with an antigen or an anchor peptide, a strong immune enhancing action was exhibited.

As a result, the ribonucleic acid of the present invention alone has the effect of reducing the mortality rate due to the virus, and thus can serve as a means for achieving the effect of prolonging the lifespan of an urgent infectious disease.

Commercial Influenza Split Vaccine Antigen Reduction Effect by hsRNA

The amount of antigen used was compared by administering each of the cases configured as follows to female Balb/c mice (FIG. 10A ).

(i) tetravalent inactivated influenza vaccine (4IIV (QIV), Vaxigrip, Sanofi Pasteur)

(ii) Mixing of 4IIV and NVT (NA)

(iii) Mixing of 4IIV and NVTII (NA+SE)

As a result, compared to the case of (i) inoculating the antigen alone, it was confirmed that the amount of antigen used in (ii) can be reduced by about 5 times and (iii) by about 25 times (FIG. 10B). This effect lasted up to at least 17 weeks after injection (FIG. 10C). From the above results, it can be seen that when NA is complexed to an antigen, the amount of vaccine antigen required to secure the necessary immunity can be significantly reduced.

The increase of these neutralizing antibodies was closely related to an increase in hemagglutinin inhibition (HAI) titer by 40 or more, and thus it was judged to provide individual defense. That is, when using NVT or NVT II, the HAI titer value, which is a protective immunity index (COP) against influenza virus, increased by about three times and met the'HAI titer≥40', which is a protective immunity index that decreases infection by 50% (Fig. ).

Antigen reduction effect of commercial meningitis vaccine by hsRNA

While administering the Menactra vaccine to mice, NA was used as an adjuvant (FIG. 11A).

Menactra vaccine is a vaccine against Neisseria meningitidis groups (A, C, Y and W-135), which are Meningococcus. Specifically, it is a vaccine in which the polysaccharide antigen is covalently linked to CRM197, which is a diphtheria toxin. The novel vaccine formulation complexed with the meningitis vaccine to NVT II of the present invention induced Menactra-specific antibodies 5 times or more strongly (FIGS. 11b and 11c).

Immunogenic Restoration Effect of NVT II

The 2018/2019 seasonal tetravalent influenza vaccine Vaxigrip (QIV) was left at 37°C for 5 weeks to completely lose its immunogenicity, and then mixed with NVT II and injected intramuscularly (i.m.). After 3 weeks, the amount of IgG induced was measured, and after 18 weeks, HAI assay was performed (FIG. 12A).

As a result, it can be seen that the neutralizing antibody against Vaxigrip was remarkably increased and immunogenicity was restored (FIG. 12B). In addition, there was an antigen reduction effect of at least 10 times. It is consistent with the above results that the HAI titer, which is a protective immunity index for H1N1 and IBV, increased to 40 or more at week 18 (Fig. 12c).

In another experiment, when only Vaxigrip was left alone at 37°C for 5 weeks, antigenicity was completely lost, whereas when Vaxigrip was first mixed with NVTII and left at 37°C for 5 weeks, antigenicity was completely maintained and the HAI titer was 40 or higher. Was increased to.

Antigen-independent tumor regression effect when hsRNA alone is used

To confirm that NA induces tumor regression and increases survival even in the absence of cancer antigens, B16F10-OVA melanoma cells were subcutaneously transplanted into 7-week-old female C57BL/6 mice, and then NA was injected intratumorally from day 6 (it ) Or intramuscularly, administered three times at 2-day intervals (FIG. 13A).

As a result, tumor growth was more inhibited in mice administered with NA compared to the control group and survived longer (FIGS. 13 b and 13c ).

The effect of hsRNA alone on the regression of colon cancer and lung cancer

After subcutaneous transplantation of CT26 colorectal cancer and LL/2 lung cancer into 7-week-old female C57BL/6 mice, NVT alone was used in subcutaneous tumor tissue (it) or intramuscular (im) once every two days. After administration, the regression of the cancer mass was confirmed.

As a result, it was confirmed that primary colon cancer and lung cancer were suppressed (FIG. 14).

The effect of hsRNA alone on immune enhancing effect on triple negative breast cancer (TNBC) regression and metastasis inhibition

A 7-week-old female C57BL/6 mouse was transplanted with a 4T1 triple-negative breast cancer (TNBC) cell line subcutaneously from mammary adipose tissue, and then administered 9 times at 2-day intervals from day 8 into tumor tissue (it) alone (Fig. 15A). ).

As a result, primary breast cancer regressed by 60% (FIG. 15B), and this regression was consistent with the remarkably reduced distant metastasis to the lung (FIG. 15C).

Evaluation of anti-metastatic activity of hsRNA alone or hsRNA complexed TAA vaccine

After administering the following cancer vaccine composition to C57BL/6 mice, B16F10/Ova melanoma cells were transplanted into the spleen, and the degree of metastasis from the spleen to the liver was investigated after administration of the same vaccine composition on the third day (Fig. ).

(i) PBS (physiological saline)

(ii) OVA

(iii) NA

(iv) OVA+NA

Except in the case of OVA+NA treatment, all mice started to die from day 14 and died within 18 days, whereas the OVA+NA treatment group started to die from day 24 and all mice died within 28 days. As a result of measuring the size and weight of the liver and spleen of each mouse, it was confirmed that the NA treatment group and OVA + NA treatment group mice had a lower degree of metastasis compared to the control and OVA alone treatment group mice (Figs. 16b and 16c). . In addition, the number of tumor nodules metastasized to the liver on the 18th day was measured. The metastasis of melanoma cells from the spleen to the liver was inhibited by about 50% by NA alone and by 90% or more in the case of OVA+NA complexed with a cancer antigen (eg, OVA) in NA (FIG. 16D).

These results indicate that when NA alone or NA complexed OVA+NA vaccine was administered intraperitoneally into the spleen, metastasis of melanoma cells to the liver was significantly reduced.

Inhibitory Effects of hRNA and OX40 Antibody Complexes on Primary and Distant Melanoma

B16F10/Ova melanoma cells were ^{injected subcutaneously into the left flank of C57BL/6 mice in an amount of 5x10 5} cells, and cancerous masses were grown for 6 days after implantation of the tumor in the mice. On the 6th, 8th, 10th, and 12th days after tumor transplantation, the following cancer vaccine compositions were each administered 4 times into the primary cancer tissues (FIG. 17A). Thereafter, the size of the cancer tissue was measured until the 22nd day, and the degree of growth of the cancer tissue was investigated.

(i) PBS (physiological saline)

(ii) PD-1 Ab

(iii) IR cells (cancer cells irradiated with radiation)

(iv) IR cells + NA + OX40 Ab

As a result, only the PD-1 antibody, an immune checkpoint anticancer antibody, inhibited the primary cancer growth by about 50%, and when only irradiated cancer cells (IR Cells) were administered as a cancer antigen, the primary cancer growth was suppressed by about 20%. On the other hand, when the cancer vaccine composition (IR cell + NA + OX40 Ab) containing NA and OX40 antibodies together with IR cells was administered, the growth of primary cancer was inhibited by about 90% or more (FIG. 17B).

In addition, even when only the cancer vaccine composition (NA + OX40 Ab) containing NA and OX40 antibodies is administered without using the antigen of IR cells, the growth of primary cancer is suppressed at almost the same level as when using the IR cell antigen. Became (Fig. 17c).

The mouse experimental group was selected, and on the 15th day, two allogeneic cancers (melanoma (B16F10/Ova) and T-cell lymphoma (EG7/Ova)) were implanted in the second right abdomen on the 15th day (2nd implantation). After that, the degree of growth of the cancer tissue was measured for 16 days in a state in which the vaccine composition for treatment was no longer administered.

As a result, in the case of the group treated with the cancer vaccine composition (NA + OX40 Ab) of the present invention, the secondary transplanted distant cancer hardly grew in all groups. On the other hand, when other isotype Abs instead of OX40 Ab were used together with NA, cancer rapidly grew after about 10 days and the corresponding mouse died suddenly. Therefore, the size of the mass was not indicated in the figure (Fig. 17D).

In summary, PD-1 antibody alone inhibited the growth of primary melanoma by about 50%, and IR cells alone inhibited the growth of primary melanoma by about 20%, but the composition of the present invention, IR cells + NA + OX40 Ab, inhibited the growth of primary melanoma. It was suppressed more than 90%.

In addition, even when only the NA + OX40 Ab composition was administered without IR cells, primary cancer and secondary transplanted distant cancer were suppressed. This shows that cancer immunity induced in primary cancer by the NA + OX40 Ab composition also affects newly transplanted cancers.

Inhibitory effect of hsRNA and PD-1 antibody or OX40 complex on primary and distant melanoma

B16F10/Ova melanoma cells were implanted by simultaneously subcutaneous injection into the right and left abdominal cavity of C57BL/6 mice in an amount of ^{5x10 5 cells, and then cancerous masses were grown for about 5 days.} Thereafter, the cancer vaccine composition constituted as follows into the cancer tissue of the right abdominal cavity was administered four times at intervals of 2 days, respectively (FIG. 18A). The size of the cancer tissue was measured, and the degree of growth of each of the right cancer (cancer administered with the vaccine composition, Vaccinated Tumor) and the left cancer (remote cancer, Remote Tumor) tissue was investigated.

(i) PBS (physiological saline)

(ii) NA

(iii) NA + OX40 Antibody (OX40 Ab) (clone OX-86, Rat IgG1, InvivoGen)

(iv) NA + PD-1 antibody (PD-1 Ab) (clone RMP1-14, Rat IgG2a/λ, InvoGen)

As a result, the inhibitory effect of the cancer vaccine composition (NA + PD-1 Ab) containing hsRNA and PD-1 antibody on primary and distant cancers is a cancer vaccine composition containing hsRNA and OX40 antibody (NA + OX40 Ab). ) Was similar to or better. In addition, the inhibitory effect on primary cancer at the site injected with the cancer vaccine composition (NA + PD-1 Ab and NA + OX40 Ab) of the present invention was better than that of distant cancer without injection of the vaccine composition, and primary cancer And it was confirmed that it shows the effect of inhibiting both distant cancer (FIGS. 18B and 18C ).

In addition, when only PD-1 antibody was treated, it was confirmed that only about 50% of the growth of primary cancer was inhibited, but when hsRNA and PD1 antibody were treated together (NA + PD-1 Ab), it had an inhibitory effect of about 90% or more. It showed an inhibitory effect similar to or better than that of NA + OX40 Ab.

From this, it was confirmed that when the hsRNA and the PD-1 antibody were treated together, refractory cancer that did not respond to the immune checkpoint inhibitor of the PD-1 antibody was converted into a reactive cancer, thereby exhibiting a more excellent anticancer effect.

<110> NA Vaccine Institute <120> NOVEL RIBONUCLEIC ACID AND PHARMACEUTICAL COMPOSITION BASED ON THE SAME <130> NAVI19P-0001-KR-PRI2 <160> 93 <170> KoPatentIn 3.0 <210> 1 <211> 123 <212> RNA <213> Artificial Sequence <220> <223> NA1001 <400> 1 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgcccuaua gugagucgua 120 uua 123 <210> 2 <211> 173 <212> RNA <213> Artificial Sequence <220> <223> NA1501 <400> 2 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcccuaua gugagucgua uua 173 <210> 3 <211> 223 <212> RNA <213> Artificial Sequence <220> <223> NA2001 <400> 3 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auacccuaua gugagucgua uua 223 <210> 4 <211> 323 <212> RNA <213> Artificial Sequence <220> <223> NA3001 <400> 4 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uaucccuaua gugagucgua uua 323 <210> 5 <211> 423 <212> RNA <213> Artificial Sequence <220> <223> NA4001 <400> 5 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caacccuaua gugagucgua 420 uua 423 <210> 6 <211> 523 <212> RNA <213> Artificial Sequence <220> <223> NA5001 <400> 6 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guucccuaua gugagucgua uua 523 <210> 7 <211> 623 <212> RNA <213> Artificial Sequence <220> <223> NA6001 <400> 7 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guuauccgcu cacaauucca cacaacauac gagccggaag 540 cauaaagugu aaagccuggg gugccuaaug agugagcuaa cucacauuaa uugcguugcg 600 cuccccuaua gugagucgua uua 623 <210> 8 <211> 723 <212> RNA <213> Artificial Sequence <220> <223> NA7001 <400> 8 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guuauccgcu cacaauucca cacaacauac gagccggaag 540 cauaaagugu aaagccuggg gugccuaaug agugagcuaa cucacauuaa uugcguugcg 600 cucacugccc gcuuuccagu cgggaaaccu gucgugccag cugcauuaau gaaucggcca 660 acgcgcgggg agaggcgguu ugcguauugg gcgcucuucc gcucccuaua gugagucgua 720 uua 723 <210> 9 <211> 823 <212> RNA <213> Artificial Sequence <220> <223> NA8001 <400> 9 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guuauccgcu cacaauucca cacaacauac gagccggaag 540 cauaaagugu aaagccuggg gugccuaaug agugagcuaa cucacauuaa uugcguugcg 600 cucacugccc gcuuuccagu cgggaaaccu gucgugccag cugcauuaau gaaucggcca 660 acgcgcgggg agaggcgguu ugcguauugg gcgcucuucc gcuuccucgc ucacugacuc 720 gcugcgcucg gucguucggc ugcggcgagc gguaucagcu cacucaaagg cgguaauacg 780 guuauccaca gaaucagggg auacccuaua gugagucgua uua 823 <210> 10 <211> 123 <212> RNA <213> Artificial Sequence <220> <223> NA1002 <400> 10 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uuccccuaua gugagucgua 120 uua 123 <210> 11 <211> 173 <212> RNA <213> Artificial Sequence <220> <223> NA1502 <400> 11 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aaccccuaua gugagucgua uua 173 <210> 12 <211> 223 <212> RNA <213> Artificial Sequence <220> <223> NA2002 <400> 12 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gaucccuaua gugagucgua uua 223 <210> 13 <211> 323 <212> RNA <213> Artificial Sequence <220> <223> NA3002 <400> 13 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggacccuaua gugagucgua uua 323 <210> 14 <211> 423 <212> RNA <213> Artificial Sequence <220> <223> NA4002 <400> 14 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucccuaua gugagucgua 420 uua 423 <210> 15 <211> 523 <212> RNA <213> Artificial Sequence <220> <223> NA5002 <400> 15 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucgcccuaua gugagucgua uua 523 <210> 16 <211> 623 <212> RNA <213> Artificial Sequence <220> <223> NA6002 <400> 16 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucguauucuc cuucuugcug cccuuucuga gggaaggacu 540 guuguugaca auuuacugag uagugacgac auucauuaca ugcuuggugc guugaaaaca 600 cuucccuaua gugagucgua uua 623 <210> 17 <211> 723 <212> RNA <213> Artificial Sequence <220> <223> NA7002 <400> 17 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucguauucuc cuucuugcug cccuuucuga gggaaggacu 540 guuguugaca auuuacugag uagugacgac auucauuaca ugcuuggugc guugaaaaca 600 cuuggacuuc auguugaaga ugacaaugaa aaccaacgag caauugugga agguuguggu 660 gggcaguuuc cugucgguaa aaagucugag gaagaaaucc aaccccuaua gugagucgua 720 uua 723 <210> 18 <211> 823 <212> RNA <213> Artificial Sequence <220> <223> NA8002 <400> 18 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucguauucuc cuucuugcug cccuuucuga gggaaggacu 540 guuguugaca auuuacugag uagugacgac auucauuaca ugcuuggugc guugaaaaca 600 cuuggacuuc auguugaaga ugacaaugaa aaccaacgag caauugugga agguuguggu 660 gggcaguuuc cugucgguaa aaagucugag gaagaaaucc aacuauuccu uggaaaugca 720 ggaacagcaa ugcguccguu gacagcagca guuacuguag cuggaggaca uucaagauau 780 guucuugaug gaguuccuag gaucccuaua gugagucgua uua 823 <210> 19 <211> 336 <212> RNA <213> Artificial Sequence <220> <223> R1 <400> 19 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugcccu auagugaguc guauua 336 <210> 20 <211> 414 <212> RNA <213> Artificial Sequence <220> <223> R2 <400> 20 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccuau agugagucgu auua 414 <210> 21 <211> 483 <212> RNA <213> Artificial Sequence <220> <223> R3 <400> 21 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccuaua gugagucgua 480 uua 483 <210> 22 <211> 605 <212> RNA <213> Artificial Sequence <220> <223> R4 <400> 22 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuacccua uagugagucg 600 uauua 605 <210> 23 <211> 681 <212> RNA <213> Artificial Sequence <220> <223> R5 <400> 23 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccuauagu gagucguauu a 681 <210> 24 <211> 750 <212> RNA <213> Artificial Sequence <220> <223> R6 <400> 24 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccuauagug agucguauua 750 <210> 25 <211> 839 <212> RNA <213> Artificial Sequence <220> <223> R7 <400> 25 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac ccuauaguga gucguauua 839 <210> 26 <211> 902 <212> RNA <213> Artificial Sequence <220> <223> R8 <400> 26 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gacccuauag ugagucguau 900 ua 902 <210> 27 <211> 1049 <212> RNA <213> Artificial Sequence <220> <223> R9 <400> 27 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gacccugcug aaaccgggug 900 acaccaucau cuucgaagcu aacgguaacc ugaucgcucc gauguacgcu uucgcucugu 960 cucgugguuu cgguucuggu aucaucaccu cuaacgcuuc uaugcacgaa ugcaacacca 1020 aaugccagac ccuauaguga gucguauua 1049 <210> 28 <211> 1170 <212> RNA <213> Artificial Sequence <220> <223> R10 <400> 28 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gacccugcug aaaccgggug 900 acaccaucau cuucgaagcu aacgguaacc ugaucgcucc gauguacgcu uucgcucugu 960 cucgugguuu cgguucuggu aucaucaccu cuaacgcuuc uaugcacgaa ugcaacacca 1020 aaugccagac cccgcugggu gcuaucaacu cuucucugcc guaccagaac auccacccgg 1080 uuaccaucgg ugaaugcccg aaauacguuc guucugcuaa acugcguaug guuaccgguc 1140 ugcguaacau cccuauagug agucguauua 1170 <210> 29 <211> 1665 <212> RNA <213> Artificial Sequence <220> <223> R11 <400> 29 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gacccugcug aaaccgggug 900 acaccaucau cuucgaagcu aacgguaacc ugaucgcucc gauguacgcu uucgcucugu 960 cucgugguuu cgguucuggu aucaucaccu cuaacgcuuc uaugcacgaa ugcaacacca 1020 aaugccagac cccgcugggu gcuaucaacu cuucucugcc guaccagaac auccacccgg 1080 uuaccaucgg ugaaugcccg aaauacguuc guucugcuaa acugcguaug guuaccgguc 1140 ugcguaacau cccgucuauc cagucucgug gucuguucgg ugcuaucgcu gguuucaucg 1200 aaggugguug gaccgguaug aucgacgguu gguacgguua ccaccaccag aacgaacagg 1260 guucugguua cgcugcugac cagaaaucua cccagaacgc uaucaacggu aucaccaaca 1320 aaguuaacac cguuaucgaa aaaaugaaca uccaguucac cgcuguuggu aaagaauuca 1380 acaaacugga aaaacguaug gaaaaccuga acaaaaaagu ugacgacggu uuccuggaca 1440 ucuggaccua caacgcugaa cugcugguuc ugcuggaaaa cgaacguacc cuggacuucc 1500 acgacucuaa cguuaaaaac cuguacgaaa aaguuaaauc ucagcugaaa aacaacgcua 1560 aagaaaucgg uaacgguugc uucgaauucu accacaaaug cgacaacgaa ugcauggaau 1620 cuguucguaa cgguaccuac gacuacccua uagugagucg uauua 1665 <210> 30 <211> 482 <212> RNA <213> Artificial Sequence <220> <223> NA,NVT,VP1,VP10 <400> 30 gggcgauaau acaguuuugg acucaggugu gagauuuuau gaucaggacu augaaggaca 60 aauaacccca auggaauaug uaacuggguu guauaacuuu uggucagggc caauagaguu 120 acguuuugau uuuguuucaa augcguuuca cacuggaaca gugauuauau cagcggagua 180 uaaucgauca ucuacuaaua cggaugagug ucagucacac ucaacuuaua cuaaaacguu 240 ccacuuggga gaacaaaaau caguacauuu cacugugccu uauauauaug auacuguuau 300 gcggagaaau acggcuagcg ccuauuuacc gguaacugau uaugauaagg cagauaaugu 360 uaguagggcg caggcuacgg ggauuagagc agaaucuaaa augagaguga aagugagauc 420 gcccuauagu gagucguauu agucgacugc agaggccugc augcaagcuu ggcguaauca 480 ug 482 <210> 31 <211> 493 <212> RNA <213> Artificial Sequence <220> <223> NVT IV,NVT 4,VP20 <400> 31 gggcgauaau acaguuuugg acucaggugu gagauuuuau gaucaggacu augaaggaca 60 aauaacccca auggaauaug uaacuggguu guauaacuuu uggucagggc caauagaguu 120 acguuuugau uuuguuucaa augcguuuca cacuggaaca gugauuauau cagcggagua 180 uaaucgauca ucuacuaaua cggaugagug ucagucacac ucaacuuaua cuaaaacguu 240 ccacuuggga gaacaaaaau caguacauuu cacugugccu uauauauaug auacuguuau 300 gcggagaaau acggcuagcg ccuauuuacc gguaacugau uaugauaagg cagauaaugu 360 uaguagggcg caggcuacgg ggauuagagc agaaucuaaa augagaguga aagugagauc 420 gcccuauagu gagucguauu agucgacugc agaggccugc augcaagcuu uguuguuguu 480 guuguuguug uug 493 <210> 32 <211> 123 <212> RNA <213> Artificial Sequence <220> <223> NA1001 <400> 32 gggcgggcuu gucugcuccc ggcauccgcu uacagacaag cugugaccgu cuccgggagc 60 ugcauguguc agagguuuuc accgucauca ccgaaacgcg cgacccuaua gugagucgua 120 uua 123 <210> 33 <211> 173 <212> RNA <213> Artificial Sequence <220> <223> NA1501 <400> 33 gggcauaguu aagccagccc cgacacccgc caacacccgc ugacgcgccc ugacgggcuu 60 gucugcuccc ggcauccgcu uacagacaag cugugaccgu cuccgggagc ugcauguguc 120 agagguuuuc accgucauca ccgaaacgcg cgacccuaua gugagucgua uua 173 <210> 34 <211> 223 <212> RNA <213> Artificial Sequence <220> <223> NA2001 <400> 34 ggguauuuca caccgcauau ggugcacucu caguacaauc ugcucugaug ccgcauaguu 60 aagccagccc cgacacccgc caacacccgc ugacgcgccc ugacgggcuu gucugcuccc 120 ggcauccgcu uacagacaag cugugaccgu cuccgggagc ugcauguguc agagguuuuc 180 accgucauca ccgaaacgcg cgacccuaua gugagucgua uua 223 <210> 35 <211> 323 <212> RNA <213> Artificial Sequence <220> <223> NA3001 <400> 35 gggauagcga agaggcccgc accgaucgcc cuucccaaca guugcgcagc cugaauggcg 60 aauggcgccu gaugcgguau uuucuccuua cgcaucugug cgguauuuca caccgcauau 120 ggugcacucu caguacaauc ugcucugaug ccgcauaguu aagccagccc cgacacccgc 180 caacacccgc ugacgcgccc ugacgggcuu gucugcuccc ggcauccgcu uacagacaag 240 cugugaccgu cuccgggagc ugcauguguc agagguuuuc accgucauca ccgaaacgcg 300 cgacccuaua gugagucgua uua 323 <210> 36 <211> 423 <212> RNA <213> Artificial Sequence <220> <223> NA4001 <400> 36 ggguuggcac uggccgucgu uuuacaacgu cgugacuggg aaaacccugg cguuacccaa 60 cuuaaucgcc uugcagcaca ucccccuuuc gccagcuggc guaauagcga agaggcccgc 120 accgaucgcc cuucccaaca guugcgcagc cugaauggcg aauggcgccu gaugcgguau 180 uuucuccuua cgcaucugug cgguauuuca caccgcauau ggugcacucu caguacaauc 240 ugcucugaug ccgcauaguu aagccagccc cgacacccgc caacacccgc ugacgcgccc 300 ugacgggcuu gucugcuccc ggcauccgcu uacagacaag cugugaccgu cuccgggagc 360 ugcauguguc agagguuuuc accgucauca ccgaaacgcg cgacccuaua gugagucgua 420 uua 423 <210> 37 <211> 523 <212> RNA <213> Artificial Sequence <220> <223> NA5001 <400> 37 gggaacaauu ucacacagga aacagcuaug accaugauua cgaacucguc cucgguuccc 60 ggcgauccuc uggagauaau cguccacuug ccgucaugcu ucuuuggcac uggccgucgu 120 uuuacaacgu cgugacuggg aaaacccugg cguuacccaa cuuaaucgcc uugcagcaca 180 ucccccuuuc gccagcuggc guaauagcga agaggcccgc accgaucgcc cuucccaaca 240 guugcgcagc cugaauggcg aauggcgccu gaugcgguau uuucuccuua cgcaucugug 300 cgguauuuca caccgcauau ggugcacucu caguacaauc ugcucugaug ccgcauaguu 360 aagccagccc cgacacccgc caacacccgc ugacgcgccc ugacgggcuu gucugcuccc 420 ggcauccgcu uacagacaag cugugaccgu cuccgggagc ugcauguguc agagguuuuc 480 accgucauca ccgaaacgcg cgacccuaua gugagucgua uua 523 <210> 38 <211> 623 <212> RNA <213> Artificial Sequence <220> <223> NA6001 <400> 38 ggggagcgca acgcaauuaa ugugaguuag cucacucauu aggcacccca ggcuuuacac 60 uuuaugcuuc cggcucguau guugugugga auugugagcg gauaacaauu ucacacagga 120 aacagcuaug accaugauua cgaacucguc cucgguuccc ggcgauccuc uggagauaau 180 cguccacuug ccgucaugcu ucuuuggcac uggccgucgu uuuacaacgu cgugacuggg 240 aaaacccugg cguuacccaa cuuaaucgcc uugcagcaca ucccccuuuc gccagcuggc 300 guaauagcga agaggcccgc accgaucgcc cuucccaaca guugcgcagc cugaauggcg 360 aauggcgccu gaugcgguau uuucuccuua cgcaucugug cgguauuuca caccgcauau 420 ggugcacucu caguacaauc ugcucugaug ccgcauaguu aagccagccc cgacacccgc 480 caacacccgc ugacgcgccc ugacgggcuu gucugcuccc ggcauccgcu uacagacaag 540 cugugaccgu cuccgggagc ugcauguguc agagguuuuc accgucauca ccgaaacgcg 600 cgacccuaua gugagucgua uua 623 <210> 39 <211> 723 <212> RNA <213> Artificial Sequence <220> <223> NA7001 <400> 39 gggagcggaa gagcgcccaa uacgcaaacc gccucucccc gcgcguuggc cgauucauua 60 augcagcugg cacgacaggu uucccgacug gaaagcgggc agugagcgca acgcaauuaa 120 ugugaguuag cucacucauu aggcacccca ggcuuuacac uuuaugcuuc cggcucguau 180 guugugugga auugugagcg gauaacaauu ucacacagga aacagcuaug accaugauua 240 cgaacucguc cucgguuccc ggcgauccuc uggagauaau cguccacuug ccgucaugcu 300 ucuuuggcac uggccgucgu uuuacaacgu cgugacuggg aaaacccugg cguuacccaa 360 cuuaaucgcc uugcagcaca ucccccuuuc gccagcuggc guaauagcga agaggcccgc 420 accgaucgcc cuucccaaca guugcgcagc cugaauggcg aauggcgccu gaugcgguau 480 uuucuccuua cgcaucugug cgguauuuca caccgcauau ggugcacucu caguacaauc 540 ugcucugaug ccgcauaguu aagccagccc cgacacccgc caacacccgc ugacgcgccc 600 ugacgggcuu gucugcuccc ggcauccgcu uacagacaag cugugaccgu cuccgggagc 660 ugcauguguc agagguuuuc accgucauca ccgaaacgcg cgacccuaua gugagucgua 720 uua 723 <210> 40 <211> 823 <212> RNA <213> Artificial Sequence <220> <223> NA8001 <400> 40 ggguaucccc ugauucugug gauaaccgua uuaccgccuu ugagugagcu gauaccgcuc 60 gccgcagccg aacgaccgag cgcagcgagu cagugagcga ggaagcggaa gagcgcccaa 120 uacgcaaacc gccucucccc gcgcguuggc cgauucauua augcagcugg cacgacaggu 180 uucccgacug gaaagcgggc agugagcgca acgcaauuaa ugugaguuag cucacucauu 240 aggcacccca ggcuuuacac uuuaugcuuc cggcucguau guugugugga auugugagcg 300 gauaacaauu ucacacagga aacagcuaug accaugauua cgaacucguc cucgguuccc 360 ggcgauccuc uggagauaau cguccacuug ccgucaugcu ucuuuggcac uggccgucgu 420 uuuacaacgu cgugacuggg aaaacccugg cguuacccaa cuuaaucgcc uugcagcaca 480 ucccccuuuc gccagcuggc guaauagcga agaggcccgc accgaucgcc cuucccaaca 540 guugcgcagc cugaauggcg aauggcgccu gaugcgguau uuucuccuua cgcaucugug 600 cgguauuuca caccgcauau ggugcacucu caguacaauc ugcucugaug ccgcauaguu 660 aagccagccc cgacacccgc caacacccgc ugacgcgccc ugacgggcuu gucugcuccc 720 ggcauccgcu uacagacaag cugugaccgu cuccgggagc ugcauguguc agagguuuuc 780 accgucauca ccgaaacgcg cgacccuaua gugagucgua uua 823 <210> 41 <211> 123 <212> RNA <213> Artificial Sequence <220> <223> NA1002 <400> 41 ggggaaaaug guuaugguag guagauugaa gggagauggg guuaggcaau uaagcagaau 60 ggaguccaau gucagcuuca ccaaccaccu uuugucggaa uuccccuaua gugagucgua 120 uua 123 <210> 42 <211> 173 <212> RNA <213> Artificial Sequence <220> <223> NA1502 <400> 42 gggguucuug gaguuguuug aagaccaaau uguuuuuuug agaaaauuuu gaagaaaaug 60 guuaugguag guagauugaa gggagauggg guuaggcaau uaagcagaau ggaguccaau 120 gucagcuuca ccaaccaccu uuugucggaa uuccccuaua gugagucgua uua 173 <210> 43 <211> 223 <212> RNA <213> Artificial Sequence <220> <223> NA2002 <400> 43 gggaucugug ccauuucuug ugaaaaacuu caguuuuucc acucucucuc uguguucuug 60 gaguuguuug aagaccaaau uguuuuuuug agaaaauuuu gaagaaaaug guuaugguag 120 guagauugaa gggagauggg guuaggcaau uaagcagaau ggaguccaau gucagcuuca 180 ccaaccaccu uuugucggaa uuccccuaua gugagucgua uua 223 <210> 44 <211> 323 <212> RNA <213> Artificial Sequence <220> <223> NA3002 <400> 44 ggguccaaag aagagagaau uugaaacaag aggacccuuu uguguuuuag aaagauugga 60 ggaauucaga cuaagggucu guaucccuug ugccaugcua cuaaucugug ccauuucuug 120 ugaaaaacuu caguuuuucc acucucucuc uguguucuug gaguuguuug aagaccaaau 180 uguuuuuuug agaaaauuuu gaagaaaaug guuaugguag guagauugaa gggagauggg 240 guuaggcaau uaagcagaau ggaguccaau gucagcuuca ccaaccaccu uuugucggaa 300 uuccccuaua gugagucgua uua 323 <210> 45 <211> 423 <212> RNA <213> Artificial Sequence <220> <223> NA4002 <400> 45 gggaugcaga aauccuaaaa gaugacuucc ucaccacccu caaaacugaa ucuuucuuaa 60 acaccccuaa ugauuuugcu gaaauuuggg uuaguuucuu ugauccaaag aagagagaau 120 uugaaacaag aggacccuuu uguguuuuag aaagauugga ggaauucaga cuaagggucu 180 guaucccuug ugccaugcua cuaaucugug ccauuucuug ugaaaaacuu caguuuuucc 240 acucucucuc uguguucuug gaguuguuug aagaccaaau uguuuuuuug agaaaauuuu 300 gaagaaaaug guuaugguag guagauugaa gggagauggg guuaggcaau uaagcagaau 360 ggaguccaau gucagcuuca ccaaccaccu uuugucggaa uuccccuaua gugagucgua 420 uua 423 <210> 46 <211> 523 <212> RNA <213> Artificial Sequence <220> <223> NA5002 <400> 46 gggcgauugg aaagggauuu cgaaccgggu aauuuaacag uaccagauau aucuuugaug 60 gguucuagca caaucucaug ggguuucucu gcaguagcca cugaugcaga aauccuaaaa 120 gaugacuucc ucaccacccu caaaacugaa ucuuucuuaa acaccccuaa ugauuuugcu 180 gaaauuuggg uuaguuucuu ugauccaaag aagagagaau uugaaacaag aggacccuuu 240 uguguuuuag aaagauugga ggaauucaga cuaagggucu guaucccuug ugccaugcua 300 cuaaucugug ccauuucuug ugaaaaacuu caguuuuucc acucucucuc uguguucuug 360 gaguuguuug aagaccaaau uguuuuuuug agaaaauuuu gaagaaaaug guuaugguag 420 guagauugaa gggagauggg guuaggcaau uaagcagaau ggaguccaau gucagcuuca 480 ccaaccaccu uuugucggaa uuccccuaua gugagucgua uua 523 <210> 47 <211> 623 <212> RNA <213> Artificial Sequence <220> <223> NA6002 <400> 47 gggaaguguu uucaacgcac caagcaugua augaaugucg ucacuacuca guaaauuguc 60 aacaacaguc cuucccucag aaagggcagc aagaaggaga auacgauugg aaagggauuu 120 cgaaccgggu aauuuaacag uaccagauau aucuuugaug gguucuagca caaucucaug 180 ggguuucucu gcaguagcca cugaugcaga aauccuaaaa gaugacuucc ucaccacccu 240 caaaacugaa ucuuucuuaa acaccccuaa ugauuuugcu gaaauuuggg uuaguuucuu 300 ugauccaaag aagagagaau uugaaacaag aggacccuuu uguguuuuag aaagauugga 360 ggaauucaga cuaagggucu guaucccuug ugccaugcua cuaaucugug ccauuucuug 420 ugaaaaacuu caguuuuucc acucucucuc uguguucuug gaguuguuug aagaccaaau 480 uguuuuuuug agaaaauuuu gaagaaaaug guuaugguag guagauugaa gggagauggg 540 guuaggcaau uaagcagaau ggaguccaau gucagcuuca ccaaccaccu uuugucggaa 600 uuccccuaua gugagucgua uua 623 <210> 48 <211> 723 <212> RNA <213> Artificial Sequence <220> <223> NA7002 <400> 48 gggguuggau uucuuccuca gacuuuuuac cgacaggaaa cugcccacca caaccuucca 60 caauugcucg uugguuuuca uugucaucuu caacaugaag uccaaguguu uucaacgcac 120 caagcaugua augaaugucg ucacuacuca guaaauuguc aacaacaguc cuucccucag 180 aaagggcagc aagaaggaga auacgauugg aaagggauuu cgaaccgggu aauuuaacag 240 uaccagauau aucuuugaug gguucuagca caaucucaug ggguuucucu gcaguagcca 300 cugaugcaga aauccuaaaa gaugacuucc ucaccacccu caaaacugaa ucuuucuuaa 360 acaccccuaa ugauuuugcu gaaauuuggg uuaguuucuu ugauccaaag aagagagaau 420 uugaaacaag aggacccuuu uguguuuuag aaagauugga ggaauucaga cuaagggucu 480 guaucccuug ugccaugcua cuaaucugug ccauuucuug ugaaaaacuu caguuuuucc 540 acucucucuc uguguucuug gaguuguuug aagaccaaau uguuuuuuug agaaaauuuu 600 gaagaaaaug guuaugguag guagauugaa gggagauggg guuaggcaau uaagcagaau 660 ggaguccaau gucagcuuca ccaaccaccu uuugucggaa uuccccuaua gugagucgua 720 uua 723 <210> 49 <211> 823 <212> RNA <213> Artificial Sequence <220> <223> NA8002 <400> 49 gggauccuag gaacuccauc aagaacauau cuugaauguc cuccagcuac aguaacugcu 60 gcugucaacg gacgcauugc uguuccugca uuuccaagga auaguuggau uucuuccuca 120 gacuuuuuac cgacaggaaa cugcccacca caaccuucca caauugcucg uugguuuuca 180 uugucaucuu caacaugaag uccaaguguu uucaacgcac caagcaugua augaaugucg 240 ucacuacuca guaaauuguc aacaacaguc cuucccucag aaagggcagc aagaaggaga 300 auacgauugg aaagggauuu cgaaccgggu aauuuaacag uaccagauau aucuuugaug 360 gguucuagca caaucucaug ggguuucucu gcaguagcca cugaugcaga aauccuaaaa 420 gaugacuucc ucaccacccu caaaacugaa ucuuucuuaa acaccccuaa ugauuuugcu 480 gaaauuuggg uuaguuucuu ugauccaaag aagagagaau uugaaacaag aggacccuuu 540 uguguuuuag aaagauugga ggaauucaga cuaagggucu guaucccuug ugccaugcua 600 cuaaucugug ccauuucuug ugaaaaacuu caguuuuucc acucucucuc uguguucuug 660 gaguuguuug aagaccaaau uguuuuuuug agaaaauuuu gaagaaaaug guuaugguag 720 guagauugaa gggagauggg guuaggcaau uaagcagaau ggaguccaau gucagcuuca 780 ccaaccaccu uuugucggaa uuccccuaua gugagucgua uua 823 <210> 50 <211> 336 <212> RNA <213> Artificial Sequence <220> <223> R1 <400> 50 gggcacaguu uaccguugug agagucuucc agcagguuaa cagagugggu aacgguaacg 60 uuuuuuucca gaacgguguc aacggugucg guagaguugu uagcguggua accgaugcag 120 auggugucag cgucagcagc agccagagcg cacagcagaa ccagcagguu agcuuucaug 180 agcucggcuc ccccggaacc gccaccggcc uuucccucua gguaugccac cacgcugggu 240 uucuccagca gugauguuac cauccgacuu accguuaguu uugccuuguu uaggcaagcg 300 ugcagguugu cgaggucccu auagugaguc guauua 336 <210> 51 <211> 414 <212> RNA <213> Artificial Sequence <220> <223> R2 <400> 51 gggucgcauu ccggguuacc cagcagccaa ccagcgaugu ugcauuuacc cagcugcagc 60 ggagcgauac cuuucagacg gcacaguuua ccguugugag agucuuccag cagguuaaca 120 gaguggguaa cgguaacguu uuuuuccaga acggugucaa cggugucggu agaguuguua 180 gcgugguaac cgaugcagau ggugucagcg ucagcagcag ccagagcgca cagcagaacc 240 agcagguuag cuuucaugag cucggcuccc ccggaaccgc caccggccuu ucccucuagg 300 uaugccacca cgcuggguuu cuccagcagu gauguuacca uccgacuuac cguuaguuuu 360 gccuuguuua ggcaagcgug cagguugucg aggucccuau agugagucgu auua 414 <210> 52 <211> 483 <212> RNA <213> Artificial Sequence <220> <223> R3 <400> 52 ggguagcaga uaccguuuuc agaguucggg guuucaacga uguaagacca agaacgaacc 60 ggcagcagcg ggucgcauuc cggguuaccc agcagccaac cagcgauguu gcauuuaccc 120 agcugcagcg gagcgauacc uuucagacgg cacaguuuac cguugugaga gucuuccagc 180 agguuaacag aguggguaac gguaacguuu uuuuccagaa cggugucaac ggugucggua 240 gaguuguuag cgugguaacc gaugcagaug gugucagcgu cagcagcagc cagagcgcac 300 agcagaacca gcagguuagc uuucaugagc ucggcucccc cggaaccgcc accggccuuu 360 cccucuaggu augccaccac gcuggguuuc uccagcagug auguuaccau ccgacuuacc 420 guuaguuuug ccuuguuuag gcaagcgugc agguugucga ggucccuaua gugagucgua 480 uua 483 <210> 53 <211> 605 <212> RNA <213> Artificial Sequence <220> <223> R4 <400> 53 ggguaacacc guugguguug ugguucggcc aagaagauuc uuucgggaag auuucgaaac 60 guucgaaaga agaaacagaa gacagcuguu cacgcaguuc uucguagucg augaagucac 120 ccggguagca gauaccguuu ucagaguucg ggguuucaac gauguaagac caagaacgaa 180 ccggcagcag cgggucgcau uccggguuac ccagcagcca accagcgaug uugcauuuac 240 ccagcugcag cggagcgaua ccuuucagac ggcacaguuu accguuguga gagucuucca 300 gcagguuaac agagugggua acgguaacgu uuuuuuccag aacgguguca acggugucgg 360 uagaguuguu agcgugguaa ccgaugcaga uggugucagc gucagcagca gccagagcgc 420 acagcagaac cagcagguua gcuuucauga gcucggcucc cccggaaccg ccaccggccu 480 uucccucuag guaugccacc acgcuggguu ucuccagcag ugauguuacc auccgacuua 540 ccguuaguuu ugccuuguuu aggcaagcgu gcagguuguc gaggucccua uagugagucg 600 uauua 605 <210> 54 <211> 681 <212> RNA <213> Artificial Sequence <220> <223> R5 <400> 54 ggguaagaac cuucuuuuuc ggucagccac agcagguuac gguagaaaga agauuuaccu 60 ucgugagagc aagcagcggu aacaccguug guguuguggu ucggccaaga agauucuuuc 120 gggaagauuu cgaaacguuc gaaagaagaa acagaagaca gcuguucacg caguucuucg 180 uagucgauga agucacccgg guagcagaua ccguuuucag aguucggggu uucaacgaug 240 uaagaccaag aacgaaccgg cagcagcggg ucgcauuccg gguuacccag cagccaacca 300 gcgauguugc auuuacccag cugcagcgga gcgauaccuu ucagacggca caguuuaccg 360 uugugagagu cuuccagcag guuaacagag uggguaacgg uaacguuuuu uuccagaacg 420 gugucaacgg ugucgguaga guuguuagcg ugguaaccga ugcagauggu gucagcguca 480 gcagcagcca gagcgcacag cagaaccagc agguuagcuu ucaugagcuc ggcucccccg 540 gaaccgccac cggccuuucc cucuagguau gccaccacgc uggguuucuc cagcagugau 600 guuaccaucc gacuuaccgu uaguuuugcc uuguuuaggc aagcgugcag guugucgagg 660 ucccuauagu gagucguauu a 681 <210> 55 <211> 750 <212> RNA <213> Artificial Sequence <220> <223> R6 <400> 55 ggguggugga uaccccacag aaccagaacu ucuuuaccuu uuuuguuaac guaagaguuu 60 uucaguuucg gguaagaacc uucuuuuucg gucagccaca gcagguuacg guagaaagaa 120 gauuuaccuu cgugagagca agcagcggua acaccguugg uguugugguu cggccaagaa 180 gauucuuucg ggaagauuuc gaaacguucg aaagaagaaa cagaagacag cuguucacgc 240 aguucuucgu agucgaugaa gucacccggg uagcagauac cguuuucaga guucgggguu 300 ucaacgaugu aagaccaaga acgaaccggc agcagcgggu cgcauuccgg guuacccagc 360 agccaaccag cgauguugca uuuacccagc ugcagcggag cgauaccuuu cagacggcac 420 aguuuaccgu ugugagaguc uuccagcagg uuaacagagu ggguaacggu aacguuuuuu 480 uccagaacgg ugucaacggu gucgguagag uuguuagcgu gguaaccgau gcagauggug 540 ucagcgucag cagcagccag agcgcacagc agaaccagca gguuagcuuu caugagcucg 600 gcucccccgg aaccgccacc ggccuuuccc ucuagguaug ccaccacgcu ggguuucucc 660 agcagugaug uuaccauccg acuuaccguu aguuuugccu uguuuaggca agcgugcagg 720 uugucgaggu cccuauagug agucguauua 750 <210> 56 <211> 839 <212> RNA <213> Artificial Sequence <220> <223> R7 <400> 56 gggugaaacg acgguuguag uuagagguaa caacagaaac guaagcguuu ucguucuggu 60 acagguucug cuguucuuua gaguucggcg ggugguggau accccacaga accagaacuu 120 cuuuaccuuu uuuguuaacg uaagaguuuu ucaguuucgg guaagaaccu ucuuuuucgg 180 ucagccacag cagguuacgg uagaaagaag auuuaccuuc gugagagcaa gcagcgguaa 240 caccguuggu guugugguuc ggccaagaag auucuuucgg gaagauuucg aaacguucga 300 aagaagaaac agaagacagc uguucacgca guucuucgua gucgaugaag ucacccgggu 360 agcagauacc guuuucagag uucgggguuu caacgaugua agaccaagaa cgaaccggca 420 gcagcggguc gcauuccggg uuacccagca gccaaccagc gauguugcau uuacccagcu 480 gcagcggagc gauaccuuuc agacggcaca guuuaccguu gugagagucu uccagcaggu 540 uaacagagug gguaacggua acguuuuuuu ccagaacggu gucaacggug ucgguagagu 600 uguuagcgug guaaccgaug cagauggugu cagcgucagc agcagccaga gcgcacagca 660 gaaccagcag guuagcuuuc augagcucgg cucccccgga accgccaccg gccuuucccu 720 cuagguaugc caccacgcug gguuucucca gcagugaugu uaccauccga cuuaccguua 780 guuuugccuu guuuaggcaa gcgugcaggu ugucgagguc ccuauaguga gucguauua 839 <210> 57 <211> 902 <212> RNA <213> Artificial Sequence <220> <223> R8 <400> 57 ggguccagua guaguucaua cgaccagccu ggucacgaac uuucggacgu ucagcgauuu 60 ccggggugaa acgacgguug uaguuagagg uaacaacaga aacguaagcg uuuucguucu 120 gguacagguu cugcuguucu uuagaguucg gcggguggug gauaccccac agaaccagaa 180 cuucuuuacc uuuuuuguua acguaagagu uuuucaguuu cggguaagaa ccuucuuuuu 240 cggucagcca cagcagguua cgguagaaag aagauuuacc uucgugagag caagcagcgg 300 uaacaccguu gguguugugg uucggccaag aagauucuuu cgggaagauu ucgaaacguu 360 cgaaagaaga aacagaagac agcuguucac gcaguucuuc guagucgaug aagucacccg 420 gguagcagau accguuuuca gaguucgggg uuucaacgau guaagaccaa gaacgaaccg 480 gcagcagcgg gucgcauucc ggguuaccca gcagccaacc agcgauguug cauuuaccca 540 gcugcagcgg agcgauaccu uucagacggc acaguuuacc guugugagag ucuuccagca 600 gguuaacaga guggguaacg guaacguuuu uuuccagaac ggugucaacg gugucgguag 660 aguuguuagc gugguaaccg augcagaugg ugucagcguc agcagcagcc agagcgcaca 720 gcagaaccag cagguuagcu uucaugagcu cggcuccccc ggaaccgcca ccggccuuuc 780 ccucuaggua ugccaccacg cuggguuucu ccagcaguga uguuaccauc cgacuuaccg 840 uuaguuuugc cuuguuuagg caagcgugca gguugucgag gucccuauag ugagucguau 900 ua 902 <210> 58 <211> 1049 <212> RNA <213> Artificial Sequence <220> <223> R9 <400> 58 gggucuggca uuugguguug cauucgugca uagaagcguu agaggugaug auaccagaac 60 cgaaaccacg agacagagcg aaagcguaca ucggagcgau cagguuaccg uuagcuucga 120 agaugauggu gucacccggu uucagcaggg uccaguagua guucauacga ccagccuggu 180 cacgaacuuu cggacguuca gcgauuuccg gggugaaacg acgguuguag uuagagguaa 240 caacagaaac guaagcguuu ucguucuggu acagguucug cuguucuuua gaguucggcg 300 ggugguggau accccacaga accagaacuu cuuuaccuuu uuuguuaacg uaagaguuuu 360 ucaguuucgg guaagaaccu ucuuuuucgg ucagccacag cagguuacgg uagaaagaag 420 auuuaccuuc gugagagcaa gcagcgguaa caccguuggu guugugguuc ggccaagaag 480 auucuuucgg gaagauuucg aaacguucga aagaagaaac agaagacagc uguucacgca 540 guucuucgua gucgaugaag ucacccgggu agcagauacc guuuucagag uucgggguuu 600 caacgaugua agaccaagaa cgaaccggca gcagcggguc gcauuccggg uuacccagca 660 gccaaccagc gauguugcau uuacccagcu gcagcggagc gauaccuuuc agacggcaca 720 guuuaccguu gugagagucu uccagcaggu uaacagagug gguaacggua acguuuuuuu 780 ccagaacggu gucaacggug ucgguagagu uguuagcgug guaaccgaug cagauggugu 840 cagcgucagc agcagccaga gcgcacagca gaaccagcag guuagcuuuc augagcucgg 900 cucccccgga accgccaccg gccuuucccu cuagguaugc caccacgcug gguuucucca 960 gcagugaugu uaccauccga cuuaccguua guuuugccuu guuuaggcaa gcgugcaggu 1020 ugucgagguc ccuauaguga gucguauua 1049 <210> 59 <211> 1170 <212> RNA <213> Artificial Sequence <220> <223> R10 <400> 59 gggauguuac gcagaccggu aaccauacgc aguuuagcag aacgaacgua uuucgggcau 60 ucaccgaugg uaaccgggug gauguucugg uacggcagag aagaguugau agcacccagc 120 ggggucuggc auuugguguu gcauucgugc auagaagcgu uagaggugau gauaccagaa 180 ccgaaaccac gagacagagc gaaagcguac aucggagcga ucagguuacc guuagcuucg 240 aagaugaugg ugucacccgg uuucagcagg guccaguagu aguucauacg accagccugg 300 ucacgaacuu ucggacguuc agcgauuucc ggggugaaac gacgguugua guuagaggua 360 acaacagaaa cguaagcguu uucguucugg uacagguucu gcuguucuuu agaguucggc 420 ggguggugga uaccccacag aaccagaacu ucuuuaccuu uuuuguuaac guaagaguuu 480 uucaguuucg gguaagaacc uucuuuuucg gucagccaca gcagguuacg guagaaagaa 540 gauuuaccuu cgugagagca agcagcggua acaccguugg uguugugguu cggccaagaa 600 gauucuuucg ggaagauuuc gaaacguucg aaagaagaaa cagaagacag cuguucacgc 660 aguucuucgu agucgaugaa gucacccggg uagcagauac cguuuucaga guucgggguu 720 ucaacgaugu aagaccaaga acgaaccggc agcagcgggu cgcauuccgg guuacccagc 780 agccaaccag cgauguugca uuuacccagc ugcagcggag cgauaccuuu cagacggcac 840 aguuuaccgu ugugagaguc uuccagcagg uuaacagagu ggguaacggu aacguuuuuu 900 uccagaacgg ugucaacggu gucgguagag uuguuagcgu gguaaccgau gcagauggug 960 ucagcgucag cagcagccag agcgcacagc agaaccagca gguuagcuuu caugagcucg 1020 gcucccccgg aaccgccacc ggccuuuccc ucuagguaug ccaccacgcu ggguuucucc 1080 agcagugaug uuaccauccg acuuaccguu aguuuugccu uguuuaggca agcgugcagg 1140 uugucgaggu cccuauagug agucguauua 1170 <210> 60 <211> 1665 <212> RNA <213> Artificial Sequence <220> <223> R11 <400> 60 ggguagucgu agguaccguu acgaacagau uccaugcauu cguugucgca uuugugguag 60 aauucgaagc aaccguuacc gauuucuuua gcguuguuuu ucagcugaga uuuaacuuuu 120 ucguacaggu uuuuaacguu agagucgugg aaguccaggg uacguucguu uuccagcaga 180 accagcaguu cagcguugua gguccagaug uccaggaaac cgucgucaac uuuuuuguuc 240 agguuuucca uacguuuuuc caguuuguug aauucuuuac caacagcggu gaacuggaug 300 uucauuuuuu cgauaacggu guuaacuuug uuggugauac cguugauagc guucugggua 360 gauuucuggu cagcagcgua accagaaccc uguucguucu ggugguggua accguaccaa 420 ccgucgauca uaccggucca accaccuucg augaaaccag cgauagcacc gaacagacca 480 cgagacugga uagacgggau guuacgcaga ccgguaacca uacgcaguuu agcagaacga 540 acguauuucg ggcauucacc gaugguaacc ggguggaugu ucugguacgg cagagaagag 600 uugauagcac ccagcggggu cuggcauuug guguugcauu cgugcauaga agcguuagag 660 gugaugauac cagaaccgaa accacgagac agagcgaaag cguacaucgg agcgaucagg 720 uuaccguuag cuucgaagau gaugguguca cccgguuuca gcagggucca guaguaguuc 780 auacgaccag ccuggucacg aacuuucgga cguucagcga uuuccggggu gaaacgacgg 840 uuguaguuag agguaacaac agaaacguaa gcguuuucgu ucugguacag guucugcugu 900 ucuuuagagu ucggcgggug guggauaccc cacagaacca gaacuucuuu accuuuuuug 960 uuaacguaag aguuuuucag uuucggguaa gaaccuucuu uuucggucag ccacagcagg 1020 uuacgguaga aagaagauuu accuucguga gagcaagcag cgguaacacc guugguguug 1080 ugguucggcc aagaagauuc uuucgggaag auuucgaaac guucgaaaga agaaacagaa 1140 gacagcuguu cacgcaguuc uucguagucg augaagucac ccggguagca gauaccguuu 1200 ucagaguucg ggguuucaac gauguaagac caagaacgaa ccggcagcag cgggucgcau 1260 uccggguuac ccagcagcca accagcgaug uugcauuuac ccagcugcag cggagcgaua 1320 ccuuucagac ggcacaguuu accguuguga gagucuucca gcagguuaac agagugggua 1380 acgguaacgu uuuuuuccag aacgguguca acggugucgg uagaguuguu agcgugguaa 1440 ccgaugcaga uggugucagc gucagcagca gccagagcgc acagcagaac cagcagguua 1500 gcuuucauga gcucggcucc cccggaaccg ccaccggccu uucccucuag guaugccacc 1560 acgcuggguu ucuccagcag ugauguuacc auccgacuua ccguuaguuu ugccuuguuu 1620 aggcaagcgu gcagguuguc gaggucccua uagugagucg uauua 1665 <210> 61 <211> 475 <212> RNA <213> Artificial Sequence <220> <223> NA,NVT,VP1,VP10 <400> 61 gggcgaucuc acuuucacuc ucauuuuaga uucugcucua auccccguag ccugcgcccu 60 acuaacauua ucugccuuau cauaaucagu uaccgguaaa uaggcgcuag ccguauuucu 120 ccgcauaaca guaucauaua uauaaggcac agugaaaugu acugauuuuu guucucccaa 180 guggaacguu uuaguauaag uugaguguga cugacacuca uccguauuag uagaugaucg 240 auuauacucc gcugauauaa ucacuguucc agugugaaac gcauuugaaa caaaaucaaa 300 acguaacucu auuggcccug accaaaaguu auacaaccca guuacauauu ccauuggggu 360 uauuuguccu ucauaguccu gaucauaaaa ucucacaccu gaguccaaaa cuguauuauc 420 gcccuauagu gagucguauu aggauccgau aucuagaugc auucgcgagg uaccg 475 <210> 62 <211> 499 <212> RNA <213> Artificial Sequence <220> <223> NVT IV,NVT 4,VP20 <400> 62 gggcgaucuc acuuucacuc ucauuuuaga uucugcucua auccccguag ccugcgcccu 60 acuaacauua ucugccuuau cauaaucagu uaccgguaaa uaggcgcuag ccguauuucu 120 ccgcauaaca guaucauaua uauaaggcac agugaaaugu acugauuuuu guucucccaa 180 guggaacguu uuaguauaag uugaguguga cugacacuca uccguauuag uagaugaucg 240 auuauacucc gcugauauaa ucacuguucc agugugaaac gcauuugaaa caaaaucaaa 300 acguaacucu auuggcccug accaaaaguu auacaaccca guuacauauu ccauuggggu 360 uauuuguccu ucauaguccu gaucauaaaa ucucacaccu gaguccaaaa cuguauuauc 420 gcccuauagu gagucguauu aggauccgau aucuagaugc auucgcgagg uaccguuguu 480 guuguuguug uuguuguug 499 <210> 63 <211> 106 <212> RNA <213> Artificial Sequence <220> <223> NA1001 <400> 63 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgccc 106 <210> 64 <211> 156 <212> RNA <213> Artificial Sequence <220> <223> NA1501 <400> 64 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augccc 156 <210> 65 <211> 206 <212> RNA <213> Artificial Sequence <220> <223> NA2001 <400> 65 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccc 206 <210> 66 <211> 306 <212> RNA <213> Artificial Sequence <220> <223> NA3001 <400> 66 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauccc 306 <210> 67 <211> 406 <212> RNA <213> Artificial Sequence <220> <223> NA4001 <400> 67 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaccc 406 <210> 68 <211> 506 <212> RNA <213> Artificial Sequence <220> <223> NA5001 <400> 68 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guuccc 506 <210> 69 <211> 606 <212> RNA <213> Artificial Sequence <220> <223> NA6001 <400> 69 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guuauccgcu cacaauucca cacaacauac gagccggaag 540 cauaaagugu aaagccuggg gugccuaaug agugagcuaa cucacauuaa uugcguugcg 600 cucccc 606 <210> 70 <211> 706 <212> RNA <213> Artificial Sequence <220> <223> NA7001 <400> 70 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guuauccgcu cacaauucca cacaacauac gagccggaag 540 cauaaagugu aaagccuggg gugccuaaug agugagcuaa cucacauuaa uugcguugcg 600 cucacugccc gcuuuccagu cgggaaaccu gucgugccag cugcauuaau gaaucggcca 660 acgcgcgggg agaggcgguu ugcguauugg gcgcucuucc gcuccc 706 <210> 71 <211> 806 <212> RNA <213> Artificial Sequence <220> <223> NA8001 <400> 71 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guuauccgcu cacaauucca cacaacauac gagccggaag 540 cauaaagugu aaagccuggg gugccuaaug agugagcuaa cucacauuaa uugcguugcg 600 cucacugccc gcuuuccagu cgggaaaccu gucgugccag cugcauuaau gaaucggcca 660 acgcgcgggg agaggcgguu ugcguauugg gcgcucuucc gcuuccucgc ucacugacuc 720 gcugcgcucg gucguucggc ugcggcgagc gguaucagcu cacucaaagg cgguaauacg 780 guuauccaca gaaucagggg auaccc 806 <210> 72 <211> 106 <212> RNA <213> Artificial Sequence <220> <223> NA1002 <400> 72 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucccc 106 <210> 73 <211> 156 <212> RNA <213> Artificial Sequence <220> <223> NA1502 <400> 73 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacccc 156 <210> 74 <211> 206 <212> RNA <213> Artificial Sequence <220> <223> NA2002 <400> 74 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauccc 206 <210> 75 <211> 306 <212> RNA <213> Artificial Sequence <220> <223> NA3002 <400> 75 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaccc 306 <210> 76 <211> 406 <212> RNA <213> Artificial Sequence <220> <223> NA4002 <400> 76 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug cauccc 406 <210> 77 <211> 506 <212> RNA <213> Artificial Sequence <220> <223> NA5002 <400> 77 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucgccc 506 <210> 78 <211> 606 <212> RNA <213> Artificial Sequence <220> <223> NA6002 <400> 78 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucguauucuc cuucuugcug cccuuucuga gggaaggacu 540 guuguugaca auuuacugag uagugacgac auucauuaca ugcuuggugc guugaaaaca 600 cuuccc 606 <210> 79 <211> 706 <212> RNA <213> Artificial Sequence <220> <223> NA7002 <400> 79 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucguauucuc cuucuugcug cccuuucuga gggaaggacu 540 guuguugaca auuuacugag uagugacgac auucauuaca ugcuuggugc guugaaaaca 600 cuuggacuuc auguugaaga ugacaaugaa aaccaacgag caauugugga agguuguggu 660 gggcaguuuc cugucgguaa aaagucugag gaagaaaucc aacccc 706 <210> 80 <211> 806 <212> RNA <213> Artificial Sequence <220> <223> NA8002 <400> 80 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucguauucuc cuucuugcug cccuuucuga gggaaggacu 540 guuguugaca auuuacugag uagugacgac auucauuaca ugcuuggugc guugaaaaca 600 cuuggacuuc auguugaaga ugacaaugaa aaccaacgag caauugugga agguuguggu 660 gggcaguuuc cugucgguaa aaagucugag gaagaaaucc aacuauuccu uggaaaugca 720 ggaacagcaa ugcguccguu gacagcagca guuacuguag cuggaggaca uucaagauau 780 guucuugaug gaguuccuag gauccc 806 <210> 81 <211> 319 <212> RNA <213> Artificial Sequence <220> <223> R1 <400> 81 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccc 319 <210> 82 <211> 397 <212> RNA <213> Artificial Sequence <220> <223> R2 <400> 82 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgaccc 397 <210> 83 <211> 466 <212> RNA <213> Artificial Sequence <220> <223> R3 <400> 83 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuaccc 466 <210> 84 <211> 588 <212> RNA <213> Artificial Sequence <220> <223> R4 <400> 84 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccc 588 <210> 85 <211> 664 <212> RNA <213> Artificial Sequence <220> <223> R5 <400> 85 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 accc 664 <210> 86 <211> 733 <212> RNA <213> Artificial Sequence <220> <223> R6 <400> 86 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca ccc 733 <210> 87 <211> 822 <212> RNA <213> Artificial Sequence <220> <223> R7 <400> 87 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cc 822 <210> 88 <211> 885 <212> RNA <213> Artificial Sequence <220> <223> R8 <400> 88 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gaccc 885 <210> 89 <211> 1032 <212> RNA <213> Artificial Sequence <220> <223> R9 <400> 89 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gacccugcug aaaccgggug 900 acaccaucau cuucgaagcu aacgguaacc ugaucgcucc gauguacgcu uucgcucugu 960 cucgugguuu cgguucuggu aucaucaccu cuaacgcuuc uaugcacgaa ugcaacacca 1020 aaugccagac cc 1032 <210> 90 <211> 1153 <212> RNA <213> Artificial Sequence <220> <223> R10 <400> 90 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gacccugcug aaaccgggug 900 acaccaucau cuucgaagcu aacgguaacc ugaucgcucc gauguacgcu uucgcucugu 960 cucgugguuu cgguucuggu aucaucaccu cuaacgcuuc uaugcacgaa ugcaacacca 1020 aaugccagac cccgcugggu gcuaucaacu cuucucugcc guaccagaac auccacccgg 1080 uuaccaucgg ugaaugcccg aaauacguuc guucugcuaa acugcguaug guuaccgguc 1140 ugcguaacau ccc 1153 <210> 91 <211> 1648 <212> RNA <213> Artificial Sequence <220> <223> R11 <400> 91 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gacccugcug aaaccgggug 900 acaccaucau cuucgaagcu aacgguaacc ugaucgcucc gauguacgcu uucgcucugu 960 cucgugguuu cgguucuggu aucaucaccu cuaacgcuuc uaugcacgaa ugcaacacca 1020 aaugccagac cccgcugggu gcuaucaacu cuucucugcc guaccagaac auccacccgg 1080 uuaccaucgg ugaaugcccg aaauacguuc guucugcuaa acugcguaug guuaccgguc 1140 ugcguaacau cccgucuauc cagucucgug gucuguucgg ugcuaucgcu gguuucaucg 1200 aaggugguug gaccgguaug aucgacgguu gguacgguua ccaccaccag aacgaacagg 1260 guucugguua cgcugcugac cagaaaucua cccagaacgc uaucaacggu aucaccaaca 1320 aaguuaacac cguuaucgaa aaaaugaaca uccaguucac cgcuguuggu aaagaauuca 1380 acaaacugga aaaacguaug gaaaaccuga acaaaaaagu ugacgacggu uuccuggaca 1440 ucuggaccua caacgcugaa cugcugguuc ugcuggaaaa cgaacguacc cuggacuucc 1500 acgacucuaa cguuaaaaac cuguacgaaa aaguuaaauc ucagcugaaa aacaacgcua 1560 aagaaaucgg uaacgguugc uucgaauucu accacaaaug cgacaacgaa ugcauggaau 1620 cuguucguaa cgguaccuac gacuaccc 1648 <210> 92 <211> 424 <212> RNA <213> Artificial Sequence <220> <223> NA,NVT,VP1,VP10 <400> 92 gggcgauaau acaguuuugg acucaggugu gagauuuuau gaucaggacu augaaggaca 60 aauaacccca auggaauaug uaacuggguu guauaacuuu uggucagggc caauagaguu 120 acguuuugau uuuguuucaa augcguuuca cacuggaaca gugauuauau cagcggagua 180 uaaucgauca ucuacuaaua cggaugagug ucagucacac ucaacuuaua cuaaaacguu 240 ccacuuggga gaacaaaaau caguacauuu cacugugccu uauauauaug auacuguuau 300 gcggagaaau acggcuagcg ccuauuuacc gguaacugau uaugauaagg cagauaaugu 360 uaguagggcg caggcuacgg ggauuagagc agaaucuaaa augagaguga aagugagauc 420 gccc 424 <210> 93 <211> 424 <212> RNA <213> Artificial Sequence <220> <223> NVT IV, NVT 4, VP20 <400> 93 gggcgauaau acaguuuugg acucaggugu gagauuuuau gaucaggacu augaaggaca 60 aauaacccca auggaauaug uaacuggguu guauaacuuu uggucagggc caauagaguu 120 acguuuugau uuuguuucaa augcguuuca cacuggaaca gugauuauau cagcggagua 180 uaaucgauca ucuacuaaua cggaugagug ucagucacac ucaacuuaua cuaaaacguu 240 ccacuuggga gaacaaaaau caguacauuu cacugugccu uauauauaug auacuguuau 300 gcggagaaau acggcuagcg ccuauuuacc gguaacugau uaugauaagg cagauaaugu 360 uaguagggcg caggcuacgg ggauuagagc agaaucuaaa augagaguga aagugagauc 420 gccc 424

Claims (38)

In the mixed structure ribonucleic acid (hsRNA) comprising double-stranded ribonucleic acid (dsRNA) and single-stranded ribonucleic acid (ssRNA), the double-stranded ribonucleic acid (dsRNA) has complementarity, and the single-stranded ribonucleic acid (ssRNA) is A mixed structure ribonucleic acid (hsRNA) selected from the following, characterized in that it is located at both 3'-ends of the double-stranded ribonucleic acid (dsRNA):
(1) a mixed structure ribonucleic acid comprising a double-stranded ribonucleic acid and a single-stranded ribonucleic acid formed as a result of complementary binding between the nucleotide sequence of SEQ ID NO: 30 and the nucleotide sequence of SEQ ID NO: 61; And
(2) A mixed structure ribonucleic acid comprising a double-stranded ribonucleic acid and a single-stranded ribonucleic acid formed as a result of complementary bonding of the nucleotide sequence of SEQ ID NO: 31 and the nucleotide sequence of SEQ ID NO: 62.
A vaccine composition for preventing or treating viral or bacterial infection, comprising the mixed structure ribonucleic acid (hsRNA) and immunogen of claim 1, wherein the mixed structure ribonucleic acid (hsRNA) acts as a TLR3 and TLR7/8 ligand.
The method of claim 2, wherein the immunogen comprises a trivalent seasonal influenza vaccine, a tetravalent seasonal influenza vaccine, an inactivated influenza vaccine, or a Neisseria meningitidis groups A, C, Y and W-135 vaccine. , Virus or bacterial infection prevention or treatment vaccine composition.
The vaccine composition for preventing or treating viral or bacterial infection according to claim 2, which comprises an additional adjuvant.
The method of claim 4, wherein the additional adjuvant is one from the group consisting of oil-in-water emulsion adjuvant, aluminum salt, Freund adjuvant, and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane). Vaccine composition for preventing or treating viral or bacterial infection, characterized in that it is selected above.
A vaccine composition for preventing or treating cancer comprising the mixed structure ribonucleic acid (hsRNA) of claim 1.
The vaccine composition for preventing or treating cancer according to claim 6, further comprising an immunogen.
The method of claim 7, wherein the immunogen is one or more from the group consisting of synthetic peptides, recombinant antigens including all or part of them, inactivated or living attenuated organisms, those derived from cells, and DNA or mRNA encoding antigenic proteins. Characterized in that selected, cancer prevention or treatment vaccine composition.
The vaccine composition for preventing or treating cancer according to claim 6 or 7, characterized in that it contains an additional adjuvant.
The method of claim 9, wherein the additional adjuvant is one from the group consisting of oil-in-water emulsion adjuvant, aluminum salt, Freund adjuvant, and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane). A vaccine composition for preventing or treating cancer, characterized in that it is selected above.
The mixed structure ribonucleic acid (hsRNA) of claim 1; And a pharmaceutical composition for preventing or treating cancer comprising an OX40 antibody or a PD-1 antibody.
The pharmaceutical composition for preventing or treating cancer according to claim 11, wherein the OX40 antibody is an OX40 agonist antibody.
The pharmaceutical composition for preventing or treating cancer according to claim 11, wherein the PD-1 antibody is a PD-1 antagonist antibody. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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