Artificial virus capsid

Abstract

To provide an artificial virus capsid having specific targeting capability.SOLUTION: An artificial virus capsid presenting nucleic acid aptamer on a surface, which is formed of a self assembly of a plurality of sub-units each of which containing: β-annulus peptide of a tomato bushy stunt virus; and nucleic acid aptamer having a length of 40 nucleotides or more connected to a C-terminal side of β-annulus peptide.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to the field of drug delivery carriers. More specifically, the present disclosure relates to artificial virus capsids.

Background Art

[0002] The shell, i.e., the capsid, of a natural spherical (including polyhedral) virus is a protein nanocapsule having a uniform size of about 20 to 100 nm depending on the type of virus, and plays a role of protecting the genetic material of the virus from the outside. A virus capsid is typically formed by the regular association of a large number of (for example, about 60) identical protein subunits.

[0003] Natural virus capsids can encapsulate various substances to be delivered in addition to the virus genome in the internal cavity. Due to the uniformity of the shape and size of the virus capsid itself and the high efficiency of introduction into cells, their potential as delivery materials or vaccine materials for low-molecular-weight pharmaceuticals, nucleic acid pharmaceuticals, protein pharmaceuticals, etc. has attracted attention in recent years. However, natural virus capsids have problems such as usually requiring live cells for preparation, being difficult to modify or decorate the capsid structure by design, and often causing serious side effects due to potential toxicity, and are considered difficult to use as drug carriers. For this purpose, artificial carriers for drug delivery systems (DDS) such as polymer micelles have been developed as alternatives to virus capsids, but these do not always have the same advantages as virus capsids, and problems such as insufficient introduction efficiency remain.

[0004] The inventors of the present application have previously succeeded in creating spherical artificial virus capsids by self - assembling β - Annulus peptides derived from the protein forming the icosahedral internal skeleton of the capsid of tomato bushy stunt virus in vitro (Non - Patent Documents 1 - 4). The β - Annulus peptide is a peptide with a minimum unit length of 24 amino acids, corresponding to a part of the capsid protein consisting of 388 amino acids. The β - Annulus peptide spontaneously self - assembles in water to form spherical and hollow nanocapsids, i.e., artificial virus capsids, with a diameter of about 30 - 50 nm. In this nanocapsid, the β - Annulus peptide has its N - terminus facing the inside of the hollow capsule and its C - terminus facing the outside of the hollow capsule. It has been demonstrated that various substances can be encapsulated in this artificial virus capsid (either in the form of free molecules or in a form that binds or interacts with the N - terminus of the β - Annulus peptide). Such encapsulated substances are also called guests, and depending on the type of guest, the diameter of the artificial virus capsid can be slightly enlarged. Also, by linking molecules such as gold nanoparticles, single - stranded DNA with a length of 20 nucleotides, coiled - coil - forming peptides, human serum albumin, ribonuclease S, etc., near the C - terminus of the β - Annulus peptide, it has been demonstrated that artificial virus capsids with their outer surfaces modified by these molecules can be formed.

[0005] In particular, Non - Patent Document 4 describes that a single - stranded DNA with a length of 20 nucleotides (polydeoxyadenosine (dA 20 ) or polydeoxythymidine (dT 20 )) linked to a cysteine residue substituting glycine at the second position from the C - terminus of the β - Annulus peptide was self - assembled in vitro to form a DNA - modified artificial virus capsid.

Prior Art Documents

Non - Patent Documents

[0006]

Non - Patent Document 1

[0007] Embodiments of the present disclosure provide artificial viral capsids with specific targeting capabilities. [Means for solving the problem]

[0008] The inventors have discovered that a nucleic acid aptamer having a length of 40 nucleotides or more can be linked to the C-terminus of a β-annulus peptide and self-assembled into an artificial viral capsid with high uniformity, and that the modified artificial viral capsid thus produced can be efficiently and specifically delivered (e.g., introduced into the interior of a specific target cell) via the aptamer portion.

[0009] Non-Patent Document 4 describes a 20-nucleotide polydeoxyadenosine (dA 20 ) or polydeoxythymidine (dT 20) was described as being linked to a cysteine residue in which the second glycine from the C-terminus of the β-annulus peptide was replaced, but the nucleic acid aptamer used in the embodiments of the present application is a nucleic acid having a length more than twice that and has a significantly large negative charge. Despite the presence of a strong negative charge repulsion presumed between such long nucleic acids, the ability to self-assemble the artificial virus capsid was an unexpected discovery. Also, the embodiments of the present disclosure are the first to achieve and demonstrate cell-specific delivery of the artificial virus capsid.

[0010] The present disclosure includes at least the following embodiments. [1] A β-annulus peptide of tomato bushy stunt virus, and A nucleic acid aptamer having a length of 40 nucleotides or more, linked to the C-terminal side of the β-annulus peptide, An artificial virus capsid formed by self-assembly of a plurality of subunits, including a subunit containing and presenting a nucleic acid aptamer on the surface. [2] The artificial virus capsid according to [1], wherein the nucleic acid aptamer is a nucleic acid aptamer having any one of the sequences of SEQ ID NOs: 2 to 9. [3] In the β-annulus peptide, a cysteine or a cysteine residue-containing peptide is linked to the C-terminal side, and the nucleic acid aptamer is linked to the β-annulus peptide via a first thiol group derived from the cysteine. The artificial virus capsid according to [1] or [2]. [4] The nucleic acid aptamer is a synthetic nucleic acid aptamer having an amino group or a second thiol group at the 5'-end, and the β-annulus peptide and the nucleic acid aptamer are linked by a linker connecting between the first thiol group and the amino group or the second thiol group. The artificial virus capsid according to [3]. [5] The artificial virus capsid according to any one of [1] to [4], further comprising a subunit containing a β-annular peptide not linked to the nucleic acid aptamer, which is mixed with the subunit. [6] A pharmaceutical composition comprising the artificial virus capsid according to any one of [1] to [5]. [7] A carrier composition for drug delivery comprising the artificial virus capsid according to any one of [1] to [5]. [8] The composition according to [7] for delivering a drug to cancer cells. [9] The composition according to [8], wherein the cancer cell is lymphoma. [Advantages of the Invention]

[0011] The technology of nucleic acid aptamers is a technology for selecting, isolating and utilizing nucleic acid molecules that specifically bind to a target of interest from a library of random sequences. If nucleic acid aptamers with a length of 40 nucleotides or more can be used, the number of options for specific targets will increase exponentially significantly. According to the embodiments of the present disclosure, a very diverse selection of nucleic acid aptamers with a length of 40 nucleotides or more can be utilized to impart target specificity to artificial virus capsids. Compared with natural virus capsids, artificial virus capsids can be easily associated in vitro and have high potential for use as delivery materials or vaccine materials for encapsulating drug delivery. The embodiments of the present disclosure, which can impart any target specificity thereto, are significantly more advantageous than conventional drug delivery means and can be a breakthrough in the field of drug delivery systems (DDS). In particular, according to the embodiments of the present disclosure, the drug to be delivered encapsulated in the artificial virus capsid can be efficiently delivered to specific target cells such as cancer cells or into the interior of specific target cells. [Brief Description of the Drawings]

[0012]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0013] In one aspect, the present disclosure provides an artificial virus capsid presenting a nucleic acid aptamer on its surface, formed by self-assembly of a plurality of subunits, each subunit containing a β-annulus peptide of the tomato bushy stunt (TBSV) virus and a nucleic acid aptamer having a length of 40 nucleotides or more linked to the C-terminal side of the β-annulus peptide. In other words, this artificial virus capsid is an artificial virus capsid having nucleic acid aptamer modification on its outer surface.

[0014] The β-annular peptide of the TBSV virus (hereinafter simply referred to as "β-annular peptide") is known and reviewed in Non-Patent Document 1. The β-annular peptide consists of the amino acid sequence INHVGGTGGAIMAPVAVTRQLVGS (SEQ ID NO: 1), which is the smallest unit capable of forming an artificial virus capsid by self-assembly in water. In the present embodiment, the nucleic acid aptamer is linked to the C-terminal side of the β-annular peptide, but this does not necessarily mean that the nucleic acid aptamer is directly linked or bound to the S residue at the C-terminal of the above sequence. For example, additional amino acid(s) may be added to the C-terminal of the above sequence, and the nucleic acid aptamer may be linked to any of the added amino acids. One end of a linker other than an amino acid / peptide may be bound to the C-terminal of the above sequence or to any of the added amino acids, and the nucleic acid aptamer may be linked to the other end of the linker. That is, being linked to the C-terminal side can mean linkage to the C-terminal itself of SEQ ID NO: 1 or linkage to the portion added to the C-terminal. Synthesizing the peptide to have a functional group useful for linker binding or the linker itself at the C-terminal is within the skill of those in the art.

[0015] It has been proven in past research that additional amino acid sequences can be added to the C-terminal and / or N-terminal of SEQ ID NO: 1 to form an artificial virus capsid. In the embodiments of the present disclosure, for example, 1 to 20 residues, 1 to 10 residues, or 1 to 5 residues of amino acids may be added to the C-terminal of SEQ ID NO: 1. In the embodiments of the present disclosure, for example, 1 to 20 residues, 1 to 10 residues, or 1 to 5 residues of amino acids may be added to the N-terminal of SEQ ID NO: 1. All of these are understood to contain the β-annular peptide itself consisting of 24 residues. The additional peptide on the N-terminal side can be useful for promoting encapsulation of the encapsulated substance by interacting or binding with the encapsulated substance.

[0016] In this embodiment, the "subunit" is a unit molecule that forms an artificial virus capsid by self-assembly. Each subunit contains at least a β-annular peptide. However, the multiple subunits that self-assemble into one artificial virus capsid are not necessarily completely identical to each other. For example, a mode in which a subunit modified with a nucleic acid aptamer and a subunit without a nucleic acid aptamer coexist is also contemplated. An artificial virus capsid formed by the self-assembly of multiple subunits composed of subunits modified with nucleic acid aptamers is also contemplated. Self-assembly means, as described in Non-Patent Documents 1 to 4, an event in which multiple β-annular peptide-containing subunits spontaneously combine in water or an aqueous solution to form a substantially spherical artificial virus capsid, and a structure formed in such a manner. The diameter (measured by DLS (dynamic light scattering method)) of each artificial virus capsid formed by self-assembly can vary depending on the presence and type of guest, but is typically 30 to 100 nm. One artificial virus capsid is estimated to contain about 60 subunits. The pH of the aqueous solution during self-assembly is typically 5 to 9, and can be, for example, 6 to 8, or 6.5 to 7.5. The concentration of the subunits during self-assembly can be, for example, 1 to 50 μM, 3 to 40 μM, or 5 to 30 μM.

[0017] Surface presentation of a nucleic acid aptamer means that the nucleic acid aptamer bound to the artificial virus capsid is exposed on the outer surface of the artificial virus capsid. In the artificial virus capsid of β-annular peptide, which is inherently a hollow nanocapsule, the C-terminus of the β-annular peptide faces the outside of the nanocapsule. Therefore, in this embodiment, surface presentation is achieved by linking a nucleic acid aptamer to the C-terminal side of the β-annular peptide.

[0018] As is understood by those skilled in the art, a nucleic acid aptamer means a nucleic acid molecule that can specifically bind to a target substance. The technology of nucleic acid aptamers itself is well known to those skilled in the art. Nucleic acid aptamers are nucleic acid molecules selected from a library of random sequences by repeatedly screening for the ability to bind to a target substance (a procedure called SELEX: Systematic evolution of ligands by exponential enrichment), or nucleic acid molecules obtained by partially modifying the sequences of such selected nucleic acid molecules (such as truncation and / or chemical modification). Nucleic acid aptamers can specifically bind to a target substance with a high binding affinity represented by a dissociation constant value Kd of less than micromolar (preferably less than 100 nanomolar, more preferably less than 10 nanomolar). Since aptamers are obtained or prepared to bind to a desired target substance, inevitably, each aptamer has a known specific target. Therefore, those skilled in the art can not only clearly recognize nucleic acid aptamers but also recognize their corresponding targets. However, for example, for nucleic acid aptamers selected to target specific cells, it may not have reached the molecular-level target identification of which molecule in the cell the aptamer binds to.

[0019] In the context of this disclosure, the term "specific" used to describe an aptamer or an artificial virus capsid binding or targeting a target means that the aptamer or artificial virus capsid non-randomly binds or targets a specific single substance, or a specific plurality of substances having common or similar structures. As is understood by those skilled in the art, the specific binding (docking) of a nucleic acid aptamer to a target substance is different from hybridization to a complementary nucleic acid, and the specific target of a nucleic acid aptamer is usually a non-nucleic acid substance. However, nucleic acid aptamers that bind (dock) to nucleic acid substances in a manner other than hybridization are not excluded.

[0020] Nucleic acid aptamers can be DNA, RNA, or combinations thereof, such as single-stranded DNA, single-stranded RNA, or combinations thereof. The term nucleic acid aptamer also includes those containing one or more nucleic acid modifications or unnatural nucleic acid moieties (such as unnatural backbone moieties and unnatural sugar moieties) known to those skilled in the art. For example, various nucleic acid modifications and unnatural nucleic acid moieties that can be used in aptamers for nuclease resistance and other purposes are known (Zhou et al., Nat Rev Drug Discov, 2017, 16(3): 181-202). Examples include, but are not limited to, 2'-fluorination, 2'-amination, 2'-O-methylation, 3' modification with inverted thymidine, phosphorothioate, LNA, and polyethylene glycol (PEG) modification.

[0021] There are also nucleic acid aptamers with a length of 10 nucleotides or less, but the nucleic acid aptamers in this embodiment are particularly those having a length of 40 nucleotides or more, that is, nucleic acid aptamers containing 40 residues or more of nucleotides. The length of the nucleic acid aptamer is typically 200 nucleotides or less, more typically 100 nucleotides or less, or 60 nucleotides or less, but may be longer than these. If it is 40 nucleotides or longer, for example, compared to the case of 20 nucleotides or shorter, the complexity of the SELEX library is significantly improved, and the diversity of specific target options is significantly increased. On the other hand, when the nucleic acid, which is a polyanion, has a length of 40 nucleotides or more, the negative charge becomes extremely large. The inventors surprisingly found that despite the strong mutual repulsion due to such a large negative charge, a β-annulus peptide modified at the C-terminal side with a nucleic acid aptamer having a length of 40 nucleotides or more can self-assemble into an artificial virus capsid, and rather, its self-assembly can even be promoted compared to the case of an unmodified β-annulus peptide.

[0022] In certain embodiments of the present disclosure, the nucleic acid aptamer is a nucleic acid aptamer having any one of the sequences of SEQ ID NOs: 2-9. SEQ ID NOs: 2-9 are the sequences of nucleic acid aptamers isolated and identified by Tang et al., Anal. Chem. 2007, 79, 4900-4907 based on the criterion of binding to Ramos cells, which are B cell lymphomas, and these nucleic acid aptamers bind to B cell- and / or T cell-derived lymphomas with high affinity and selectivity. The inventors have found that the artificial virus capsid of this embodiment presenting these nucleic acid aptamers specifically binds to lymphoma cells and that the artificial virus capsid and / or its inclusion can be efficiently taken up into the cells, as compared to the capsid without the nucleic acid aptamer. This is an important advancement from the perspective of cancer treatment or, more generally, drug delivery to specific cells using artificial virus capsids, but it was not necessarily predictable. Tang et al. described isolating these nucleic acid aptamers by a screening procedure in which a random DNA library of SELEX was bound to Ramos cells, washed to remove unbound DNA, and the DNA bound to the cell surface was eluted by heating and the recovered DNA was amplified for use in the next round of selection. It is of great significance to have found that the artificial virus capsid presenting the nucleic acid aptamer is not only bound to the surface of lymphoma cells but also taken up into the cells.

[0023] SEQ ID NOs: 2, 4, 5, 6, 7, and 8 may be particularly suitable for targeting B lymphoma, and SEQ ID NOs: 2, 3, 5, 8, and 9 may be particularly suitable for targeting T lymphoma. The nucleic acid aptamer in this embodiment may be a nucleic acid aptamer consisting of SEQ ID NOs: 2 to 9, but additional nucleic acid sequences may be added to the 5'-side and / or 3'-side. In fact, the nucleic acid aptamer of Tang et al. was also originally isolated and identified from a nucleic acid population in the state of a long sequence with primer sequences for DNA amplification added to both ends. Thus, it is typical in general nucleic acid aptamers that additional nucleic acid sequences can be added to the 5'-side and / or 3'-side of the core sequence responsible for target binding ability. An artificial virus capsid presenting a nucleic acid aptamer containing or consisting of SEQ ID NO: 6 is an example of a particularly preferred embodiment.

[0024] As described above, in the β-annular peptide in the embodiment of the present disclosure, additional amino acid(s) may be added to the C-terminus of SEQ ID NO: 1. Non-Patent Document 4 describes ligating a 20-nucleotide-long nucleic acid to a cysteine residue in which glycine, the second internal residue from the C-terminus, is substituted, rather than the C-terminus itself of the β-annular peptide. However, in the present disclosure, by ligating a nucleic acid aptamer to a portion further added to the C-terminus, particularly a peptide further added to the C-terminus, it has been found that the nucleic acid aptamer can be presented on an artificial virus capsid with a narrower particle size distribution (i.e., higher uniformity). Thus, it is speculated that the portion added to the C-terminus may serve as a spacer suitable for self-assembling into an artificial virus capsid while presenting the nucleic acid aptamer.

[0025] In one embodiment, a cysteine or a cysteine residue-containing peptide is linked to the C-terminus of the β-annular peptide. This linkage is preferably a linkage by a peptide bond, that is, an extension of the peptide. As a result, a β-annular peptide-containing peptide longer than 24 residues is formed. It is preferable that there are, for example, 1 to 10 amino acids, 2 to 5 amino acids, or 3 amino acids between the C-terminus of the β-annular peptide represented by SEQ ID NO: 1 and the above-mentioned cysteine residue. The cysteine residue is preferably not located at the outermost C-terminus of the entire β-annular peptide-containing peptide. A specific example of a suitable cysteine residue-containing peptide is GGGCG (SEQ ID NO: 10).

[0026] The nucleic acid aptamer can be linked to the β-annular peptide via a first thiol group derived from this cysteine. As is well known to those skilled in the art, the thiol group can react efficiently with, for example, a maleimide group to form a thiosuccinimide linkage. Alternatively, it can also react with another thiol group to form a disulfide linkage. Based on ordinary knowledge, those skilled in the art can link an appropriate linker or an aptamer-binding linker to the β-annular peptide by these thiol group reactions.

[0027] The 24-residue β-annular peptide, which is the minimum unit for self-assembly, originally contains no cysteine residues and has no free thiol groups. Therefore, the first thiol group derived from the cysteine residue added in this way can be easily utilized for site-specific ligation of nucleic acid aptamers. Alternatively, a cysteine or a cysteine residue-containing peptide can be ligated to the N-terminus of the β-annular peptide, and the thiol group derived from the cysteine can be used for ligation to the capsid inclusion. It should be noted that terms such as "first" and "second" in the present disclosure are merely for convenience of expression to refer to different objects and do not necessarily mean a specific order. For example, the term "first thiol group" in the previous paragraph and this paragraph is merely a term for convenience of distinction from thiol groups provided by molecules other than the β-annular peptide-containing peptide and does not mean a specific position in the amino acid sequence.

[0028] In a specific embodiment where a cysteine or a cysteine residue-containing peptide providing the first thiol group is ligated to the C-terminus of the β-annular peptide, a synthetic nucleic acid aptamer having an amino group or a second thiol group at the 5'-end may be used as the nucleic acid aptamer. Synthesizing a nucleic acid having these groups at the 5'-end (i.e., these groups are bonded or ligated) is within the ordinary skill of those in the art. In this embodiment, the β-annular peptide and the nucleic acid aptamer can be ligated by a linker connecting between the first thiol group provided from the β-annular peptide side and the amino group or the second thiol group provided from the nucleic acid aptamer side.

[0029] A linker, as commonly understood by those skilled in the art, is a chemical structure that connects one chemical moiety (e.g., a peptide) to another chemical moiety (e.g., a nucleic acid) by a series of covalent bonds. The linker is typically a hydrocarbon-based linker. The hydrocarbon backbone of the hydrocarbon-based linker may contain non-carbon atoms substituted or inserted in the backbone. Linkers capable of connecting between a thiol group and an amino group are well known to those skilled in the art (N-(4-maleimidobutyryloxy)sulfosuccinimide (Sulfo-GMBS) is an example thereof), and those skilled in the art can also design and synthesize them as appropriate. When such a linker is, for example, "connecting between a thiol group and an amino group", or "linked to a β-annular peptide via a thiol group", etc., it means that the reactions of these mentioned reactive groups are utilized for the formation of the linking moiety, and it will be understood by those skilled in the art that the thiol group and the amino group do not remain in their original forms after the linkage.

[0030] Linkers capable of connecting between a thiol group and a thiol group are also well known to those skilled in the art (bis-maleimides such as 1,8-bis-maleimidodiethylene glycol are examples thereof), and those skilled in the art can also design and synthesize them as appropriate. When directly reacting a thiol group with a thiol group to form a disulfide linkage, the linker between the two will be a single covalent bond.

[0031] Other reactive groups known to those skilled in the art can also be used, that is, other reactive groups can be linked to the C-terminal side of the β-annular peptide and / or the 5' or 3' end of the nucleic acid aptamer to link the β-annular peptide and the nucleic acid aptamer. In any case, the length of the linker between the β-annular peptide-containing peptide and the nucleic acid aptamer can be, for example, 2-100 Å, 2-50 Å, or 5-20 Å, but is not limited thereto. In one example, one end of the linker can first be linked to the 5' or 3' end of the nucleic acid aptamer, and then the other end of the linker can be linked to the C-terminal side of the β-annular peptide. In another example, one end of the linker can first be linked to the C-terminal side of the β-annular peptide, and then the other end of the linker can be linked to the 5' or 3' end of the nucleic acid aptamer. In yet another example, the linking of the nucleic acid aptamer and the β-annular peptide to both ends of the linker can be carried out simultaneously.

[0032] As described above, all subunits may be β-annular peptides linked to nucleic acid aptamers. However, in another embodiment, in addition to the subunits of β-annular peptides linked to nucleic acid aptamers, subunits of β-annular peptides not linked to nucleic acid aptamers may further be included. The artificial virus capsid according to the embodiment of the present disclosure includes at least one subunit of a β-annular peptide linked to a nucleic acid aptamer. The molar ratio of the β-annular peptide linked to the nucleic acid aptamer is preferably 10% or more, and may be 30% or more, 50% or more, 70% or more, or 90% or more, and may even be 100%.

[0033] The subunit of the β-annular peptide not linked to the nucleic acid aptamer may be, for example, the β-annular peptide alone. In another embodiment, the subunit of the β-annular peptide not linked to the nucleic acid aptamer may have an encapsulated substance of the artificial virus capsid linked to its N-terminus, or a moiety (e.g., an amino acid or a peptide) for binding or interacting with the encapsulated substance may be linked to its N-terminus.

[0034] In another aspect, the present disclosure provides a pharmaceutical composition comprising the artificial virus capsid described above. In other words, an artificial virus capsid for use as a medicine is provided by the present disclosure. Embodiments of a treatment method including administering the artificial virus capsid or pharmaceutical composition of the present disclosure to a subject are also contemplated. In another aspect, a drug delivery carrier composition comprising the artificial virus capsid described above is provided. In other words, an artificial virus capsid for use as a drug delivery carrier is provided by the present disclosure. A pharmaceutical composition can be prepared by encapsulating a therapeutically effective amount of a drug inside the capsid of the drug delivery carrier composition. Embodiments of a method for delivering a drug to a specific target site within a subject, such as a target tissue, a target cell, or a target molecule, including preparing an artificial virus capsid containing an effective amount of the drug or a pharmaceutical composition containing the same, and / or administering the artificial virus capsid or pharmaceutical composition to the subject are also contemplated.

[0035] These compositions can include pharmaceutically acceptable carriers, excipients, additives, etc. in addition to the artificial virus capsid. An example of a suitable carrier is water. The composition can contain salts at a concentration suitable for, for example, injection into the human body. Those skilled in the art can appropriately determine and select a suitable administration route for delivering an effective amount of the artificial virus capsid or pharmaceutical composition to the site or region where the target substance of the nucleic acid aptamer is present. Examples of administration routes include, but are not limited to, oral, intravenous, intraarterial, intramuscular, subcutaneous, transdermal, intraperitoneal, intrathecal, rectal, vaginal, ocular, and inhalation.

[0036] The artificial virus capsid and compositions according to embodiments of the present disclosure can be useful in vitro, ex vivo, and in vivo. Accordingly, the subject to which the artificial virus capsid or composition is administered is typically an animal individual, such as a mammalian individual, particularly a human individual or a human patient such as a cancer patient, although in vitro tissues or cultured cells of these animals may also be the subject of administration.

[0037] Drug delivery carrier compositions can have different specific targets depending on the type of nucleic acid aptamer. In one example, the specific target is cancer cells. That is, the drug delivery carrier composition can be for delivering a drug to cancer cells. The drug delivery carrier composition can be for delivering a drug inside cancer cells. In certain embodiments, the cancer cells are lymphoma. The lymphoma can be B-cell lymphoma and / or T-cell lymphoma. The lymphoma can be, for example, Burkitt lymphoma.

[0038] In the present disclosure, the symbol "~" indicating a numerical range means including the numerical values described before and after it as the lower limit value and the upper limit value, respectively. When a plurality of possible numerical ranges such as "A~B" and "C~D" are separately described, a numerical range obtained by combining one lower limit or upper limit with the other lower limit or upper limit (for example, "A~D", "C~B") is also understood to be disclosed in this specification. Also, when a combination of a first element and a second element is described and a plurality of possible options are described for each of the first element and the second element, all possible combinations of each option of the first element and each option of the second element are understood to be disclosed in this specification. In the present disclosure, expressions such as "containing X" and "including X" are understood to include the "consisting of X" aspect as well, unless it is explicitly stated that the "consisting of X" aspect is excluded.

Examples

[0039] Hereinafter, specific embodiments will be described in detail by showing examples, but these are examples and the invention of the present disclosure is not limited to these examples.

[0040] [Example 1. Preparation of an artificial virus capsid presenting a nucleic acid aptamer on the surface] Among the nucleic acid aptamers described by Tang et al., Anal. Chem. 2007, 79, 4900-4907, an example using the 43-nucleotide-long DNA aptamer TE02 is shown. TE02 consists of the sequence 5’-TAGGCAGTGGTTTGACGTCCGCATGTTGGGAATAGCCACGCCT-3’ (SEQ ID NO: 6). In this example, this TE02 aptamer was custom-synthesized (by Bex Co., Ltd.) in a form where an amino group (-NH2) was linked via a -(CH2)6-linker at the 5’ end. This form of the TE02 aptamer is called TE02-NH2. Subsequently, a maleimide group was linked to TE02-NH2 according to the following synthetic scheme. The resulting compound is called TE02-maleimide.

[0041]

Chemical formula

[0042] Specifically, to 20 μL of a 0.80 mM stock solution of TE02-NH2, 0.9 mg (120 equiv.) of Sulfo-GMBS crosslinker (Mw 382.28, Dojindo Laboratories) and 60 μL of 0.1 M aqueous NaHCO3 solution were added, and the mixture was allowed to stand in an incubator at 25 °C for 4 hours (final concentration 200 μM TE02-NH2, 24.0 mM Sulfo-GMBS). Then, dialysis was performed with 1000 mL of ion-exchanged water (Spectra / por7, cutoff Mw: 1000, manufactured by SPECTRUM). The ion-exchanged water was replaced 1, 2, 3, and 20 hours after the start of dialysis, and stirring was carried out for a total of 24 hours. After dialysis, by UV measurement, adenine (ε = 15400 M -1 cm -1 ), thymine (ε = 9300 M -1 cm -1 ), guanine (ε = 11800 M -1 cm -1 ), and cytosine (ε = 7400 M -1 cm -1The concentration was determined from the absorption coefficient of () and the absorbance at 260 nm derived from DNA according to Lambert-Beer's law.

[0043] The UV measurement conditions are as follows. Bandwidth: 1.5 nm Response: Fast Measurement wavelength range: 700 - 200 nm Data acquisition interval: 1.0 nm Scanning speed: 400 nm / min Temperature: 25°C Optical path length: 10 mm

[0044] C = 0.1154×50 / [(15400×8)+(9300×11)+(11800×14)+(7400×10)] = 12.4 μM 12.4 μM × 900 μL = 11.2 nmol From the above calculation, the yield of TE02-maleimide was determined to be 11.2 nmol, which is a yield of 70.0% with respect to the theoretical yield of 16 nmol. TE02-maleimide was separated and purified from unreacted TE02-NH2 by reverse-phase HPLC (CH3CN / NH4HCO2 aq.; CH3CN 5% → 100% (95 min)), and TE02-maleimide (Mw 13635) was confirmed by MALDI-TOF-MS (matrix: 3-HPA).

[0045] Subsequently, a β-annular peptide with a TE02 aptamer linked to the C-terminus was synthesized according to the following scheme. β-annular-GGGCG peptide (Mw 2637) has a GGGCG peptide in such a form that the C-terminus of the 24-amino acid-long minimum unit (SEQ ID NO: 1) of the β-annular peptide is extended. This C (cysteine) residue provides an SH group (thiol group). This SH group was reacted with the maleimide group of TE02-maleimide to link the β-annular peptide and the TE02 aptamer. The resulting product is called β-annular-aptamer, or particularly β-annular-TE02.

[0046]

Chemical formula

[0047] Specifically, first, TE02-maleimide (11.2 nmol) placed in an Eppendorf tube was lyophilized, and then 100 μL of 0.2 M sodium phosphate buffer (pH 7.0) was added thereto. In another Eppendorf tube, 0.467 mg of β-annulus-GGGCG peptide was weighed, and 400 μL of ion-exchanged water and 500 μL of acetonitrile were added thereto. Then, the contents of these two Eppendorf tubes were mixed (final concentration of TE02-maleimide: 11.2 μM, final concentration of β-annulus-GGGCG peptide: 177 μM). After stirring with a vortex, it was shaken at 40 °C for 70 hours, and fractionation was performed by reverse-phase HPLC.

[0048] The fractionation conditions are as follows. Sample: After centrifuging the reaction sample, the supernatant was used. Sample injection volume: 1000 μL Column: Inertsil WP300 C18 (5 mm, 20×250 mm) Mobile phase solvent: 95% aqueous 0.1 M ammonium formate solution (0 min) → 0% (95 min), acetonitrile 5% (0 min) → 100% (95 min) linear gradient Flow rate: 10.0 mL / min Detection: 260 nm

[0049] Regarding the fractionation solution, after evaporating the solvent with an evaporator, 300 μL of ion-exchanged water was added, and the concentration was determined by UV measurement under the same conditions as above. C = 0.3883×10 / [(15400×8)+(9300×11)+(11800×14)+(7400×10)] = 8.36 μM 8.36 μM×300 μL = 2.51 nmol By the above calculation, the yield of β - annulus - aptamer was determined to be 2.51 nmol, which is a yield of 22.4% with respect to the theoretical yield of 11.2 nmol. β - annulus - TE02 was separated and purified from unreacted TE02 - maleimide by reverse - phase HPLC (CH3CN / NH4HCO2aq.; CH3CN 5% → 100% (95 min)), and β - annulus - TE02 was confirmed by MALDI - TOF - MS (matrix: 3 - HPA).

[0050] Subsequently, an aqueous solution of β - annulus - aptamer was prepared to be 5 - 50 μM in 10 mM phosphate buffer (pH 7.0). After irradiating this aqueous solution with ultrasonic waves for 5 minutes, it was allowed to stand for 30 minutes. By the above operations, the β - annulus peptide moiety self - assembled, and an artificial virus capsid presenting a 43 - nucleotide - long TE02 aptamer on the surface was obtained.

[0051] [Example 2. Characterization of artificial virus capsid particles presenting nucleic acid aptamers on the surface] 2 - 1. Particle size measurement by dynamic light scattering (DLS) The measurement conditions of DLS are as follows. Cell: ZEN2112 - Low volume glass cuvette (12 μL) Temperature: 25 °C Solvent: 10 mM phosphate buffer (pH 7.0)

[0052] 2 - 2. Transmission electron microscope (TEM) observation 5 μL of the artificial virus capsid obtained in Example 1 was dropped onto a grid (C - SMART Hydrophilic TEM grids, manufactured by Alliance Biosystems) and allowed to stand for 1 minute. Then, the droplet was removed using filter paper, and 5 μL of a staining solution (2% Na3(PW 12 O 40 )(H2O) n ) was dropped and allowed to stand for 1 minute, and then the droplet was removed again. Subsequently, after drying under reduced pressure overnight, TEM observation was performed (acceleration voltage 80 kV).

[0053] 2-3. Results The results of DLS measurement are shown in Fig. 1(a). When β-annulus-aptamer was self-assembled at a concentration of 5 μM or 25 μM, particle sizes with an average diameter of 53 nm or 55 nm were obtained, respectively. From this particle size and narrow distribution, it was clear that the artificial virus capsid was properly formed, and this conclusion was confirmed by the TEM observation shown in Fig. 1(b). The particle size distributions of 53±12 nm and 55±16 nm obtained here are significantly narrower than the 98±63 nm obtained in Non-Patent Document 4, that is, an artificial virus capsid with higher uniformity was obtained. In this regard, it was considered preferable to ligate the nucleic acid aptamer to the residue further added to the C-terminus rather than the internal residue near the C-terminus of the β-annulus peptide.

[0054] On the other hand, when β-annulus-aptamer was self-assembled at a concentration of 50 μM, the average diameter measured by DLS was 222 nm. Considering it together with the TEM image, it was considered that aggregation of the artificial virus capsids had occurred (the left end of Fig. 1a, the left end of Fig. 1b).

[0055] The critical association concentration of β-annulus-GGGCG peptide without aptamer modification is estimated to be 29 μM. The fact that the β-annulus-aptamer could associate even at a low concentration of 5 μM below it suggests that the self-assembly stabilization by the aptamer-modified β-annulus was rather promoted despite the long nucleic acid of more than 40 nucleotides and the accompanying large negative charge and electrical repulsion.

[0056] [Example 3. CD Spectrum Measurement of Artificial Virus Capsid with Nucleic Acid Aptamer Presented on the Surface] 10 mM phosphate buffer (pH 7.0) was added to TE02-NH2 or β-annulus-TE02 to prepare them to be 20 μM each. CD (circular dichroism) spectrum measurement was performed using these samples. The measurement conditions are as follows. Solvent: 10 mM phosphate buffer (pH 7.0) Cell: Quartz cell with an optical path length of 0.1 cm Temperature: 25°C, Sample volume: 200 μL each, Measurement wavelength range: 200 - 400 nm Scanning speed: 200 nm / min Number of integrations: 32 times

[0057] For the CD measurement with temperature change, the measurement wavelength was set to 270 nm, and the measurement temperature range was set to -10°C to 90°C.

[0058] The results are shown in Figure 2. These CD measurement results indicate that even when the nucleic acid aptamer is presented on the artificial virus capsid based on β-annular peptide, the spectrum of B-type DNA is maintained, and the original secondary structure of the nucleic acid aptamer is substantially maintained. It was also observed that the denaturation point of the aptamer presented on this artificial virus capsid increased compared to the same aptamer existing alone (Figure 2c; it increased from 40°C for the aptamer alone to 45°C on the artificial virus capsid). The denaturation point corresponds to the "inflection point" of the melting curve as represented in Figure 2(c), and mathematically, it is the point where the differential value is maximized. The increase in the denaturation point suggests that the structural stability of the nucleic acid aptamer has been improved by being presented on the artificial virus capsid.

[0059] [Example 4. Uptake of β-annulars-TE02 artificial virus capsid by cancer cells] This example describes an experiment demonstrating that the artificial virus capsid presenting the TE02 aptamer on its surface is taken into the target cancer cells via this cancer cell-specific aptamer.

[0060] 4-1. Preparation of TAMRA-β-annulars-TE02 artificial virus capsid For this purpose, first, an attempt was made to prepare an artificial virus capsid that presents the TE02 aptamer on its surface and encapsulates the fluorescent dye TAMRA (carboxytetramethylrhodamine) inside. TAMRA is useful for visualizing the uptake of the artificial virus capsid into cells.

[0061] A peptide with a cysteine residue added to the N-terminus of the β-annular peptide was synthesized, and a commercially available TAMRA-maleimide (Mw 2890) was reacted with the thiol group of the cysteine to synthesize TAMRA-β-annular peptide. β-annular-TE02 prepared in Example 1 and TAMRA-β-annular were mixed at a concentration ratio of 10:1, and these two types of subunits were self-assembled, that is, co-assembled, in the same procedure as in Example 1.

[0062] More specifically, 10 μL each of a 50 μM aqueous solution of β-annular-TE02 and a 5 μM aqueous solution of TAMRA-β-annular were taken and mixed to prepare a mixed aqueous solution with final concentrations of 25 μM and 2.5 μM, respectively. This was irradiated with ultrasonic waves for 5 minutes and allowed to stand. DLS measurements were performed in the same manner as in Example 2 at 0 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes after standing. At any time point, a particle size of about 70 to 80 nm was obtained as the main peak of the particle size distribution.

[0063] 4-2. Administration of the artificial virus capsid to lymphoma cells TE02 is an aptamer identified as binding to human Burkitt lymphoma cells (Tang et al. supra). In this experiment, the Daudi cell line derived from Burkitt lymphoma was used.

[0064] As described in 4-1 above, an artificial virus capsid in which β-annular-TE02 and TAMRA-β-annular were mixed at 10:1, and a control artificial virus capsid in which a non-aptamer-modified β-annular peptide and TAMRA-β-annular were mixed at 10:1 were each prepared and diluted 5-fold with cell culture medium.

[0065] Daudi cells (cell density: 1.0×10 624 μL of the cells (100 μL) were added to a culture dish, and 30 μL of the solution of the artificial virus capsid was taken and added to the dish. After incubating at 37°C for 1 hour, 6 μL of Hoechst 33342 (100 μg / mL), a cell nucleus staining fluorescent dye, was added and incubated for 10 minutes, and then confocal laser scanning microscopy (CLSM) observation was performed. The conditions of CLSM were Ex: 553 nm, Em: 577 nm, laser intensity: 15% for TAMRA (rhodamine), and Ex: 346 nm, Em: 460 nm, laser intensity: 15% for Hoechst 33342.

[0066] The results of detecting the signal of TAMRA by CLSM are shown in Fig. 3. Although not shown, it has been confirmed by cell nucleus staining with Hoechst 33342 and bright-field microscopy that one live cell is located in each panel of Fig. 3. When a control artificial virus capsid without an aptamer was administered, the rhodamine fluorescence signal in the cells was substantially unchanged from the background (lower panel of Fig. 3), whereas when an artificial virus capsid presenting the TE02 aptamer was administered, a rhodamine fluorescence signal derived from TAMRA was clearly observed on the cell surface and inside most of the lymphoma cells (upper panel of Fig. 3). This result demonstrates that the artificial virus capsid binds to the cells that are the specific targets of the aptamer and is efficiently taken up into the cells through the presence of the cell-specific nucleic acid aptamer presented on the surface of the artificial virus capsid.

Claims

**Claim 1**: A β-annulus peptide having the amino acid sequence of SEQ ID NO: 1, and a nucleic acid aptamer having a length of 40 nucleotides or more, linked to the C-terminal side of the β-annulus peptide A plurality of subunits formed by self-assembly of subunits containing An artificial virus capsid presenting a nucleic acid aptamer on the surface, In the β-annulus peptide, a cysteine residue-containing peptide is linked to the C-terminal side of SEQ ID NO: 1 by a peptide bond, and 2 to 5 amino acids are present between the C-terminal of SEQ ID NO: 1 and the cysteine residue, and the nucleic acid aptamer is linked to the β-annulus peptide via a first thiol group derived from the cysteine residue. Artificial virus capsid. **Claim 2** The artificial virus capsid according to claim 1, wherein the nucleic acid aptamer is a nucleic acid aptamer having any one of the sequences of SEQ ID NOs: 2 to 9. **Claim 3** The nucleic acid aptamer is a synthetic nucleic acid aptamer having an amino group or a second thiol group at the 5'-end, and the β-annulus peptide and the nucleic acid aptamer are linked by a linker connecting between the first thiol group and the amino group or the second thiol group. The artificial virus capsid according to claim 1 or 2. **Claim 4** The artificial virus capsid according to any one of claims 1 to 3, further comprising a subunit containing a β-annulus peptide not linked to the nucleic acid aptamer, which is mixed with the subunit. **Claim 5** A pharmaceutical composition comprising the artificial virus capsid according to any one of claims 1 to 4. **Claim 6** A carrier composition for drug delivery comprising the artificial virus capsid according to any one of claims 1 to 4. **Claim 7** The composition according to claim 6, for delivering a drug to cancer cells. **Claim 8** The composition according to claim 7, wherein the cancer cells are lymphoma.

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