Aptamer for adeno-associated virus serotype 9 and use thereof

Abstract

To provide an aptamer capable of specifically binding to adeno-associated virus serotype 9 (AAV9) and usable for detection or purification of AAV9 or efficient drug delivery of the virus.SOLUTION: An aptamer for adeno-associated virus serotype 9 (AAV9), comprising (a) or (b): (a) a nucleotide sequence represented by SEQ ID No. 1: 5'-GUGCGUACAUUUGUUGUAAUACACAAAUGCCACC-3' (uracil (U) may be thymine (T)); and (b) a nucleotide sequence with one to several nucleotides substituted, deleted, inserted and / or added from the nucleotide sequence of (a).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an aptamer that specifically binds to adeno-associated virus serotype 9 (AAV9) and uses thereof. [Background technology]

[0002] Adeno-associated virus serotype 9 (AAV9) is a type of non-pathogenic adeno-associated virus that is highly safe, with low immunogenicity and other characteristics. Due to this, it is a viral vector that is being used not only as a research tool but is also gaining increasing medical applications. In fact, with the approval of Zolgensma, a gene therapy drug for spinal muscular atrophy, in Japan in 2019, expectations are growing for its application to other genetic diseases.

[0003] To realize the wide-ranging medical applications of AAV9, it is important to develop AAV9-binding molecules that can be used for its detection, purification, and effective delivery of the virus to expression sites. Camelid-derived VHH antibodies, which have high binding affinity and are stable to heat and acid, are widely used for affinity purification of AAV vectors. However, development of affinity ligands that specifically and tightly bind to AAV9 while enabling elution of virus particles under mild conditions, as well as AAV9 delivery molecules with low immunogenicity and high specificity as alternatives to antibodies, has lagged behind.

[0004] Aptamers are single-stranded nucleic acids that bind to target molecules with high affinity and specificity by forming a unique secondary structure. They are relatively small molecules, can be chemically synthesized and modified, and have advantages such as high thermal stability and low immunogenicity. Furthermore, some aptamers for receptors on the cell membrane are taken up into cells via endocytosis, and have been used for intracellular delivery of membrane-impermeable drugs and for crossing the blood-brain barrier (BBB) ​​(e.g., Non-Patent Documents 1 and 2).

[0005] As an aptamer for a viral vector, a DNA aptamer for adenovirus serotype 2 (AdV2) has been reported (Patent Document 1), but no aptamer for AAV9 is known (it should be noted that the inventors have confirmed that the AdV2 aptamer does not bind to AAV9). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2020 / 126435 [Non-patent literature]

[0007] [Non-Patent Document 1] Xie, S. et al., J. Am. Chem. Soc. 2023, 145, 7677-7691 [Non-patent document 2] Bukari, B. et al., Biomedicines 2020, 8, 120; doi:10.3390 / biomedicines8050120 Summary of the Invention [Problem to be solved by the invention]

[0008] The first object of the present invention is to provide a novel binding molecule that specifically and tightly binds to AAV9 while enabling AAV9 particles to be eluted under mild conditions, and to provide a means for efficiently detecting or purifying AAV9 using the binding molecule. The second objective of the present invention is to provide target cell-directed molecules, including AAV9-binding molecules, which have low immunogenicity and high affinity and specificity for AAV9 as drug delivery molecules instead of antibodies, and to provide a means for using such molecules to deliver and express AAV9 and therapeutic substances encapsulated therein into target cells. [Means for solving the problem]

[0009] The present inventors conducted extensive research to solve the above problems and successfully identified a consensus primary structure by using the SELEX method to select a group of aptamers with similar nucleotide sequences that exhibit high binding affinity to AAV9. They also predicted the secondary structures of these aptamers and determined the common partial secondary structure that is presumed to contribute to binding to AAV9. Based on the most frequently found aptamer sequence, they gradually shortened the sequence to retain the partial secondary structure, and successfully isolated a 35-nucleotide aptamer with high binding affinity.

[0010] The aptamer was immobilized on a sensor chip, and its binding to AAV9 and elution of the virus particles were evaluated by surface plasmon resonance (SPR). AAV9 was captured on the solid-phase support and eluted from the support using a biochemically mild eluent. This binding and elution of AAV9 could be repeated. Furthermore, when the binding to other AAV serotypes was examined, it was revealed that the aptamer specifically bound to AAV9, but not to any of the other serotypes.

[0011] Furthermore, the present inventors have constructed a bispecific aptamer by linking an AAV9 aptamer to a receptor aptamer that enables receptor-mediated intracellular delivery and transcytosis, and confirmed its ability to bind to AAV9 and selective delivery to organs where the receptor-expressing cells are present. This indicates that AAV9 can be delivered into target cells by forming a complex (bispecific aptamer) between the AAV9 aptamer and a cell membrane-permeable aptamer for a membrane receptor expressed in the target cells.

[0012] The present inventors have conducted further research based on these findings and have completed the present invention, which provides the following:

[0013] [Section 1] Either (a) or (b) of the following: (a) SEQ ID NO: 1: 5'-GUGCGUACAUUUGUUGUAAUACACAAAUGCCACC-3' (However, uracil (U) may be replaced by thymine (T)) (b) A nucleotide sequence in which one or more nucleotides are substituted, deleted, inserted, and / or added in the nucleotide sequence of (a) above. An aptamer against adeno-associated virus serotype 9 (AAV9), comprising: [Section 2] Item 2. The aptamer according to Item 1, wherein the nucleotide sequence (a) or (b) is represented by the following formula: 5'-GUGYGUACAUUUGUUGUAAUACRC1ARRA1UGC1R1Y1N-3' (SEQ ID NO: 23) (In the formula, Y represents C or U, Y represents C, U, or is absent, each R represents independently A or G, R represents A, G, or is absent, A represents A or is absent, each C represents independently C or is absent, and N represents A, G, C, or U.) [Section 3] Either (a) or (b) of the following: (a) a nucleotide sequence represented by any one of SEQ ID NOs: 2 to 19 (wherein uracil (U) may be replaced with thymine (T)); (b) A nucleotide sequence in which one or more nucleotides are substituted, deleted, inserted, and / or added in the nucleotide sequence of (a) above. An aptamer against AAV9, comprising: [Section 4] Item 4. The aptamer according to any one of Items 1 to 3, which has a length of 65 nucleotides or less. [Section 5] A complex comprising the aptamer according to any one of Items 1 to 4 and a functional substance selected from the group consisting of an affinity substance, a labeling substance, an enzyme, a drug delivery vehicle, and a drug. [Section 6] A reagent for detecting or purifying AAV9, comprising the aptamer according to any one of Items 1 to 4 or the complex according to Item 5. [Section 7] A method for detecting or purifying AAV9, characterized by using the aptamer according to any one of Items 1 to 4 or the complex according to Item 5. [Section 8] A pharmaceutical composition comprising the complex according to Item 5, which comprises a drug delivery vehicle, and AAV9. [Section 9] Item 9. The pharmaceutical composition according to Item 8, wherein the drug delivery vehicle is an aptamer for a surface molecule of a target cell. [Section 10] Item 10. The pharmaceutical composition according to Item 9, wherein the surface molecule is involved in crossing the blood-brain barrier. [Effects of the Invention]

[0014] The aptamer of the present invention is a nucleic acid or a derivative thereof that replaces an antibody, and therefore has the advantages of being a smaller molecule than an antibody or a fragment thereof, having low immunogenicity, and having high binding specificity to AAV9. The aptamer of the present invention specifically and strongly binds to AAV9 and can be eluted under mild conditions, allowing for the purification of AAV9 with higher activity and purity. The complex of the aptamer of the present invention and a drug delivery vehicle allows for efficient delivery of therapeutic AAV9 to the desired target tissue, thereby enhancing the efficacy of AAV gene therapy. [Brief explanation of the drawings]

[0015] [Figure 1] 1 shows the predicted secondary structure of the AAV9 aptamer HIT15_AAV9_c3_1 (SEQ ID NO: 5) of the present invention. [Figure 2] 1 shows the predicted secondary structure of the AAV9 aptamer HIT15_AAV9_c3_1_44 (SEQ ID NO: 3) of the present invention. [Figure 3] 1 shows the predicted secondary structure of the AAV9 aptamer HIT15_AAV9_c3_1_35 (SEQ ID NO: 2) of the present invention. [Figure 4]This figure shows a combination of charts showing the time-dependent change in surface plasmon resonance (SPR) signal when aptamers (HIT15_AAV9_c3_1, HIT15_AAV9_c3_1_44, or HIT15_AAV9_c3_1_35) with polyriboadenine (rA16) attached to the 3' end were immobilized on a sensor chip (Sensor Chip SA (Cytiva, USA)) with biotinylated polydeoxyribonucleotide (d(T)16) immobilized on the sensor chip, and AAV9 dissolved in binding buffer was injected. The vertical axis represents the SPR signal, expressed as a relative value (RU). The horizontal axis represents the elapsed time (unit: seconds). In Figure 4, the SPR signal when the RNA aptamer bound to the sensor chip was set to 0. The solid line represents the time-dependent change in the SPR signal for the binding of HIT15_AAV9_c3_1 to AAV9. The dotted line indicates the time course of the SPR signal for binding of HIT15_AAV9_c3_1_44 to AAV9. The dashed line indicates the time course of the SPR signal for binding of HIT15_AAV9_c3_1_35 to AAV9. As shown in Figure 4, the maximum signals for HIT15_AAV9_c3_1, HIT15_AAV9_c3_1_44, and HIT15_AAV9_c3_1_35 were approximately 2200 RU, approximately 2000 RU, and approximately 1300 RU, respectively. The concentration of AAV9 in the binding buffer was 5 ng / μL, and the flow rate and time for the injection of the binding buffer containing AAV9 were 10 μL per minute for 2 minutes. For both aptamers, the SPR signal remained constant for approximately 400 seconds after reaching saturation. [Figure 5]This chart shows the time course of SPR signal when AAV9 dissolved in binding buffer was injected onto a sensor chip on which the aptamer (HIT15_AAV9_c3_1_35) was immobilized using hybridization between the d(T)16 and rA16 regions. The maximum SPR signal for the aptamer (HIT15_AAV9_c3_1_35) in this example was approximately 1800 RU. The AAV9 concentration in the binding buffer was 10 ng / μL, and the flow rate and time for injection of the binding buffer containing AAV9 dissolved therein were 10 μL per minute for 2 minutes. In this example, 1× SELEX buffer was used as the binding buffer for the RNA aptamer (HIT15_AAV9_c3_1_35) and AAV9. 1× SELEX buffer supplemented with 10 mM EDTA and 50 mM arginine was used as the elution buffer for dissociating and eluting AAV9 from the RNA aptamer. The vertical downward arrows in the chart indicate the time points at which the binding buffer or elution buffer containing the RNA aptamer (HIT15_AAV9_c3_1_35) or AAV9 was started to be injected into the sensor chip. [Figure 6] This is a combination of charts showing the time course of SPR signal when binding buffer containing AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV(DJ / 8), AAV(DJ), AAV9, or AAV9 (lab-made) was injected onto a sensor chip immobilized with the aptamer (HIT15_AAV9_c3_1_35) via hybridization between the d(T)16 and rA16 regions. The vertical axis represents the SPR signal, expressed as a relative value (RU). In Figure 6, the SPR signal when the RNA aptamer bound to the sensor chip was set to 0. The horizontal axis represents the elapsed time (unit: seconds). The solid line represents the chart for AAV9 (lab-made), the double line represents AAV9, and the dashed-dotted line represents AAV2. The line types for the other AAVs are as described in Figure 6. The concentration of each AAV in the binding buffer was 10 ng / μL, and the flow rate and time for injecting the binding buffer in which each AAV was dissolved were 10 μL per minute for 2 minutes. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides an aptamer that specifically binds to AAV9 (hereinafter also referred to as the "aptamer of the present invention"). An aptamer is a nucleic acid molecule that has binding activity to a specific target molecule. The aptamer of the present invention may be RNA, DNA, modified nucleic acid, or a mixture thereof. Furthermore, the aptamer of the present invention may be linear, cyclic, or stem-loop shaped, but preferably has a stem-loop structure as described below.

[0017] In one embodiment, the aptamer of the present invention can bind to AAV9, thereby binding AAV9 to a solid support or purifying AAV9 by affinity chromatography. Furthermore, by binding a functional substance to AAV9, the aptamer of the present invention can qualitatively detect the presence or absence of AAV9, quantitatively detect the amount of AAV9 present, or provide a new delivery method for AAV9.

[0018] The aptamer of the present invention is not particularly limited as long as it is an aptamer that specifically binds to AAV9, and may bind to any part of AAV9.

[0019] The length of the aptamer of the present invention is not particularly limited and can usually be about 200 nucleotides or less, for example, about 100 nucleotides or less, preferably about 70 nucleotides or less, more preferably 65 nucleotides or less, even more preferably 50 nucleotides or less, and particularly preferably about 45 nucleotides or less. A smaller total number of nucleotides facilitates chemical synthesis and mass production, and is also cost-effective. It is also believed to be easier to chemically modify, more stable in vivo, and less toxic. The lower limit of the length of the aptamer of the present invention is not particularly limited, as long as it contains the consensus sequence described below and can adopt a characteristic stem-loop structure, but is preferably 34 nucleotides or more. In view of the above, in a particularly preferred embodiment of the present invention, the length of the aptamer of the present invention is 34 to 45 nucleotides.

[0020] The aptamer of the present invention is (a) SEQ ID NO: 1: 5'-GUGCGUACAUUUGUUGUAAUACACAAAUGCCACC-3' (wherein uracil (U) may be replaced by thymine (T)), or (b) A nucleotide sequence in which one or more nucleotides are substituted, deleted, inserted, and / or added in the nucleotide sequence of (a) above. Includes.

[0021] Here, the number of nucleotides to be substituted, deleted, inserted or added is not particularly limited as long as they still bind to AAV9 after the substitution, deletion, insertion or addition, but may be, for example, 1 to about 10, preferably 1 to 6, more preferably 1 to 5, even more preferably 1 to 4, even more preferably 1 to 3, and most preferably 1 or 2. The site at which the nucleotides are substituted, deleted, inserted or added is also ... or 2, as shown by the following formula: 5'-GUGYGUACAUUUGUUGUAAUACRC1ARRA1UGC1R1Y1N-3' (In the formula, Y represents C or U, Y1 represents C, U, or not present, each R represents independently A or G, R1 represents A, G, or not present, A1 represents A or not present, each C1 represents independently C or not present, and N represents A, G, C, or U.) (However, uracil (U) may be thymine (T).) Preferably, the ambiguously represented bases in the above formula (I) contain mutations that satisfy these definitions.

[0022] Furthermore, the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1 has been found to have the following secondary structure in secondary structure prediction using RNAfold (see Mol. Biol. 6, 26 (2011), etc.):

[0023] [ka]

[0024] It has been shown that the AAV9 aptamer can have a characteristic stem-bulge-stem-loop structure represented by the formula: This predicted secondary structure is common to many AAV9 aptamers screened by SELEX. Therefore, if the consensus sequence contains a substitution, deletion, insertion, or addition at a position other than the ambiguous base, it is desirable to introduce the mutation so that the predicted secondary structure is maintained. Such mutations can be easily selected using an RNA secondary structure prediction algorithm such as RNAfold.

[0025] In another preferred embodiment, the aptamer of the present invention comprises: (a) a nucleotide sequence represented by any one of SEQ ID NOs: 2 to 19 (wherein uracil (U) may be replaced with thymine (T)); or (b) A nucleotide sequence in which one or more nucleotides are substituted, deleted, inserted, and / or added in the nucleotide sequence of (a) above. Includes. The number of nucleotides substituted, deleted, inserted, or added is not particularly limited as long as they still bind to AAV9 after the substitution, deletion, insertion, or addition, but may be, for example, 1 to about 10, preferably 1 to 6, more preferably 1 to 5, even more preferably 1 to 4, even more preferably 1 to 3, and most preferably 1 or 2. The site at which the nucleotides are substituted, deleted, inserted, or added is also not particularly limited as long as they still bind to AAV9 after the substitution, deletion, insertion, or addition, but, as above, preferably includes a mutation in accordance with the definition of an ambiguously defined base in the consensus sequence. However, RYN3 at the 3' end of the consensus sequence may be deleted as long as the stem structure is maintained.

[0026] Furthermore, it has been shown that each of the nucleic acids consisting of the nucleotide sequences represented by SEQ ID NOs: 2, 3, and 5 can have the predicted secondary structures shown in Figures 3, 2, and 1, respectively, in secondary structure prediction using RNAfold. Therefore, it is desirable that substitutions, deletions, insertions, or additions in the nucleotide sequence represented by SEQ ID NO: 2 be introduced so that the predicted secondary structure described in Figure 3 is maintained, substitutions, deletions, insertions, or additions in the nucleotide sequence represented by SEQ ID NOs: 3 or 4 be introduced so that the predicted secondary structure described in Figure 2 is maintained, and substitutions, deletions, insertions, or additions in the nucleotide sequences represented by SEQ ID NOs: 5 to 19 be introduced so that the predicted secondary structure described in Figure 1 is maintained.

[0027] The aptamer of the present invention may also be a conjugate of multiple aptamers of any one of the above types, or a conjugate containing one or more of each of two or more types of aptamers selected from the above types. These conjugates can also bind to AAV9. The linkage can be performed in tandem. A linker may be used for the linkage. Examples of linkers include nucleotide chains (e.g., 1 to about 20 nucleotides), non-nucleotide chains (e.g., -(CH2) n -linker, -(CH2CH2O) n -linker, hexaethylene glycol linker, TEG linker, peptide-containing linker, -SS- bond-containing linker, -CONH- bond-containing linker, and -OPO3- bond-containing linker). The term "plurality" in the above-mentioned multiple conjugates is not particularly limited as long as it is two or more, and may be, for example, two, three, or four.

[0028] Each nucleotide contained in the aptamer of the present invention may be the same or different, and may be a nucleotide containing a hydroxy group at the 2' position of the ribose (e.g., the ribose of a pyrimidine nucleotide, the ribose of a purine nucleotide) (i.e., a natural ribonucleotide), or a nucleotide in which the hydroxy group at the 2' position of the ribose has been replaced (modified) with any atom or group (sometimes referred to herein as a "modified nucleotide").

[0029] Examples of such an arbitrary atom or group include a hydrogen atom, a fluorine atom, or an -O-alkyl group (e.g., an -O-Me group), an -O-acyl group (e.g., an -O-CHO group), an amino group (e.g., an -NH2 group), etc. The aptamer of the present invention may also include at least one type (e.g., one, two, three, or four types) of nucleotides that contain, at the 2'-position of ribose, a hydroxy group or at least two types (e.g., two, three, or four types) of atoms or groups selected from the group consisting of any of the above-mentioned atoms or groups, for example, a hydrogen atom, a fluorine atom, and an -O-Me group.

[0030] In the aptamers of the present invention, all pyrimidine nucleotides may be nucleotides in which the 2'-position of the ribose is a fluorine atom, or the fluorine atom may be the same or different, unsubstituted, or substituted with any of the atoms or groups described above, preferably an atom or group selected from the group consisting of a hydrogen atom, a hydroxyl group, and a methoxy group. In particular, when the method for producing the aptamers of the present invention using the laboratory-produced T7 RNA polymerase Y639F mutant used in the Examples is applied, an aptamer in which the 2'-position of the ribose of the pyrimidine nucleotide is fluorinated can be obtained. Aptamers in which the fluorine atom is substituted with other atoms or groups described above can be produced by the methods described below.

[0031] In the aptamer of the present invention, all purine nucleotides may be nucleotides in which the 2'-position of ribose is a hydroxy group, or the hydroxy group may be the same or different and either unsubstituted or substituted with any of the above-mentioned atoms or groups, preferably an atom or group selected from the group consisting of a hydrogen atom, a methoxy group, and a fluorine atom. Aptamers in which the hydroxy group is substituted with another of the above-mentioned atoms or groups can be produced by the method described below.

[0032] In the aptamer of the present invention, all pyrimidine nucleotides may be nucleotides in which the fluorine atom at the 2' position of ribose is replaced with any of the atoms or groups described above, for example, the same atom or group selected from the group consisting of a hydrogen atom, a hydroxy group, and an -O-Me group. In the aptamer of the present invention, all purine nucleotides may be nucleotides in which the hydroxy group at the 2' position of ribose is replaced with any of the atoms or groups described above, for example, the same atom or group selected from the group consisting of a hydrogen atom, a fluorine atom, and an -O-Me group.

[0033] In a preferred embodiment, each pyrimidine nucleotide contained in the aptamer of the present invention is a nucleotide containing a fluorine atom at the 2'-position of ribose, and each purine nucleotide is a nucleotide containing a hydroxy group at the 2'-position of ribose. In another embodiment, the fluorine atom at the 2'-position of ribose of each of the pyrimidine nucleotides may be independently substituted with an atom or group selected from the group consisting of a hydrogen atom, a hydroxy group, and a methoxy group, and the hydroxy group at the 2'-position of ribose of each of the purine nucleotides may be independently substituted with an atom or group selected from the group consisting of a hydrogen atom, a methoxy group, and a fluorine atom.

[0034] In this specification, the modification of the sugar group in the nucleotide is explained assuming that the nucleotide constituting the aptamer is RNA (i.e., assuming that the sugar group is ribose), but this does not mean that DNA is excluded from the nucleotides constituting the aptamer, and it can be interpreted as a modification to DNA as appropriate. For example, when the nucleotide constituting the aptamer is DNA, the replacement of the hydroxyl group at the 2'-position of ribose with X can be interpreted as the replacement of the hydrogen atom at the 2'-position of deoxyribose with X.

[0035] In the aptamers of the present invention, one or several phosphodiester bonds in the nucleotides, for example, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 nucleotides, may be modified or substituted with any substituent. For example, the phosphodiester bond may be substituted with a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond, or the like. Here, for example, "the nucleotide is substituted with a phosphorothioate bond" means that the phosphate group at the bond site between adjacent nucleotides is sulfurized, i.e., the phosphodiester bond is modified to a phosphorothioate bond.

[0036] In the aptamer of the present invention, one or several nucleotides, for example, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 nucleotides, may be substituted with a bridged nucleic acid (BNA) or a locked nucleic acid (LNA) for the purpose of stabilizing the aptamer and improving its activity. Here, the term "bridged nucleic acid" refers to a nucleic acid having a structure that increases the binding affinity to a complementary sequence and acquires nuclease resistance by restricting the degree of freedom of the nucleic acid through intramolecular crosslinking, and examples thereof include, but are not limited to, 2',4'-BNA (LNA) and 2'-O,4'-C-ethylene-bridged nucleic acid (ENA).

[0037] The aptamers of the present invention may be modified in the sugar residue (e.g., ribose) of each nucleotide to enhance binding to AAV9, stability, drug delivery, etc. Examples of sites of modification in the sugar residue include those in which the oxygen atoms at the 2', 3', and / or 4' positions of the sugar residue are replaced with other atoms. Examples of types of modification include fluorination, O-alkylation (e.g., O-methylation, O-ethylation), O-allylation, S-alkylation (e.g., S-methylation, S-ethylation), S-allylation, and amination (e.g., -NH2). Other examples include 4'-SRNA in which the oxygen at the 4' position is replaced with sulfur, LNA (Locked Nucleic Acid) in which the 2' and 4' positions are crosslinked via a methylene, and 3'-N-phosphoroamidate nucleic acid in which the hydroxyl group at the 3' position is replaced with an amino group. The aptamers of the present invention may be produced with certain modifications to the oxygen atom at the 2' position of the ribose of pyrimidine nucleotides due to their production method. For example, when a production method using a laboratory-produced T7 RNA polymerase Y639F mutant is applied, an aptamer is preferably produced in which the 2' position of the ribose of all pyrimidine nucleotides is fluorinated. Therefore, by subsequently modifying the sugar residues of the obtained aptamer, it is possible to produce various variations of aptamers with the same base sequence but enhanced activity. For these reasons, the aptamers of the present invention may preferably be aptamers in which the sugar residue of at least one nucleotide is modified. Such modifications of sugar residues can be carried out by methods known per se (see, for example, Sproat et al., (1991), Nucl. Acid. Res. 19, 733-738; Cotton et al., (1991), Nucl. Acid. Res. 19, 2629-2635; Hobbs et al., (1973), Biochemistry 12, 5138-5145). Specifically, based on an aptamer in which the hydroxyl groups at the 2'-position of ribose of all pyrimidine nucleotides are substituted with fluoro groups, an aptamer can be produced in which the hydroxyl group at the 2'-position of ribose is substituted with an atom or group selected from the group consisting of a hydrogen atom, a hydroxyl group, and a methoxy group.

[0038] The aptamers of the present invention may also be modified (e.g., chemically substituted) at nucleic acid bases (e.g., purines, pyrimidines) to enhance binding to AAV9, prevent multimerization, stability, drug delivery, etc. Examples of such modifications include modification of the 5-position pyrimidine, modification of the 6- and / or 8-position purines, modification with exocyclic amines, substitution with 4-thiouridine, and substitution with 5-bromo- or 5-iodo-uracil. Furthermore, the phosphate group contained in the aptamer of the present invention may be modified to provide resistance to nucleases and hydrolysis. For example, the P(O)O group may be substituted with P(O)S (thioate), P(S)S (dithioate), P(O)N(R)R' (amidate), P(O)R, P(O)OR, CO or CH2 (formacetal) or 3'-amine (-NH-CH2-CH2-), where each R or R' is independently H or substituted or unsubstituted alkyl (e.g., methyl, ethyl). Examples of the linking group include -O-, -N-, and -S-, and adjacent nucleotides can be bound via these linking groups. Modifications may also include 3' and 5' modifications such as capping.

[0039] Modifications can also be carried out by adding polyethylene glycol (PEG), amino acids, peptides, inverted dT, nucleic acids, nucleosides, myristoyl, lithocolic-oleyl, docosanyl, lauroyl, stearoyl, palmitoyl, oleoyl, linoleoyl, other lipids, steroids, cholesterol, caffeine, vitamins, dyes, fluorescent substances, anticancer drugs, toxins, enzymes, radioactive substances, biotin, etc. to the termini. For details of such modifications, see, for example, U.S. Patent Nos. 5,660,985 and 5,756,703.

[0040] In particular, when modification is performed by terminal addition of PEG, the molecular weight of PEG is not particularly limited, but is preferably 1,000 to 100,000, more preferably 30,000 to 90,000. PEG may be linear or may be branched into two or more chains (multi-arm PEG). Terminal addition of PEG is useful for preventing aptamer multimerization, as described below. Such PEG is not particularly limited, and those skilled in the art can appropriately select and use commercially available or known PEG (see, for example, http: / / www.peg-drug.com / peg_product / branched.html). However, specific examples of suitable PEG to be applied to the aptamer of the present invention include bibranched AS type (functional group: -CH2-COO-NHS) PEG (Y-NHS-40K, manufactured by Jenkem) with a molecular weight of 40,000, bibranched GS type (functional group: -CO-(CH2)3-COO-NHS) PEG (SUNBRIGHT GL2-400GS, manufactured by NOF Corp.) with a molecular weight of 40,000, bibranched TS type (active group: -COO-NHS) PEG (SUNBRIGHT GL2-400TS, manufactured by NOF Corp.) with a molecular weight of 40,000, 4-branched TS type PEG (SUNBRIGHT GL4-400TS, manufactured by NOF Corp.), bibranched TS type PEG (SUNBRIGHT GL4-400TS, manufactured by NOF Corp.) with a molecular weight of 80,000, and 80,000. GL2-800TS manufactured by NOF Corp.), or 4-branched TS type PEG with a molecular weight of 80,000 (SUNBRIGHT GL4-800TS manufactured by NOF Corp.).

[0041] In this case, the aptamer of the present invention may have PEG directly attached to its terminus, but it is more preferable to attach a linker or the like having a group capable of binding to PEG to its terminus, and to attach PEG to the aptamer of the present invention via this linker.

[0042] The linker between PEG and the aptamer used in the present invention is not particularly limited, and the number of carbon chains, functional groups, and the like can be selected appropriately depending on the binding site, the type of PEG, and the like. Examples of such linkers include linkers having an amino group. Specific examples include ssH Linker (SAFC) or DMS(O)MT-AMINO-MODIFIER (GLENRESEARCH) when attached to the 5' end, and TFA Amino C-6 lcaa CPG (ChemGenes) when attached to the 3' end. When this linker is selected, the aptamer used in the present invention and PEG can be linked via the linker by adding an active group, for example, N-hydroxysuccinimide, to PEG and then reacting this with the amino group on the linker.

[0043] Commercially available PEG and linkers can be preferably used. Furthermore, reaction conditions for binding of PEG, linkers, and the aptamer used in the present invention can be appropriately determined by those skilled in the art.

[0044] The aptamers of the present invention can be chemically synthesized using the disclosures herein and methods known in the art. Aptamers bind to target substances through a variety of binding modes, including ionic bonds utilizing the negative charge of phosphate groups, hydrophobic and hydrogen bonds utilizing ribose, and hydrogen bonds and stacking bonds utilizing nucleobases. In particular, ionic bonds utilizing the negative charges of phosphate groups, which exist in equal numbers as the constituent nucleotides, are strong and bind to the positive charges of lysine and arginine present on the surface of proteins. Therefore, nucleobases not involved in direct binding with the target substance can be substituted. In particular, the stem structure is already base-paired and faces inward in the double helix structure, making it difficult for nucleobases to directly bind to the target substance. Therefore, replacing base pairs with other base pairs often does not reduce the activity of the aptamer. Base substitution is also possible in structures without base pairs, such as loop structures, as long as the nucleobases are not involved in direct binding with the target molecule. Regarding modifications at the 2'-position of ribose, although the functional group at the 2'-position of ribose may occasionally interact directly with the target molecule, it is often unrelated and can be replaced with other modified molecules. Thus, aptamers often retain their activity unless the functional group involved in direct binding to the target molecule is replaced or deleted. It is also important that the overall three-dimensional structure does not change significantly.

[0045] Aptamers can be produced using the SELEX method and its improved methods (e.g., Ellington et al., (1990), Nature, 346, 818-822; Tuerk et al., (1990), Science, 249, 505-510). In the SELEX method, increasing the number of rounds or using a competing substance enriches and selects aptamers with stronger binding affinity to the target substance. Therefore, by adjusting the number of SELEX rounds and / or changing the competitive conditions, it may be possible to obtain aptamers with different binding affinity, different binding forms, or aptamers with the same binding affinity or binding form but different nucleotide sequences. In addition, the SELEX method includes a PCR amplification process, and introducing mutations during this process, for example by using manganese ions, can enable SELEX with greater diversity.

[0046] Active aptamers selected in this way can be further improved by performing optimized SELEX, which involves creating a template in which a portion of an aptamer with a fixed sequence is randomized, or a template doped with 10-30% random sequence, and then performing SELEX again.

[0047] Aptamers obtained by SELEX are approximately 80 nucleotides in length, making them difficult to use as medicines. Therefore, it is preferable to shorten them through repeated trial and error to a length that can be easily synthesized chemically (for example, chemical synthesis is possible to a length of approximately 60 nucleotides or less, more preferably approximately 50 nucleotides or less, and even more preferably 45 nucleotides or less). The ease of subsequent minimization of aptamers obtained by SELEX depends on the primer design. Even if active aptamers are selected by SELEX, if the primers are not well designed, further development will be impossible.

[0048] Aptamers can be chemically synthesized and therefore easily modified. By predicting the secondary structure using the MFOLD program and determining the three-dimensional structure using X-ray and NMR analysis, it is possible to predict to some extent which nucleotides can be substituted or deleted, and where new nucleotides can be inserted. Aptamers with predicted new sequences can be easily chemically synthesized, and whether the aptamer retains its activity can be confirmed using existing assay systems.

[0049] If the portion of the aptamer that is important for binding to the target substance can be identified through the above-described trial and error process, adding new sequences to both ends of the sequence will often not change the activity, and the length of the new sequences is not particularly limited.

[0050] Furthermore, as already mentioned, modifications can be designed or altered to a high degree in the same way as sequences.

[0051] As described above, aptamers can be highly designed or modified. The present invention also provides a method for producing an aptamer, which allows for highly designing or modifying an aptamer containing a predetermined sequence (e.g., a sequence corresponding to a portion selected from a stem portion, an internal loop portion, a hairpin loop portion, and a single-stranded portion; hereinafter, abbreviated as "fixed sequence" as necessary).

[0052] For example, a method for producing such an aptamer is as follows:

[0053] [ka]

[0054] [In the above, (N)a represents a nucleotide chain consisting of a number of Ns, and (N)b represents a nucleotide chain consisting of b number of Ns, and each N may be the same or different and is a nucleotide selected from the group consisting of A, G, C, and T (preferably, A, G, C, and U). Each a and b may be the same or different and may be any number, for example, 1 to about 100, preferably 1 to about 50, more preferably 1 to about 30, and even more preferably 1 to about 20 or 1 to about 10], and a single type of nucleic acid molecule or multiple types of nucleic acid molecules (e.g., a library of nucleic acid molecules where the numbers of a and b differ) consisting of a nucleotide sequence represented by the formula (wherein (N)a represents a nucleotide chain consisting of a number of Ns, and (N)b represents a nucleotide chain consisting of b number of Ns, and each N may be the same or different and is a nucleotide selected from the group consisting of A, G, C, and T (preferably, A, G, C, and U). Each a and b may be the same or different and may be any number, for example, 1 to about 100, preferably 1 to about 50, more preferably 1 to about 30, and even more preferably 1 to about 20 or 1 to about 10), and a primer pair corresponding to primer sequences (i) and (ii), respectively.

[0055] The present invention also provides a complex comprising the aptamer of the present invention and a functional substance bound thereto. The bond between the aptamer and the functional substance in the complex of the present invention can be a covalent bond or a non-covalent bond. The complex of the present invention can be one in which the aptamer of the present invention is bound to one or more (e.g., two or three) functional substances of the same or different types. The functional substance is not particularly limited, as long as it can newly add some function to the aptamer of the present invention or can change (e.g., improve) some property that the aptamer of the present invention can possess. Examples of functional substances include proteins, peptides, amino acids, lipids, carbohydrates, monosaccharides, polynucleotides, and nucleotides.Examples of functional substances include affinity substances (e.g., biotin, streptavidin, polynucleotides having affinity for target complementary sequences, antibodies, glutathione sepharose, histidine), labeling substances (e.g., fluorescent substances, luminescent substances, radioisotopes), enzymes (e.g., horseradish peroxidase, alkaline phosphatase), drug delivery vehicles (e.g., liposomes, microspheres, peptides, polyethylene glycols), drugs (e.g., those used in missile therapy such as calicheamicin and duocarmycin, nitrogen mustard analogs such as cyclophosphamide, melphalan, ifosfamide, or trofosfamide, ethyleneimines such as thiotepa, nitrosoureas such as carmustine, alkylating agents such as temozolomide or dacarbazine, methotrexate, or raltitrexed). and other folate-like antimetabolites, purine analogs such as thioguanine, cladribine, or fludarabine, pyrimidine analogs such as fluorouracil, tegafur, or gemcitabine, vinca alkaloids and their analogs such as vinblastine, vincristine, or vinorelbine, etoposide, taxanes, podophyllotoxin derivatives such as docetaxel or paclitaxel, anthracyclines and analogs such as doxorubicin, epirubicin, idarubicin, and mitoxantrone, other cytotoxic antibiotics such as bleomycin and mitomycin, platinum compounds such as cisplatin, carboplatin, and oxaliplatin, pentostatin, miltefosine, estramustine, topotecan, irinotecan, and bicalutamide, toxins (e.g., ricin toxin, riatoxin, and verotoxin). These functional molecules may ultimately be removed. Furthermore, the nucleic acid may be a peptide that can be recognized and cleaved by an enzyme such as thrombin, matrix metalloproteinase (MMP) or Factor X, or a polynucleotide that can be cleaved by a nuclease or restriction enzyme.

[0056] The present invention provides an AAV9-specific binding agent. The AAV9-specific binding agent of the present invention comprises the aptamer or complex of the present invention. The binding agent can be used as a reagent for detecting or purifying AAV9.

[0057] The present invention provides a method for purifying AAV9 from a mixture of AAV9 and impurities. The method for purifying AAV9 of the present invention includes, for example, (1) preparing a solid phase substrate on which the aptamer or complex of the present invention is immobilized; (2) contacting the mixture with the immobilized solid phase substrate; (3) washing the immobilized solid phase substrate under conditions in which AAV9 binds to the immobilized solid phase substrate but the impurities do not bind to the immobilized solid phase substrate to separate the impurities from AAV9; (4) separating AAV9 by washing the immobilized solid phase substrate under conditions that dissociate AAV9 from the immobilized solid phase substrate.

[0058] The present invention provides a further method for purifying AAV9 from a mixture of AAV9 and impurities. The further method for purifying AAV9 of the present invention comprises, following step (4) of separating AAV9 in the above-described method for purifying AAV9, step (2') of contacting a new mixture of AAV9 and impurities with the immobilized solid phase substrate, and repeating step (3), step (4), and step (5) following step (2').

[0059] The present invention provides a method for detecting AAV9. The method for detecting AAV9 of the present invention can be, for example, (1) contacting AAV9 with the complex of the present invention, wherein the functional substance is a labeling substance or an enzyme; The method includes a step of qualitatively detecting the presence or absence of AAV9 using the labeling substance or enzyme, or quantitatively detecting the amount of AAV9 present.

[0060] The present invention also provides a pharmaceutical composition comprising a complex of the present invention containing a drug delivery vehicle and AAV9, wherein the AAV9 may be an oncolytic virus that has therapeutic activity against a disease itself, or may be loaded with an exogenous therapeutic gene.

[0061] In a preferred embodiment, the drug delivery vehicle can be an aptamer for a surface molecule of a target cell. The surface molecule targeted by the aptamer is not particularly limited, as long as it is highly expressed, preferably specifically expressed, in the target cell where AAV9 is delivered and introduced into the cell to exert a therapeutic effect. Examples include, but are not limited to, surface receptors involved in passing through the blood-brain barrier (BBB), such as insulin receptor, glutathione receptor, transferrin receptor, enkephalin receptor, etc. Aptamers for such surface molecules can be obtained by the SELEX method using the same method as the aptamers of the present invention, or existing ones can also be used.

[0062] The pharmaceutical composition of the present invention may be formulated with a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropyl cellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium glycol starch, sodium bicarbonate, calcium phosphate, and calcium citrate; magnesium stearate, ethylene glycol, and the like. These include, but are not limited to, lubricants such as arosil, talc, sodium lauryl sulfate, fragrances such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, orange powder, preservatives such as sodium benzoate, sodium bisulfite, methylparaben, propylparaben, stabilizers such as citric acid, sodium citrate, acetic acid, suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, dispersing agents such as surfactants, diluents such as water, saline, orange juice, and base waxes such as cocoa butter, polyethylene glycol, and white kerosene.

[0063] Suitable formulations for oral administration include solutions in which an effective amount of the ligand is dissolved in a diluent such as water, physiological saline, or orange juice; capsules, sachets, or tablets containing an effective amount of the ligand as a solid or granule; suspensions in which an effective amount of the active ingredient is suspended in a suitable dispersion medium; and emulsions in which a solution of an effective amount of the active ingredient is dispersed and emulsified in a suitable dispersion medium.

[0064] Furthermore, the pharmaceutical of the present invention can be coated by a method known per se, if necessary, for purposes such as taste masking, enteric coating, or sustained release. Examples of coating agents used for coating include hydroxypropylmethylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, polyoxyethylene glycol, Tween 80, Pluronic F68, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxymethylcellulose acetate succinate, Eudragit (manufactured by Rohm, Germany, methacrylic acid-acrylic acid copolymer), and pigments (e.g., red iron oxide, titanium dioxide, etc.). The pharmaceutical may be in either an immediate-release or sustained-release formulation. Examples of substrates for sustained-release formulations include liposomes, atelocollagen, gelatin, hydroxyapatite, and PLGA.

[0065] Suitable formulations for parenteral administration (e.g., intravenous, subcutaneous, intramuscular, topical, intraperitoneal, nasal, pulmonary, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc. Such formulations can be packaged in unit doses or multiple doses in containers such as ampoules or vials. The active ingredient and a pharmaceutically acceptable carrier can also be lyophilized and stored in a state that requires only dissolution or suspension in an appropriate sterile solvent immediately before use. In addition to injection solutions, inhalants and ointments are also possible. Inhalants are administered by inhaling the lyophilized active ingredient into fine particles using an appropriate inhalation device. Conventional surfactants, oils, flavorings, cyclodextrin or its derivatives, etc., can be appropriately blended into inhalants as needed.

[0066] Examples of surfactants include oleic acid, lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl trioleate, glyceryl monolaurate, glyceryl monooleate, glyceryl monostearate, glyceryl monoricinoate, cetyl alcohol, stearyl alcohol, polyethylene glycol 400, cetylpyridinium chloride, sorbitan trioleate (trade name Span 85), sorbitan monooleate (trade name Span 80), sorbitan monolaurate (trade name Span 20), polyoxyethylene hydrogenated castor oil (trade name HCO-60), polyoxyethylene ( Examples include 20) sorbitan monolaurate (Tween 20), polyoxyethylene (20) sorbitan monooleate (Tween 80), lecithin derived from natural sources (Epiclon), oleyl polyoxyethylene (2) ether (Brij 92), stearyl polyoxyethylene (2) ether (Brij 72), lauryl polyoxyethylene (4) ether (Brij 30), oleyl polyoxyethylene (2) ether (Genapol 0-020), and a block copolymer of oxyethylene and oxypropylene (Synperonic). Span, Tween, Epiclon, Brij, Genapol, and Synperonic are trademarks. Examples of oils include corn oil, olive oil, cottonseed oil, sunflower oil, etc. In the case of ointments, an appropriate pharmaceutically acceptable base (yellow petrolatum, white petrolatum, paraffin, plastibase, silicone, white ointment, beeswax, lard, vegetable oil, hydrophilic ointment, hydrophilic petrolatum, purified lanolin, hydrous lanolin, water-absorbing ointment, hydrophilic plastibase, macrogol ointment, etc.) is used, mixed with the active ingredient, and formulated for use.

[0067] The dosage of the pharmaceutical composition of the present invention varies depending on the type and activity of the active ingredient, the severity of the disease, the animal species to be administered, the drug tolerance, body weight, age, etc., of the subject, but it is desirable to use a dosage that has been confirmed to be safe and effective in previous AAV gene therapies.

[0068] In this specification, the conjunction "about" used to modify a numerical value means a numerical range of 90% or more and 110% or less of the numerical value. For example, when the amount of nucleic acid bound to a sensor chip in a surface plasmon resonance (SPR) method is "about 100 RU," this means that the amount of nucleic acid is within a numerical range including the lower and upper limits of 90 RU and 110 RU, respectively.

[0069] All documents mentioned herein are incorporated by reference in their entirety.

[0070] The present invention will be explained in more detail below by way of examples, but these are merely illustrative and do not limit the present invention in any way. [Example]

[0071] Example 1: Preparation of RNA aptamer that binds to AAV9 (1) RNA aptamers specifically binding to AAV9 were generated using the SELEX method, which was performed with reference to the methods of Ellington et al. (Ellington and Szostak, Nature 346, 818-822, 1990) and Tuerk et al. (Tuerk and Gold, Science 249, 505-510, 1990).

[0072] The library used for aptamer screening was constructed as follows. First, a 63-nucleotide single-stranded DNA template with primer sequences at both ends of a 30-nucleotide random sequence was chemically synthesized. The single-stranded DNA template was converted into double-stranded DNA using a complementary forward primer and DNA polymerase at the 3' end primer. RNA was then obtained by transcription using the T7 RNA polymerase Y639F mutant, which was developed in the laboratory. For the T7 RNA polymerase reaction, nucleotide triphosphates with fluorinated 2'-positions of the ribose were used as substrates for pyrimidine nucleotides (cytosine and uracil), and ribonucleotide triphosphates were used for purine nucleotides (adenine and guanine).

[0073] The N sequence in the DNA template is any combination of 30 nucleotides, resulting in a unique sequence region for the resulting aptamer. The forward primer contains the promoter sequence for T7 RNA polymerase. Theoretically, the RNA pool can be 10 14 The structure of the RNA library used for screening the aptamer of the present invention is described below.

[0074] The RNA library from which the finally selected aptamers were screened is HIT15N30. The single-stranded DNA of HIT15N30 contains a polynucleotide with 30 consecutive random nucleotides N. Primers are linked to the 5' and 3' ends of the random sequence, respectively. The reverse complementary sequences of the 5' and 3' primer sequences are the reverse and forward primers, respectively. Since the forward primer contains a promoter sequence for T7 RNA polymerase, when the forward primer hybridizes to the single-stranded DNA of HIT15N30, RNA is synthesized using the forward primer through N30 and the 5' end primer of HIT15N30 as a template. The primary structures of HIT15N30, the forward primer, and the reverse primer are as follows:

[0075] HIT15N30: 5'-GGGGTACGATGCTTGTGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGAGCGAAACTCAGCT-3' (SEQ ID NO: 20)

[0076] Forward primer: 5'- TAATACGACTCACT ATAGGGGTACGATGCTTGTGC-3' (SEQ ID NO: 21) (the underlined portion indicates the T7 promoter sequence)

[0077] Reverse primer: 5'-AGCTGAGTTTCGCTC-3' (SEQ ID NO: 22)

[0078] The AAV9 virus and RNA library were mixed and incubated at room temperature for 30 minutes. The mixture was then placed in a Vivaspin 500 ultrafiltration device (molecular weight cutoff: 100 kDa, manufactured by Sartorius AG, Germany) and centrifuged at 14,000 × g for 5 minutes using a microcentrifuge. After discarding the flow-through fraction, the Vivaspin 500 was washed with 1× SELEX buffer to remove RNA that did not bind to the AAV9 virus. This washing procedure was repeated 5 to 10 times. The 1× SELEX buffer was a mixture of 20 mM Tris-HCl (pH 7.6), 20 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, and 0.01% TWEEN 80. After washing, the fraction containing AAV9-bound RNA recovered from Vivaspin 500 was subjected to phenol-chloroform treatment and ethanol precipitation to recover only the bound RNA. The recovered RNA was amplified by reverse transcription PCR and transcribed using the T7 RNA polymerase Y639F mutant, which was developed in the laboratory, and used as the pool for the next round. This process was repeated nine times, with the above being one round. For the T7 RNA polymerase reaction, pyrimidine nucleotides (cytosine and uracil) were used as substrates, and ribonucleotide triphosphates with 2'-fluorinated ribose were used for the substrates. For purine nucleotides (adenine and guanine), ribonucleotide triphosphates were used.

[0079] After the SELEX screening was completed, the base sequences were analyzed using a next-generation sequencer. The next-generation sequencer used was iSeq100 (Illumina, USA), and the analysis was performed according to Illumina's specifications. Sequences with two or more reads were analyzed. The analysis was performed using the analysis software "FASTAptamer." Changes of six bases or less (substitutions, insertions, deletions) were processed as one cluster.

[0080] As a result of such screening, the 63-nucleotide HIT15_AAV9_c3_1 was isolated from the HIT15N30 RNA library.

[0081] HIT15_AAV9_c3_1: 5'-GGGGUACGAUGCUUGUGCGUACAUUUGUUGUAAUACACAAAUGCCACCGAGCGAAACUCAGCU-3' (SEQ ID NO: 5)

[0082] Analysis of reads in the same cluster as HIT15_AAV9_c3_1 revealed 14 other AAV9 aptamer sequences.

[0083] (i) 5'-GGGGUACGAUGCUUGUGCGUACAUUUGUUGUAAUACGCAAAUGCCACCGAGCGAAACUCAGCU-3' (SEQ ID NO: 6) (ii) 5'-GGGGUACGAUGCUUGUGCGUACAUUUGUUGUAAUACACAAAAUGCCACCGAGCGAAACUCAGCU-3' (SEQ ID NO: 7) (iii) 5'-GGGGUACGAUGCUUGUGUGUACAUUUGUUGUAAUACACAAAUGCCACCGAGCGAAACUCAGCU-3' (SEQ ID NO: 8) (iv) 5'-GGGGUACGAUGCUUGUGCGUACAUUUGUUGUAAUACACAAAUGCCACUGAGCGAAACUCAGCU-3' (SEQ ID NO: 9) (v) 5'-GGGGUACGAUGCUUGUGCGUACAUUUGUUGUAAUACACAAAUGCCAUCGAGCGAAACUCAGCU-3' (SEQ ID NO: 10) (vi) 5'-GGGGUACGAUGCUUGUGCGUACAUUUGUUGUAAUACACAAAUGCCGCCGAGCGAAACUCAGCU-3' (SEQ ID NO: 11) (vii) 5'-GGGGUACGAUGCUUGUGCGUACAUUUGUUGUAAUACACAAGUGCCACCGAGCGAAACUCAGCU-3' (SEQ ID NO: 12) (viii) 5'-GGGGUACGAUGCUUGUGCGUACAUUUGUUGUAAUACAAAAUGCCACCGAGCGAAACUCAGCU-3' (SEQ ID NO: 13) (ix) 5'-GGGGUACGAUGCUUGUGCGUACAUUUGUUGUAAUACACAAAUGCCACGGAGCGAAACUCAGCU-3' (SEQ ID NO: 14) (x) 5'-GGGGUACGAUGCUUGUGCGUACAUUUGUUGUAAUACGCAAAUGCCACAGAGCGAAACUCAGCU-3' (SEQ ID NO: 15) (xi) 5'-GGGGUACGAUGCUUGUGCGUACAUUUGUUGUAAUACACAAAUGCCACGAGCGAAACUCAGCU-3' (SEQ ID NO: 16) (xii) 5'-GGGGUACGAUGCUUGUGCGUACAUUUGUUGUAAUACACAAAAUGCCACAGAGCGAAACUCAGCU-3' (SEQ ID NO: 17) (xiii) 5'-GGGGUACGAUGCUUGUGCGUACAUUUGUUGUAAUACACAGAUGCCACCGAGCGAAACUCAGCU-3' (SEQ ID NO: 18) (xiv) 5'-GGGGUACGAUGCUUGUGCGUACAUUUGUUGUAAUACACAAAUGCCGAGCGAAACUCAGCUAU-3' (SEQ ID NO: 19)

[0084] Alignment of these nucleotide sequences belonging to the same cluster resulted in the following consensus sequence: 5'-GGGGUACGAUGCUUGUGYGUACAUUUGUUGUAAUACRC1ARRA1UGC1R1Y1NGAGCGAAACUCAGCU-3' (SEQ ID NO: 24) (In the formula, Y represents C or U, Y1 represents C, U, or not present, each R represents independently A or G, R1 represents A, G, or not present, A1 represents A or not present, each C1 represents independently C or not present, and N represents A, G, C, or U.) (However, uracil (U) may be thymine (T).)

[0085] The predicted secondary structure of the AAV9 aptamer HIT15_AAV9_c3_1 (SEQ ID NO: 3) of the present invention is shown in FIG.

[0086] Example 2: Preparation of RNA aptamers that specifically bind to AAV9 (2) HIT15_AAV9_c3_1 was shortened to obtain HIT15_AAV9_c3_1_46 (46 nucleotides), HIT15_AAV9_c3_1_44 (44 nucleotides), and HIT15_AAV9_c3_1_35 (35 nucleotides). The nucleotide sequences of these fragments are as follows:

[0087] HIT15_AAV9_c3_1_46: 5'-GGGCUUGUGCGUACAUUUGUUGUAAUACACAAAUGCCACCGAGCCC-3' (SEQ ID NO: 4)

[0088] HIT15_AAV9_c3_1_44: 5'-GGGUUGUGCGUACAUUUGUUGUAAUACACAAAUGCCACCGACCC-3' (SEQ ID NO: 3)

[0089] HIT15_AAV9_c3_1_35: 5'-GGUGCGUACAUUUGUUGUAAUACACAAAUGCCACC-3' (SEQ ID NO: 2)

[0090] The predicted secondary structures of the AAV9 aptamers of the present invention, HIT15_AAV9_c3_1_44 (SEQ ID NO: 3) and HIT15_AAV9_c3_1_35 (SEQ ID NO: 2), are shown in Figures 2 and 3, respectively. The partial predicted secondary structure characteristic of HIT15_AAV9_c3_1 was also retained in these shortened aptamers.

[0091] Example 3 Quantitative evaluation of binding between AAV9 and RNA aptamer that specifically binds to AAV9 In this example, a binding buffer containing three types of RNA aptamer molecules linked to polyriboadenine (rA16) sequences was injected onto a sensor chip (Sensor Chip SA (Cytiva, USA)) on which biotinylated polydeoxyribonucleotides (d(T)16) were immobilized, and the time-dependent changes in surface plasmon resonance (SPR) signals associated with the binding of each immobilized RNA aptamer molecule to AAV9 via hybridization between d(T)16 and rA16 were observed.

[0092] In the following Examples, the binding of RNA aptamers was quantitatively measured by the SPR method using a streptavidin-immobilized sensor chip (Sensor Chip SA (Cytiva, USA)) and Biacore2000 (Cytiva, USA).

[0093] A polydeoxyribonucleotide d(T)16 biotinylated at the 5' end was immobilized on a sensor chip, and then a binding buffer containing an aptamer (HIT15_AAV9_c3_1, HIT15_AAV9_c3_1_44, or HIT15_AAV9_c3_1_35) with a polyriboadenine (rA16) sequence attached to the 3' end was injected into the immobilized sensor chip, and the aptamer was immobilized to the sensor chip via hybridization between the d(T)16 and (rA16) regions. 1x SELEX buffer was used as the binding buffer for the aptamer (HIT15_AAV9_c3_1, HIT15_AAV9_c3_1_44, or HIT15_AAV9_c3_1_35) with a polyriboadenine (rA16) sequence attached.

[0094] Figure 4 is a combination of charts showing the time course of SPR signals when AAV9 dissolved in binding buffer was injected onto aptamers (HIT15_AAV9_c3_1, HIT15_AAV9_c3_1_44, or HIT15_AAV9_c3_1_35) immobilized on a Sensor Chip SA via hybridization between the d(T)16 and (rA16) regions. The vertical axis represents the SPR signal, expressed as a relative value (RU). The horizontal axis represents the elapsed time (unit: seconds). In Figure 4, the SPR signal when the RNA aptamer bound to the sensor chip was set to 0. The solid line represents the chart for HIT15_AAV9_c3_1. The dotted line represents the chart for HIT15_AAV9_c3_1_44. The dashed line represents the chart for HIT15_AAV9_c3_1_35. As shown in Figure 4, the maximum SPR signals for HIT15_AAV9_c3_1, HIT15_AAV9_c3_1_44, and HIT15_AAV9_c3_1_35 were approximately 2200 RU, approximately 2000 RU, and approximately 1300 RU, respectively. For all aptamers, the SPR signal remained constant for approximately 400 seconds after reaching its maximum value. The concentration of AAV9 in the binding buffer was 5 ng / μL, and the flow rate and time for the injection of the binding buffer containing AAV9 were 10 μL per minute for 2 minutes. From the above results, the consensus sequence shown in the formula below was obtained as the primary structure of the aptamer important for binding to AAV9. 5'-GUGYGUACAUUUGUUGUAAUACRC1ARRA1UGC1R1Y1N-3' (SEQ ID NO: 23) (In the formula, Y represents C or U, Y1 represents C, U, or not present, each R represents independently A or G, R1 represents A, G, or not present, A1 represents A or not present, each C1 represents independently C or not present, and N represents A, G, C, or U.) (However, uracil (U) may be thymine (T).)

[0095] Example 4 Evaluation of the ability of RNA aptamers that specifically bind to AAV9 to retain AAV9 binding In this example, using a sensor chip on which an RNA aptamer has been immobilized according to Example 3, we demonstrate that the ability of the RNA aptamer to specifically bind to AAV9 is retained even when AAV9 is repeatedly bound to the solid phase in a binding buffer and dissociated from the solid phase in an elution buffer.

[0096] Figure 5 is a chart showing the change in SPR signal over time when a binding buffer containing AAV9 was injected onto a sensor chip on which the aptamer (HIT15_AAV9_c3_1_35) had been immobilized according to Example 3. The vertical axis represents the SPR signal, expressed as a relative value (RU). The horizontal axis represents the elapsed time (unit: seconds). In this example, the maximum SPR signal for the aptamer (HIT15_AAV9_c3_1_35) was approximately 1800 RU. The concentration of AAV9 in the binding buffer was 10 ng / μL, and the flow rate and time for injection of the binding buffer containing AAV9 were 10 μL per minute for 2 minutes. In this example, 1x SELEX buffer was used as the binding buffer for the RNA aptamer (HIT15_AAV9_c3_1_35) and AAV9. The elution buffer used for dissociating and eluting AAV9 from the RNA aptamer was 1x SELEX buffer supplemented with 10 mM EDTA and 50 mM arginine. The vertical downward arrows in the chart indicate the time points at which the binding buffer or elution buffer containing the RNA aptamer (HIT15_AAV9_c3_1_35) or AAV9 was injected into the sensor chip.

[0097] As shown in Figure 5, when the RNA aptamer was first injected onto the sensor chip, it bound to the sensor chip, generating an SPR signal. When AAV9 was injected onto the sensor chip, AAV9 bound to the RNA aptamer, further increasing the SPR signal. When the elution buffer was then injected onto the sensor chip, AAV9 dissociated from the RNA aptamer, rapidly reducing the SPR signal to the intensity before AAV9 was injected. Figure 5 shows that even after five cycles of AAV9 binding and dissociation, the SPR signal intensities upon AAV9 binding and dissociation remained the same. This indicates that the binding and dissociation of the immobilized RNA aptamer to AAV9 can be controlled simply by switching the buffer. Therefore, this example suggests that the solid phase immobilized with the RNA aptamer of the present invention can be used as an affinity chromatography support for AAV9 purification.

[0098] Furthermore, the elution buffer used to dissociate the binding between the RNA aptamer of the present invention and AAV9 did not affect the complementary base pairing between polyriboadenine (rA16) and polydeoxyribonucleotide (d(T)16) attached to the RNA aptamer, nor the specific binding between the biotin group attached to d(T)16 and streptavidin attached to the sensor chip. Therefore, the conditions for dissociating the binding between the RNA aptamer of the present invention and AAV9 can be said to be mild enough not to affect at least the complementary base pairing and the specific binding between biotin and streptavidin.

[0099] Example 5: Examination of AAV serotype specificity of RNA aptamers In this example, it was verified whether the RNA aptamer according to the present application binds to AAV serotypes other than AAV9.

[0100] AAVs of different serotypes (AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV(DJ / 8), AAV(DJ), and AAV9) were prepared using the AAV Serotype Testing Kit (Code#SL100886) from SingaGen Laboratories. AAV9 (lab-made) was prepared according to the literature (PMID: 36977769). The elution buffer used in this example was 1x SELEX buffer supplemented with 10 mM EDTA and 50 mM arginine.

[0101] Figure 6 is a combination of charts showing the time course of SPR signal when binding buffer containing AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV(DJ / 8), AAV(DJ), AAV9, or AAV9 (lab-made) was injected onto a sensor chip on which the aptamer (HIT15_AAV9_c3_1_35) had been immobilized according to Example 3. The vertical axis represents the SPR signal, expressed as a relative value (RU). In Figure 6, the SPR signal when the RNA aptamer bound to the sensor chip was set to 0. The horizontal axis represents the elapsed time (unit: seconds). The solid line represents the chart for AAV9 (lab-made), the double line represents AAV9, and the dashed-dotted line represents AAV2. The line types in the charts for the other AAVs are as described in Figure 6. The concentration of each AAV in the binding buffer was 10 ng / μL, and the flow rate and time for injecting the binding buffer in which each AAV was dissolved were 10 μL per minute for 2 minutes.

[0102] As shown in Figure 6, when AAV9 or AAV9 (lab-made) dissolved in binding buffer was injected onto the sensor chip with immobilized RNA aptamer, the SPR signal increased and remained constant after reaching a maximum value until the elution buffer was injected. The maximum SPR signal was approximately 2200 RU for AAV9 (lab-made) and approximately 1700 RU for AAV9. HIT15_AAV9_c3_1_35 hardly bound to AAV9 or AAV9 (lab-made). This suggests that HIT15_AAV9_c3_1_35 is an aptamer that binds very specifically to AAV9 and AAV9 (lab-made).

[0103] The above experimental results demonstrated that the aptamer of the present invention has extremely high binding specificity with AAV9, and that the aptamer of the present invention and AAV9 can be bound, dissociated, and manipulated under mild conditions that do not affect complementary base pairing and the specific binding between biotin and streptavidin. [Industrial Applicability]

[0104] The aptamers of the present invention are nucleic acids or derivatives thereof that replace the antibodies of conventional technology, and are therefore useful in that they are smaller molecules than antibodies or their fragments, have lower immunogenicity, and have high binding specificity to AAV9.

Claims

1. (a) or (b) below: (a) SEQ ID NO: 1: 5'-GUGCGUACAUUUGUUGUAAUACACAAAAUGCCACC-3' (wherein uracil (U) may be replaced by thymine (T)). (b) A nucleotide sequence in which one or several nucleotides are substituted, deleted, inserted, and / or added in the nucleotide sequence of (a) above. An aptamer against adeno-associated virus serotype 9 (AAV9), comprising:

2. The aptamer according to claim 1, wherein the nucleotide sequence (a) or (b) is represented by the following formula: 5'-GUGYGUACAUUUGUUGUAAUACRC １ ARRA １ UGC １ R １ Y 1 N-3' (SEQ ID NO: 23) (Wherein, Y is C or U, Y １ is C or U or absent, each R is independently A or G, R １ is A or G or absent, A １ is A or absent, and each C １ are independently C or absent, and N represents A, G, C, or U (however, uracil (U) may be thymine (T)).

3. (a) or (b) below: (a) a nucleotide sequence represented by any one of SEQ ID NOs: 2 to 19 (wherein uracil (U) may be replaced with thymine (T)); (b) A nucleotide sequence in which one or more nucleotides are substituted, deleted, inserted and / or added in the nucleotide sequence of (a) above (however, uracil (U) may be replaced with thymine (T)). An aptamer against AAV9, comprising:

4. The aptamer according to any one of claims 1 to 3, which has a length of 65 nucleotides or less.

5. A complex comprising the aptamer according to any one of claims 1 to 4 and a functional substance selected from the group consisting of an affinity substance, a labeling substance, an enzyme, a drug delivery vehicle, and a drug.

6. A reagent for detecting or purifying AAV9, comprising the aptamer according to any one of claims 1 to 4 or the complex according to claim 5.

7. A method for detecting or purifying AAV9, comprising using the aptamer according to any one of claims 1 to 4 or the complex according to claim 5.

8. A pharmaceutical composition comprising the complex of claim 5 comprising a drug delivery vehicle and AAV9.

9. The pharmaceutical composition of claim 8 , wherein the drug delivery vehicle is an aptamer for a surface molecule of a target cell.

10. The pharmaceutical composition of claim 9 , wherein the surface molecule is involved in crossing the blood-brain barrier.

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