Agent for introducing gene into cardiac constituent cell species

Abstract

The principal purpose of the present invention is to provide a highly safe and efficient technology for introducing a gene into a cardiac constituent cell species. The present application discloses, as one embodiment, an agent for introducing a gene into a cardiac constituent cell species, the agent containing a divalent molecule capable of binding to both a natural AAV capsid and a cardiac constituent cell species surface antigen.

Description

Gene transfer agents for cardiac cell types

[0001] As one embodiment of the present invention, the present invention provides a gene delivery agent for cardiac cell types that uses a bivalent molecule capable of binding to both the capsid of a natural adeno-associated virus (hereinafter also referred to as "AAV") and target molecules on cardiac cell types such as cardiomyocytes. The present invention is useful, for example, in fields such as gene therapy for heart diseases.

[0002] Gene therapy for heart diseases (e.g., heart failure, various cardiomyopathy, arrhythmias, etc.) has become an effective treatment method in clinical practice. In carrying out such gene therapy, viruses that deliver their own genes to host cells have been used in the field of gene therapy as a means of delivering therapeutic genes to patients. However, not all viruses are suitable for this purpose due to their pathogenicity and immunogenicity. AAV is one of the most commonly used viruses because it is not pathogenic to humans and has an excellent safety profile (Non-Patent Literature 1). Transduction of AAV into cells is initiated by the attachment of the virus to the cell via the cell surface glycan portion (hereinafter referred to as the adhesion receptor). Since the amino acid sequence of the surface loop of the capsid involved in adhesion receptor binding differs among different serotypes, the chemical properties of the adhesion receptor of a particular AAV depend on its serotype. For example, AAV serotype 2 (AAV2) binds to heparan sulfate proteoglycan (HSPG), AAV1 to sialic acid, and AAV9 to galactose at the end of the sugar chain. After attachment, when AAV binds to the second class of receptors (generally called internalized receptors), internal translocation of the virus is initiated via endocytosis. The gene product KIAA0319L (later renamed AAVR) was discovered as a universal co-receptor for the internal translocation of most AAV serotypes, with a few exceptions including AAV4 (Non-Patent Literature 2). AAVR is localized to the intracellular trans-Golgi network (TGN) under steady state, and cells lacking it cannot be infected with AAV, suggesting that retrograde transport via AAVR is essential for AAV transduction. AAVR is also recognized by a loop protruding from the surface of the AAV capsid, and its interaction mode has been elucidated by cryo-electron microscopy. Among viral vectors, AAVs have emerged as promising candidates for gene therapy vectors in recent years. This is evidenced by the fact that two drugs, Zolgensma (using AAV9 as a capsid) for spinal muscular atrophy and Hemgenix (using AAV5) for hemophilia, were approved by the FDA in 2020 and 2022, respectively.AAV is a suitable vector for gene therapy due to its relatively low immunogenicity, ability to transduce a wide range of cells including both dividing and non-dividing cells, and long-term expression induced from stable episomes. However, despite the success of drug development, current AAV vectors have many problems, and there is a strong need for fundamental improvement. The main problem is that both the infection specificity and transduction efficiency of AAV are low, which necessitates high-dose administration to achieve therapeutic effects in target organs (Non-Patent Literature 3). Such high-dose AAV administration can cause serious side effects, and in fact, multiple deaths due to liver dysfunction occurred in clinical trials for X-linked myotubular myopathy (Non-Patent Literature 4). The need for high-dose administration also means that large quantities of medical-grade viral vectors must be produced, and aside from safety, manufacturing costs are a hurdle to the medical application of AAV vectors. Therefore, there is a desire to improve specificity and transduction efficiency and reduce the vector dose, and in recent years, a method that has become popular for this purpose is to modify the capsid itself, which is the main determinant of AAV directivity and transduction characteristics (Non-Patent Literature 3). The general approach to capsid modification is to first prepare a library of AAV capsid variants by random mutagenesis, capsid shuffling, or insertion of short random peptides, administer this to animals, and select candidates with the desired directivity or characteristics. However, this conventional approach requires an enormous amount of time and effort not only for genetic engineering and production to create millions of different viruses, but also for their identification and evaluation. Moreover, even if a promising mutant capsid is obtained through such efforts, its in vivo characteristics depend on various factors, and it is often not applicable to animal species or serotypes other than those used for screening (Non-Patent Literature 5). Furthermore, a common challenge with variants obtained through in vivo selection starting from randomized capsid libraries is that the molecular mechanisms and responsible receptors underlying their characteristics, such as infection specificity and improved transduction efficiency, often remain unexplained.In contrast to the approach of probabilistically obtaining desirable mutant viruses through screening from randomized libraries as described above, rational design is an approach that highly predicts and modifies the properties of mutant viruses from the outset by utilizing existing knowledge of intermolecular interactions. A notable success of rational AAV design is the method of inserting or fusing nanobodies or small binder proteins such as DARPins to the capsid for specific cell receptors (Non-Patent Literature 6 and 7). However, this strategy has the drawback that such special binder molecules must be obtained in advance. More importantly, this method requires inserting fairly large protein domains (both nanobodies and DARPins are about 15 kDa) into the capsid subunits, and there is a very high risk of losing the structural and functional integrity of the capsid. In fact, it is known that even the insertion of extremely short random peptides (7 amino acids) can significantly alter the chemical structure of the capsid surface, making it impossible to recombinantly express most of the resulting variants. In other words, whether starting from a random library through directed evolution or utilizing rational design, it has been pointed out that if even a slight modification is made to a naturally occurring serotype of AAV, it is necessary to verify each aspect, such as the recombination production efficiency, in vivo pharmacokinetics, and whether it exhibits unexpected human toxicity, before it can be applied to actual gene therapy (Non-Patent Literature 8). As mentioned above, various modifications of AAV capsids have been attempted to overcome the shortcomings of existing gene therapy AAV vectors, namely "low target specificity" and "low transduction efficiency." Many of these attempts aim to retarget AAV to specific membrane proteins, achieving this by conferring the ability to interact with novel molecules that do not act as infection receptors for natural AAV. However, this alone is insufficient for retargeting. This is because, even after modification, AAV retains its ability to infect via "normal" adhesion receptors, and therefore still transduces cells in organs and tissues other than the therapeutic target, resulting in little substantial infection specificity.Therefore, some retargeting strategies employ methods that eliminate the innate targeting properties of the original viral vector and confer new molecular specificity to it. For example, Patent Documents 1 and 2 describe a method for obtaining a highly efficient retargeted modified virus by replacing the important amino acid region involved in binding to the cell adhesion receptor on the natural AAV capsid subunit with an artificial tag peptide sequence, thereby neutralizing the innate cell targeting properties and simultaneously conferring novel receptor binding characteristics to molecules (antibodies, etc.) that bind to the tag peptide sequence. However, in this method, the binding ability to adhesion receptors and endogenous receptors is also greatly weakened at the point when the capsid is modified to eliminate the innate targeting properties. As a result, even after acquiring targeting properties, the gene transfer ability remains at the same level or even lower than that of an unmodified wild-type virus (for example, Figures 8B and 9-2 in Patent Document 1). Furthermore, these methods still suffer from the aforementioned problems specific to modified capsids. A method using a molecule called an "adapter" has also been devised as a way to convert the targeting properties of a virus through rational design (Non-Patent Document 9). An "adapter" is a mechanism that presents an exogenous molecule on a virus that has the ability to bind to a target receptor, thereby bringing the virus closer to specific cells and altering infection specificity. Several adapter strategies have been proposed for adenoviruses (Ad) and AAVs, but most of them involve modification of the capsid (Patent Documents 1 and 2, and Non-Patent Documents 10 and 11), and few use the natural viral capsid. As for technologies that do not involve capsid modification, only soluble viral receptors (CARs) (Non-Patent Document 12) and an artificial binder protein called DARPin (Non-Patent Document 13) have been reported for Ad, and antibodies against the Cap subunit have been reported for AAVs (Non-Patent Document 14).However, because these adapters cover the site (the loop protruding from the surface) where the capsid binds to the attached receptor or internalized receptor, even if the virus acquires the ability to bind to a new target receptor through adapter binding, the overall gene transfer efficiency of the virus cannot generally be significantly increased compared to the original virus (without adapter addition).

[0003] Special table No. 2020-525020 Publication No. 2020-529196

[0004] Zhao et al, Bioeng Transl Med. 2021 Oct 20;7(1): e10258Pillay et al, Nature 530, 108-112 (2016)Rodriguez-Marquez et al, Expert Opin Biol Ther. 2021 Jun;21(6):749-766Nat Biotechnol 38, 910 (2020)Davidsson et al, Proc Natl Acad Sci US A. 2019 Dec 26;116(52):27053-27062Eichhoff et al, Mol Ther Methods Clin Dev. 2019 Sep 16;15: 211-220Munch et al, Nat Commun. 2015 Feb 10;6: 6246Margaret et al al, ACS Nano, 2020, 14, 14262-83.Reinhard et al. Nature Rev. Genetics, 2007, 8, 573-587Parrott et al, Mol. Ther. 8, 688-700 (2003)Gigout et al, Mol. Ther. 11, 856-865 (2005)Pereboev et al, Mol. Ther. 712-720 (2004)Dreier et al, Proc Natl Acad Sci USA. 2013 Mar 5;110(10): E869-77Bartlett et al, Nature Biotechnol. 17, 181-186 (1999)

[0005] As described above, no technology yet exists that simultaneously achieves two objectives: (1) using natural AAV capsids that have not been modified in any way, and (2) dramatically increasing infectivity to cardiac cell types. There has been a great need for a new technology that can deliver safe and highly efficient gene transfer agents to cardiac cell types to achieve both objectives.

[0006] The inventors of this invention have investigated a retargeting strategy using wild-type AAV for gene transfer into cardiac cell types and have completed the present invention. Specific embodiments are as follows. However, the present invention is not limited to these embodiments.

[0007] [1] A gene transfer agent for cardiac cell species comprising a molecule ("molecule") which includes a module (A) capable of binding to the capsid of AAV (adeno-associated virus) for carrying foreign genes ("AAV capsid-binding") and a module (B) capable of binding to the surface antigen of cardiac cell species ("cardiac cell species surface antigen-binding"), and which can simultaneously bind to the capsid of AAV and the surface antigen of cardiac cell species, wherein module (A) is 1) an AAV capsid-binding peptide, or 2) a scaffold protein having a structure into which the AAV capsid-binding peptide has been transplanted, and module (B) is 1) an anti-cardiac cell species surface antigen antibody, 2) a cardiac cell species surface antigen-binding peptide, or 3) a scaffold protein having a structure into which the cardiac cell species surface antigen-binding peptide has been transplanted. [2] The gene transfer agent according to [1] above, wherein the molecule is an anti-cardiac cell type surface antigen antibody having a structure in which an AAV capsid-binding peptide has been transplanted. [3] The gene transfer agent according to [1] above, wherein the molecule is a protein formed by linking a scaffold protein having a structure in which an AAV capsid-binding peptide has been transplanted and an anti-cardiac cell type surface antigen antibody. [4] The gene transfer agent according to [1] above, wherein the molecule is a protein formed by linking a scaffold protein having a structure in which an AAV capsid-binding peptide has been transplanted and a scaffold protein having a structure in which a cardiac cell type surface antigen-binding peptide has been transplanted. [5] The gene transfer agent according to [1] above, wherein the molecule is a scaffold protein having a structure in which an AAV capsid-binding peptide and a cardiomyocyte surface antigen-binding peptide have been transplanted.[6] The gene transfer agent according to [1] above, wherein the molecule is an anticardiac cell surface antigen antibody having at least one loop structure, wherein all or part of the amino acid sequence constituting at least one loop structure is replaced with all or part of an AAV capsid-binding peptide sequence, or all or part of an AAV capsid-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure, and the molecule is an anticardiac cell surface antigen antibody having an amino acid sequence into which all or part of an AAV capsid-binding peptide sequence is incorporated. [7] The gene transfer agent according to [1] above, wherein the molecule is a scaffold protein having at least one loop structure, in which all or part of the amino acid sequence constituting at least one loop structure is replaced with all or part of an AAV capsid-binding peptide sequence, or in which all or part of an AAV capsid-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure, and an AAV capsid-binding scaffold protein having an amino acid sequence into which all or part of an AAV capsid-binding peptide sequence is incorporated, and an anti-cardiac constituent cell type surface antigen antibody is linked to it.[8] A molecule is a protein formed by linking an AAV capsid-binding scaffold protein having an amino acid sequence incorporating an AAV capsid-binding peptide sequence, wherein in a scaffold protein having at least one loop structure, all or part of the amino acid sequence constituting at least one loop structure is replaced with all or part of an AAV capsid-binding peptide sequence, or all or part of an AAV capsid-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure, and an AAV capsid-binding scaffold protein having an amino acid sequence incorporating all or part of an AAV capsid-binding peptide sequence; and a cardiac cell species surface antigen-binding scaffold protein having a scaffold protein having at least one loop structure, wherein all or part of the amino acid sequence constituting at least one loop structure is replaced with all or part of a cardiac cell species surface antigen-binding peptide sequence, or all or part of a cardiac cell species surface antigen-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure, and an AAV capsid-binding scaffold protein having an amino acid sequence incorporating all or part of a cardiac cell species surface antigen-binding peptide sequence. The gene transfer agent described in [1] above. [9] The gene transfer agent described in [1] above, wherein the molecule is a scaffold protein having two or more loop structures, wherein all or part of the amino acid sequence constituting at least one loop structure is replaced with all or part of an AAV capsid-binding peptide sequence, or all or part of an AAV capsid-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure, and all or part of the amino acid sequence constituting at least one loop structure different from the above loop structure is replaced with all or part of a cardiac cell species surface antigen-binding peptide sequence, or all or part of a cardiac cell species surface antigen-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure different from the above loop structure, and the molecule is a protein having an amino acid sequence into which all or part of the AAV capsid-binding peptide sequence and the cardiac cell species surface antigen-binding peptide sequence are incorporated.

[10] The gene transfer agent according to [1] above, wherein the molecule is a protein formed by linking a biotinylated AAV capsid-binding peptide and a biotinylated cardiac cell species surface antigen-binding peptide via a protein having two or more biotin-binding sites.

[11] The gene transfer agent according to [1] above, wherein the molecule is a protein formed by linking a biotinylated AAV capsid-binding peptide and an anti-cardiac cell species surface antigen antibody via a protein having two or more biotin-binding sites.

[0008]

[12] The gene transfer agent according to any one of [1] to

[11] above, wherein the cardiac cell surface antigen is one or more antigens selected from CD29, CD46, CD47, CD49f, CD58, CD59, CD98, CD147, CD321, CD340, SSEA-4, CD172a, ITGA7 (integrin α7), and VCAM1, which are expressed on cardiac cell types.

[13] The gene transfer agent according to

[12] above, wherein the cardiac cell surface antigen is one or more antigens selected from CD49f, CD340, and CD172a, which are expressed on cardiac cell types.

[14] The gene transfer agent according to any one of [1] to

[13] above, wherein the cardiac cell surface antigen-binding peptide is IB8.

[15] A gene transfer agent according to any one of [1] to

[13] above, wherein the anticardiac constituent cell type surface antigen antibody is trastuzumab, anzurustwald, or pertuzumab.

[0009]

[16] The AAV capsid-binding peptide is a core motif sequence (I) represented by: (1) the amino acid sequence represented by Tyr-Thr-Tyr-Arg-Thr-Thr-Ile-Pro-His-Glu-Tyr-Leu-Ala (SEQ ID NO: 1), or (2) an amino acid sequence that has 80% or more homology to the amino acid sequence represented by SEQ ID NO: 1 and is AAV9 capsid-binding; or, a core motif sequence (II) represented by: Formula: Xaa1-Ser-Lys-Ser-Xaa2-Xaa3-Leu-Ala-Gln-Xaa4 (wherein Xaa1 represents Phe or Tyr, Xaa2 represents Leu, Val, Ile or Phe, Xaa3 represents any amino acid, and The gene transfer agent according to any one of

[11] to

[15] above, wherein Xaa4 is an amino acid sequence represented by (Gln, Asp, Glu, or Asn) or a sequence containing such a sequence.

[17] The gene transfer agent according to

[16] above, wherein the core motif sequence (II) is (1) an amino acid sequence represented by Tyr-Ser-Lys-Ser-Val-Cys-Leu-Ala-Gln-Asp (Sequence ID 2), or an amino acid sequence having 70% or more homology with the amino acid sequence represented by Sequence ID 2 and being AAV9 capsid-binding, or (2) an amino acid sequence represented by Phe-Ser-Lys-Ser-Leu-His-Leu-Ala-Gln-Glu (Sequence ID 3), or an amino acid sequence having 70% or more homology with the amino acid sequence represented by Sequence ID 3 and being AAV9 capsid-binding.

[18] The gene transfer agent according to any one of [1] to

[15] above, wherein the AAV capsid-binding peptide is the whole sequence or partial sequence constituting a cyclic peptide represented by the following formulas (Ia), (Ib), or (Ic).

[0010]

[0011] (In the formula, Variable region 1 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 1 below, and S represents S derived from the thiol group of Cys.)

[0012]

[0013]

[0014] (In the formula, Variable region 1 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 2 below, Variable region 2 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 3 below, and S represents S derived from the thiol group of Cys.)

[0015]

[0016]

[0017]

[0018] (In the formula, Variable region 1 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 4 below, and S represents S derived from the thiol group of Cys.)

[0019]

[0020]

[19] A gene transfer agent according to any one of [1] to

[18] above, which reduces the nonspecific infectivity of AAV by binding to AAV via module (A) and at the same time increases the specific infectivity to cardiac constituent cell types that are targets of module (B).

[20] A gene transfer agent according to any one of [1] to

[19] above, further comprising AAV having a wild-type capsid.

[21] A gene transfer agent according to any one of [1] to

[20] above, wherein the molecule is mixed with AAV having a wild-type capsid to form a complex of the two.

[22] A gene transfer agent according to

[20] or

[21] above, wherein an exogenous gene is loaded onto AAV.

[23] A gene transfer agent according to

[22] above, for infecting cardiac constituent cell types with AAV.

[24] A gene transfer agent according to

[23] above, for introducing an exogenous gene into cardiac constituent cell types.

[25] A gene transfer agent according to any one of

[20] to

[24] above, wherein the AAV is AAV9.

[0021] As one embodiment of the present invention, a gene transfer agent for cardiac constituent cell types is provided, which contains a bivalent molecule capable of binding to both the capsid of natural adeno-associated virus and the surface antigen of cardiac constituent cell types. This molecule acts as an "engager" that attracts AAV to cardiac constituent cell types expressing the surface antigen. By using a complex of this molecule with recombinant AAV containing the natural AAV capsid as a vector to act on cells, a significantly higher gene transfer efficiency is achieved in a surface antigen-dependent manner compared to when natural AAV alone is used as a vector. Furthermore, even when using natural serotype AAV, the complex with the above molecule suppresses infection and gene transfer in cells that do not express the surface antigen of cardiac constituent cell types, and also inhibits the binding of neutralizing antibodies, thereby suppressing the neutralization of AAV by antibodies.

[0022] Figure 2 illustrates six representative embodiments of the above-described molecule as a bivalent engager that binds to AAV and cardiac cell surface antigens. Figure 3 shows the evaluation results of infection efficiency by various engagers in Example A described below. Figure 4 shows the evaluation results of infection efficiency by various engagers in Example B described below. Figure 5 shows the evaluation results of infection efficiency by various engagers in Example D described below. Figure 6 shows the evaluation results of infection efficiency by various engagers in Example E described below.

[0023] The present invention will be described below with reference to specific embodiments. However, the present invention is not limited to these. Those skilled in the art can modify embodiments of the present invention in various ways without departing from the meaning of the present invention, and such modifications will also be included within the scope of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Also, unless otherwise specified herein, amino acids mean L-forms.

[0024] One embodiment of the present invention is a gene transfer agent for cardiac cell species comprising: "[1] a module (A) capable of binding to the capsid of AAV (adeno-associated virus) for carrying an exogenous gene ("AAV capsid-binding") and a module (B) capable of binding to the surface antigen of cardiac cell species ("cardiac cell species surface antigen-binding"), wherein the molecule can simultaneously bind to the capsid of AAV and the surface antigen of cardiac cell species, and module (A) is 1) an AAV capsid-binding peptide, or 2) a scaffold protein having a structure in which the AAV capsid-binding peptide is transplanted, and module (B) is 1) an anti-cardiac cell species surface antigen antibody, 2) a cardiac cell species surface antigen-binding peptide, or 3) a scaffold protein having a structure in which the cardiac cell species surface antigen-binding peptide is transplanted, the molecule ("molecule"). This embodiment will be described in detail below.

[0025] [A bivalent molecule capable of binding to both AAV capsid and cardiac cell surface antigens] The "molecule" in this embodiment is a bivalent molecule capable of simultaneously binding to the capsid of natural AAV and cardiac cell surface antigens. It is added to AAV having a natural AAV capsid to form a complex, which then acts on cells as a viral vector. The molecule comprises two functional units (modules), which are linked together to form a single unit. Various forms of linkage are included, but details will be described later. One is a module that binds to the natural (unmodified) AAV capsid (also referred to as module (A)), and the other is a module that binds to cardiac cell surface antigens to achieve retargeting (also referred to as module (B)). The molecule binds to the natural (unmodified) AAV capsid via module (A) and to the surface antigen of cardiac cell species via module (B), thereby achieving proximity between AAV and the surface antigen of cardiac cell species as an engager, and increasing the AAV's inherent ability to adhere, internalize, translocate to the nucleus, and transduce in a cardiac cell species surface antigen-dependent manner. The serotype of AAV to which the molecule is applied is not particularly limited and can be applied to all known serotypes of AAV, but it is preferably applied to AAVs of serotypes 1, 2, 5, 6, 8, 9, Rh10, etc., and is particularly preferably applied to AAV of serotype 9.

[0026] (Conditions for Module (A)) Module (A) described above is either 1) an AAV capsid-binding peptide, or 2) a scaffold protein having a structure into which the AAV capsid-binding peptide has been transplanted (more specifically, a structure in which the AAV capsid-binding peptide is crosslinked on a protein by covalent bonds, a structure in which it is tethered on a protein by non-covalent bonds, or a structure in which it is incorporated into the internal sequence of a protein, preferably a structure incorporated into the internal sequence of a protein). Module (A) is not particularly limited as long as it contains an AAV capsid-binding peptide in its molecule that reversibly binds to the natural AAV capsid. For example, a preferred embodiment is one that contains a cyclic peptide or a substructure derived from the cyclic peptide, and moreover, the cyclic peptide itself has the property of not reducing the transduction activity (the ability to adhere to cells, internalize, translocate to the nucleus, and transduce) that AAV inherently possesses.

[0027] In this specification, the term "AAV capsid-binding peptide" is not particularly limited as long as it has the property of reversibly binding to the natural AAV capsid, but for example, it may be all or part of a cyclic peptide having such property. The cyclic peptide may be selected by the RaPID method, but details will be described later. In this specification, "structure to which the AAV capsid-binding peptide has been implanted" more specifically refers to a structure in which all or part of the "AAV capsid-binding peptide" is crosslinked on a protein by covalent bonds, a structure in which it is tethered on a protein by non-covalent bonds, or a structure incorporated into the internal sequence of a protein. Incorporation into the internal sequence of a protein may be done by the LassoGraft method, but details will be described later. In this specification, "scaffold protein" is not particularly limited, but examples include immunoglobulin, fibronectin, albumin, ubiquitin, human growth hormone, alkaline phosphatase, retinol-binding protein, uteroglobin, fibrinogen, blood coagulation factors, transferrin, etc. The scaffold protein may be a partial fragment protein, and examples include partial fragment proteins of the proteins exemplified above. The scaffold protein preferably has a loop structure in its secondary structure, and a partial fragment containing a loop structure may be used as part of the structure of a full-length protein. The loop structure is not particularly limited, but means a structure having a bent structure, which is a peptide chain region that connects α-helices and / or β-sheets. Furthermore, the scaffold protein may be a protein having a loop structure or a partial fragment protein having a loop structure to which another protein is fused. If the AAV capsid-binding peptide constituting the active structure of module (A) is a cyclic peptide, it is desirable that it does not compete with the binding of adherent receptors or endogenous receptors when binding to the AAV capsid surface, and that the binding site is located in a recessed region rather than a protruding portion on the capsid surface.

[0028] (Conditions for Module (B)) Module (B) is not particularly limited as long as it contains an active structure that reversibly binds on the cell surface to cardiac constituent cell type antigens. In this specification, "cardiac constituent cell type" is not particularly limited as long as it is a cell type that makes up the heart, but specific examples include cardiomyocytes (ventricular myocytes, atrial myocytes), cardiac fibroblasts, epicardial cells, endocardial cells, smooth muscle cells, endothelial cells, cardiac macrophages, Purkinje cells, pacemaker cells, etc. In this specification, "cardiac constituent cell type surface antigen" is not particularly limited as long as it is an antigen expressed on the surface of the above cardiac constituent cell types, but examples include one or more antigens selected from CD29, CD46, CD47, CD49f, CD58, CD59, CD98, CD147, CD321, CD340, SSEA-4, CD172a, ITGA7 (integrin α7), and VCAM1. Among these, one or more antigens selected from CD49f, CD340, and CD172a are preferred.

[0029] Preferred embodiments of the above module (B) include: 1) an anti-cardiac cell species surface antigen antibody, 2) a cardiac cell species surface antigen-binding peptide, or 3) a scaffold protein having a structure in which the cardiac cell species surface antigen-binding peptide is transplanted (more specifically, a structure in which the target receptor-binding peptide is crosslinked on a protein by covalent bonds, a structure in which it is tethered on a protein by non-covalent bonds, or a structure in which it is incorporated into the internal sequence of a protein, preferably a structure in which it is incorporated into the internal sequence of a protein).

[0030] In this specification, "anti-cardiac cell type surface antigen antibody" is not particularly limited as long as it is an antibody against an antigen that shows a specific distribution in cardiac cell types that contribute to the retargeting of AAV. Furthermore, the antibody form includes not only the natural IgG type, but also antibody-like substances that have undergone various engineering modifications, such as Fab fragments, single-chain antibodies (scFv), and nanobodies. If necessary, one or more types may be used in combination. Examples of such antibodies include trastuzumab, anzurstwald, pertuzumab, mouse ErbB2 antibody H2Mab300, SIRPa antibody mab3, and ITGA7 antibody M36. In this specification, "target receptor-binding peptide" is not particularly limited as long as it has the property of reversibly binding to cardiac cell type surface antigens, but for example, it may be all or part of a cyclic peptide having such property. The cyclic peptide may be selected by the RaPID method, but details will be described later. Examples of such peptides include IB8. More specifically, in this specification, "structure to which cardiac cell type surface antigen-binding peptides are transplanted" refers to structures in which all or part of the "cardiac cell type surface antigen-binding peptides" are crosslinked on a protein by covalent bonds, tethered on a protein by non-covalent bonds, or incorporated into the internal sequence of a protein. Incorporation into the internal sequence of a protein may be performed by the LassoGraft method, but details will be described later.

[0031] (Regarding the manner in which modules (A) and module (B) are linked) In the "molecule" described herein, the structure linking module (A) and module (B) may be a peptide bond, a glycosidic bond, a disulfide bond, or any other covalent bond, or it may be a non-covalent bond with sufficient chemical stability (for example, a base pair bond or an avidin-biotin bond). Modules (A) and (B) constituting the "molecule" described herein may be two structurally independent units that can exist stably on their own, or they may be a single structurally indivisible unit that has both an AAV binding active site and a target receptor binding site. In the latter case, it appears to be one structure, but functionally it has two modules. The "molecule" described herein may contain one module (A) and one module (B), or more modules. In addition, the "molecule" described herein may have one or more structural units linked to modules (A) and (B) that have functions unrelated to them.

[0032] The above describes in detail a molecule that, in one embodiment of the present invention, plays the role of an engager for gene introduction into cardiac cell types, comprising a module (A) that can bind to the capsid of AAV (adeno-associated virus) for carrying a foreign gene ("AAV capsid-binding") and a module (B) that can bind to the surface antigen of a cardiac cell type ("cardiac cell type surface antigen-binding"), and which can simultaneously bind to the capsid of AAV and the surface antigen of a cardiac cell type, wherein module (A) is 1) an AAV capsid-binding peptide, or 2) a scaffold protein having a structure into which the AAV capsid-binding peptide has been transplanted, and module (B) is 1) an anti-cardiac cell type surface antigen antibody, 2) a cardiac cell type surface antigen-binding peptide, or 3) a scaffold protein having a structure into which the cardiac cell type surface antigen-binding peptide has been transplanted. In the above "molecule", the AAV may carry a foreign gene. A person skilled in the art can appropriately select the foreign gene, taking into consideration the disease to be treated with gene therapy and the type of cardiac cell to which the gene is to be introduced. The "molecule" containing the foreign gene can be used for gene introduction into cardiac cell types by methods commonly used in this art. Details will be described in the section below, "[Forms of Use of the Above-mentioned "Molecule" as a Gene Introduction Agent]".

[0033] Specific preferred embodiments of the "molecule" are exemplified below as follows. Embodiment A A molecule (Type V engager), which is an anti-cardiac constituent cell type surface antigen antibody having a structure grafted with an AAV capsid-binding peptide. Embodiment B A molecule (Type III engager), which is a protein formed by linking a scaffold protein having a structure grafted with an AAV capsid-binding peptide and a scaffold protein having a structure grafted with a cardiac constituent cell type surface antigen-binding peptide. Embodiment C A molecule (Type IV engager), which is a scaffold protein having a structure grafted with an AAV capsid-binding peptide and a cardiac constituent cell type surface antigen-binding peptide. Embodiment D A molecule (Type I engager), which is a protein formed by linking a biotinylated AAV capsid-binding peptide and a biotinylated cardiac constituent cell type surface antigen-binding peptide via a protein having two or more biotin binding sites. Embodiment E A molecule (Type II engager), which is a protein formed by linking a biotinylated AAV capsid-binding peptide and a biotinylated anti-cardiac constituent cell type surface antigen antibody via a protein having two or more biotin binding sites. Embodiment F A molecule (Type VI engager), which is a protein formed by linking a scaffold protein having a structure grafted with an AAV capsid-binding peptide and an anti-cardiac constituent cell type surface antigen antibody.

[0034] Specific more preferred embodiments of the molecule are specifically exemplified as follows.

[0035] Embodiment A-1 In an anti-cardiac constituent cell type surface antigen antibody having at least one loop structure, the whole or a part of the amino acid sequence constituting at least one loop structure is replaced with the whole or a part of an AAV capsid-binding peptide sequence, or the whole or a part of an AAV capsid-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure, and the molecule is an anti-cardiac constituent cell type surface antigen antibody having an amino acid sequence into which the whole or a part of the AAV capsid-binding peptide sequence is integrated.

[0036] In the present specification, "the whole or part of the amino acid sequence constituting the loop structure is replaced with the whole or part of the AAV capsid-binding peptide sequence" means that the whole or part of the amino acid sequence constituting the loop structure is deleted, and instead of the said sequence, the whole or part of the AAV capsid-binding peptide sequence is incorporated as a part of the amino acid sequence constituting the loop structure. In the present specification, "the whole or part of the AAV capsid-binding peptide sequence is inserted into the amino acid sequence constituting the loop structure" means that the whole or part of the AAV capsid-binding peptide sequence is inserted between two adjacent amino acids in the amino acid sequence constituting the loop structure, and is incorporated as a part of the amino acid sequence constituting the loop structure.

[0037] Embodiment B-1 A molecule that is a protein formed by linking: an AAV capsid-binding scaffold protein, which is a scaffold protein having at least one loop structure, wherein the whole or part of the amino acid sequence constituting at least one loop structure is replaced with the whole or part of an AAV capsid-binding peptide sequence, or the whole or part of an AAV capsid-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure, and which has an amino acid sequence incorporating the whole or part of the AAV capsid-binding peptide sequence; and a heart constituent cell type surface antigen-binding scaffold protein, which is a scaffold protein having at least one loop structure, wherein the whole or part of the amino acid sequence constituting at least one loop structure is replaced with the whole or part of a heart constituent cell type surface antigen-binding peptide sequence, or the whole or part of a heart constituent cell type surface antigen-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure, and which has an amino acid sequence incorporating the whole or part of the heart constituent cell type surface antigen-binding peptide sequence.

[0038] Embodiment C-1 A scaffold protein having two or more loop structures, wherein all or part of the amino acid sequence constituting at least one loop structure is replaced with all or part of an AAV capsid-binding peptide sequence, or all or part of an AAV capsid-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure, and all or part of the amino acid sequence constituting at least one loop structure different from the above loop structure is replaced with all or part of a cardiac cell species surface antigen-binding peptide sequence, or all or part of a cardiac cell species surface antigen-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure different from the above loop structure, and the molecule is a protein having an amino acid sequence into which all or part of the AAV capsid-binding peptide sequence and the cardiac cell species surface antigen-binding peptide sequence are incorporated.

[0039] Embodiment D-1 A molecule that is a protein formed by linking a biotinylated AAV capsid-binding peptide and a biotinylated cardiac cell type surface antigen-binding peptide via avidin or streptavidin.

[0040] Embodiment E-1 A molecule that is a protein formed by linking a biotinylated AAV capsid-binding peptide and a biotinylated cardiac cell type surface antigen antibody via avidin or streptavidin.

[0041] Embodiment F-1 A molecule comprising a scaffold protein having at least one loop structure, wherein all or part of the amino acid sequence constituting at least one loop structure is replaced with all or part of an AAV capsid-binding peptide sequence, or all or part of an AAV capsid-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure, and an AAV capsid-binding scaffold protein having an amino acid sequence into which all or part of an AAV capsid-binding peptide sequence is incorporated, and a cardiac cell type surface antigen antibody linked together.

[0042] [Cyclic Peptide] In the above-described [bivalent molecule capable of binding to both AAV capsid and cardiac cell species surface antigens] in one embodiment of the present invention, the AAV capsid-binding peptide and the cardiac cell species surface antigen-binding peptide may be the entire sequence or a partial sequence of the AAV capsid-binding cyclic peptide sequence and the cardiac cell species surface antigen-binding cyclic peptide sequence. The AAV capsid-binding cyclic peptide and the cardiac cell species surface antigen-binding cyclic peptide are described in detail below. This cyclic peptide constitutes one embodiment of the present invention as a cyclic peptide itself.

[0043] (AAV Capsid-Binding Cyclic Peptide) In this specification, one embodiment of the AAV capsid-binding cyclic peptide is a "cyclic peptide comprising an AAV capsid-binding amino acid sequence in its peptide sequence" (Embodiment (I)). Here, the AAV capsid-binding amino acid sequence is not particularly limited as long as it is an amino acid sequence that reversibly binds to the natural AAV capsid. As described above, this cyclic peptide is useful, for example, in constructing a bivalent molecule that can bind to both the AAV capsid and a molecular target. More preferred specific embodiments of Embodiment (I) are given below: Embodiment (1) to (12). Embodiment (1) The cyclic peptide according to (I) above, having a ring structure formed by a crosslinking bond linking one amino acid and another amino acid in the peptide sequence. Embodiment (2) The cyclic peptide according to (1) above, wherein the AAV capsid-binding cyclic peptide has a ring structure containing 4 to 30 amino acids. Embodiment (3) The cyclic peptide according to (1) or (2) above, wherein the crosslinking structure includes a thioether bond or a disulfide bond. Embodiment (4) The cyclic peptide according to any one of (1) to (3) above, wherein the AAV capsid-bonded cyclic peptide has a ring structure formed via a crosslinking bond that links an amino acid having functional group 1 shown in functional group groups (A) to (E) in Table 5 below ("ring-forming amino acid 1") and an amino acid having the corresponding functional group 2 ("ring-forming amino acid 2").

[0044]

[0045] (In the formula, X1 is a leaving group, and Ar represents an aromatic ring which may have substituents.) Embodiment (5) Ring-forming amino acid 1 is an amino acid having the functional group (A-1) described in Table 1, and ring-forming amino acid 2 is an amino acid having the corresponding functional group (A-2), and the ring structure is -N-CO-CH formed between the two amino acids ２ A cyclic peptide as described in (4) above, having an -S- structure. Embodiment (6) -N-CO-CH ２ The cyclic peptide described in (4) above, wherein the N in the -S- structure is derived from the amino group of ring-forming amino acid 1 selected from alanine, tyrosine, phenylalanine, tryptophan, ornithine, lysine, diaminopropionic acid, and diaminobutyric acid. Embodiment (7) -N-CO-CH ２The cyclic peptide according to (4) above, wherein S in the -S- structure is derived from the thiol group of ring-forming amino acid 2 selected from cysteine, homocysteine, mercaptonorvaline, mercaptonorleucine, 2-amino-7-mercaptoheptanoic acid, and 2-amino-8-mercaptooctanoic acid. Embodiment (8) The cyclic peptide according to any one of (1) to (7) above, wherein the AAV capsid-binding cyclic peptide is a cyclic peptide ("AAV9 capsid-binding cyclic peptide") that includes an amino acid sequence in the peptide sequence that can bind to the capsid of AAV9 (adeno-associated virus serotype 9) ("AAV9 capsid-binding"). Embodiment (9) The cyclic peptide according to (8) above, wherein the AAV9 capsid-binding cyclic peptide has a ring structure formed by: 1) a crosslinking bond connecting one amino acid located at one end of the AAV9 capsid-binding amino acid sequence or outside of that end to another amino acid located at the other end of the AAV9 capsid-binding amino acid sequence or outside of that end, or 2) a crosslinking bond connecting one amino acid located at one end of the AAV9 capsid-binding amino acid sequence or outside of that end to another amino acid located inside the AAV9 capsid-binding amino acid sequence. Embodiment (10) The AAV9 capsid-binding amino acid sequence is a core motif sequence (I) represented as follows: (1) the amino acid sequence represented by Tyr-Thr-Tyr-Arg-Thr-Thr-Ile-Pro-His-Glu-Tyr-Leu-Ala (SEQ ID NO: 1), or (2) an amino acid sequence that has 80% or more homology to the amino acid sequence represented by SEQ ID NO: 1 and is AAV9 capsid-binding; or, a core motif sequence (II) represented as follows: Formula: Xaa1-Ser-Lys-Ser-Xaa2-Xaa3-Leu-Ala-Gln-Xaa4 (wherein Xaa1 represents Phe or Tyr, Xaa2 represents Leu, Val, Ile or Phe, Xaa3 represents any amino acid, and The cyclic peptide described in (9) above, wherein Xaa4 is an amino acid sequence represented by Glu, Asp, Glu, or Asn.Embodiment (11) The cyclic peptide according to (9) above, wherein the core motif sequence (II) is (1) an amino acid sequence represented by Tyr-Ser-Lys-Ser-Val-Cys-Leu-Ala-Gln-Asp (SEQ ID NO: 2), or an amino acid sequence having 70% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and being AAV9 capsid-binding, or (2) an amino acid sequence represented by Phe-Ser-Lys-Ser-Leu-His-Leu-Ala-Gln-Glu (SEQ ID NO: 3), or an amino acid sequence having 70% or more homology with the amino acid sequence represented by SEQ ID NO: 3 and being AAV9 capsid-binding. Embodiment (12) The cyclic peptide according to any one of (8) to (11) above, wherein the active structure of the AAV9 capsid-binding cyclic peptide is selected from the cyclic peptides represented by the following formulas (Ia), (Ib), or (Ic).

[0046]

[0047] (In the formula, Variable region 1 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 6 below, and S represents S derived from the thiol group of Cys.)

[0048]

[0049]

[0050] (In the formula, Variable region 1 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 7 below, Variable region 2 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 8 below, and S represents S derived from the thiol group of Cys.)

[0051]

[0052] The preferred amino acid sequence for Variable region 1, as shown in Table 7, is SerLysSerVal (SKSV).

[0053]

[0054]

[0055] (In the formula, Variable region 1 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 9 below, and S represents S derived from the thiol group of Cys.)

[0056]

[0057] The above-mentioned AAV capsid-binding cyclic peptide can be used in either its free form or in the form of a salt thereof (preferably a pharmaceutically acceptable salt thereof). Examples of pharmaceutically acceptable salts include salts with inorganic acids such as hydrochloride, hydrobromide, sulfate, and phosphate; salts with organic acids such as acetate, fumarate, oxalate, citrate, methanesulfonate, benzenesulfonate, tosylate, and maleate; salts with bases such as alkali metal salts such as sodium and potassium salts, and alkaline earth metal salts such as calcium salts; and salts with amino acids such as glycine, lysine, arginine, histidine, ornithine, glutamate, and aspartate.

[0058] The characteristics of the ring structure and amino acid sequence of the AAV capsid-binding cyclic peptide in the embodiments of the present invention have been described in detail above. As stated above, the AAV capsid-binding peptide in the embodiments of the present invention may be the entire sequence or a partial sequence of the AAV capsid-binding cyclic peptide sequence. From this viewpoint, preferred amino acid sequences of the "AAV capsid-binding peptide" in the embodiments described as specific embodiments [1] to

[15] of the AAV retargeting strategy in the above section [Means for Solving the Problems] are described below. (Sequence A) Preferred sequences for AAV capsid-binding peptides include the following core motif sequence (I): (1) the amino acid sequence represented by Tyr-Thr-Tyr-Arg-Thr-Thr-Ile-Pro-His-Glu-Tyr-Leu-Ala (SEQ ID NO: 1), or (2) an amino acid sequence having 80% or more homology to the amino acid sequence represented by SEQ ID NO: 1 and being AAV9 capsid-binding; or the following core motif sequence (II): Formula: Xaa1-Ser-Lys-Ser-Xaa2-Xaa3-Leu-Ala-Gln-Xaa4 (wherein Xaa1 represents Phe or Tyr, Xaa2 represents Leu, Val, Ile or Phe, Xaa3 represents any amino acid, and Sequence A is an amino acid sequence represented by Xaa4 (where Xaa4 represents Gln, Asp, Glu, or Asn), or a sequence containing such a sequence. (Sequence B) A more preferred sequence for the AAV capsid-binding peptide is Sequence A, in which the core motif sequence (II) is (1) an amino acid sequence represented by Tyr-Ser-Lys-Ser-Val-Cys-Leu-Ala-Gln-Asp (SEQ ID NO: 2), or an amino acid sequence having 70% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and being AAV9 capsid-binding, or (2) an amino acid sequence represented by Phe-Ser-Lys-Ser-Leu-His-Leu-Ala-Gln-Glu (SEQ ID NO: 3), or an amino acid sequence having 70% or more homology with the amino acid sequence represented by SEQ ID NO: 3 and being AAV9 capsid-binding.(Sequence C) Another preferred sequence for the AAV capsid-binding peptide is sequence C, which is the whole or partial sequence constituting a cyclic peptide represented by the following formulas (Ia), (Ib), or (Ic).

[0059]

[0060] (In the formula, Variable region 1 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 10 below, and S represents S derived from the thiol group of Cys.)

[0061]

[0062]

[0063] (In the formula, Variable region 1 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 11 below, Variable region 2 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 12 below, and S represents S derived from the thiol group of Cys.)

[0064]

[0065] As the amino acid sequence for Variable region 1 listed in Table 16, SerLysSerVal (SKSV) is preferred.

[0066]

[0067]

[0068] (In the formula, Variable region 1 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 13 below, and S represents S derived from the thiol group of Cys.)

[0069]

[0070] (Cardiac Cell Species Surface Antigen-Binding Cyclic Peptide) In this specification, one embodiment of the cardiac cell species surface antigen-binding cyclic peptide is a "cyclic peptide containing a cardiac cell species surface antigen-binding amino acid sequence in its peptide sequence" (Embodiment (II)). Here, the cardiac cell species surface antigen-binding amino acid sequence is not particularly limited as long as it is an amino acid sequence that reversibly binds to the cardiac cell species surface antigen. As described above, this cyclic peptide is useful, for example, in constructing a bivalent molecule that can bind to both the AAV capsid and a molecular target. A more preferred specific embodiment of Embodiment (II) is the following Embodiment (a). Embodiment (a) The cyclic peptide described in (II) above, having a ring structure formed via a crosslinking bond linking one amino acid and another amino acid in the peptide sequence. Hereinafter, more preferred embodiments of the cardiac cell species surface antigen-binding cyclic peptide are embodiments similar to Embodiments (2) to (7) described above as preferred examples of the AAV-binding cyclic peptide.

[0071] The above-mentioned cardiac cell type surface antigen-binding cyclic peptides can also be used in either a free form or in the form of a salt thereof (preferably a pharmaceutically acceptable salt thereof). Examples of pharmaceutically acceptable salts include the salts described above for AAV capsid-binding cyclic peptides.

[0072] (Method for producing cyclic peptides) The cyclic peptides described above are cyclic peptides that can be produced using known peptide synthesis techniques. Examples of methods for producing cyclic peptides include chemical synthesis methods such as liquid-phase methods, solid-phase methods, and hybrid methods combining liquid-phase and solid-phase methods, as well as genetic engineering methods and translational synthesis methods using cell-free translation systems.

[0073] It is preferable to use a cyclic peptide that can be suitably produced by mRNA display methods such as the RaPID method or the TRAP method, or by phage display methods. In carrying out the present invention, it is particularly preferable to produce it by the RaPID method.

[0074] The RaPID method combines the translation and synthesis of a special cyclic peptide library with mRNA display to enrich the active species that bind to the target protein. Furthermore, the cDNA encoding the active species is recovered and amplified by PCR to transcribe the mRNA again. By repeating this process, cyclic peptides with high affinity and specificity can be identified (see International Publication No. 2011 / 049157 and Japanese Patent Publication No. 2013-46637, etc.).

[0075] The method for cyclizing peptides is not particularly limited, but for example, by including an amino acid having functional group 1 and an amino acid having the corresponding functional group 2 as shown in Table 19 below, peptides translated and synthesized by spontaneous reactions can be cyclized to form cyclic peptides. Functional groups 1 and 2 may be either at the N-terminus, or they may be positioned at the N-terminus and C-terminus, or one may be a terminal amino acid and the other a non-terminal amino acid, or both may be non-terminal amino acids. The bond formed by functional group 1 and functional group 2 can be said to be a chemical cross-linking structure for forming the molecular cyclic structure in cyclic peptides.

[0076]

[0077] (In the formula, X1 is a leaving group, and examples of leaving groups include halogen atoms such as Cl, Br, and I, while Ar is an aromatic ring which may have substituents (e.g., a benzene ring).)

[0078] Furthermore, in the phage display method, cyclic peptides can be formed by the bonding of Cys groups, thus enabling the creation of cyclic peptides having a disulfide bond between functional group 1 (-SH) and functional group 2 (HS-).

[0079] In the RaPID method, as the amino acid having the (A-1) functional group, for example, a chloroacetylated amino acid can be used. Examples of chloroacetylated amino acids include N-chloroacetyl-L-alanine, N-chloroacetyl-L-phenylalanine, N-chloroacetyl-L-tyrosine, N-chloroacetyl-Ltryptophan, N-3-(2-chloroacetamide o) benzoyl-L-phenylanine, N-3-(2-chloroacetamido)benzoyl-L-tyrosine, N-3-(2 -chloroacetamide)benzoyl-L-tryptophane, β-N-chloroacetyl-L-diaminopropanoic Examples include acid, γ-N-chloroacetyl-L-diaminobutyric acid, σ-N-chloroacetyl-L-ornithine, ε-N-chloroacetyl-L-lysine, and corresponding D-amino acid derivatives. As amino acids having the functional group of (A-1), N-chloroacetyl-L-tryptophan and N-chloroacetyl-L-tyrosine are preferably used.

[0080] Examples of amino acids having the functional group (A-2) include cysteine, homocysteine, mercaptonorvaline, mercaptonorleucine, 2-amino-7-mercaptoheptanoic acid, and 2-amino-8-mercaptooctanoic acid. As an amino acid having the functional group (A-1), cysteine ​​is preferably used.

[0081] Examples of cyclization methods using an amino acid having the functional group (A-1) and an amino acid having the functional group (A-2) include those described in Kawakami, T. et al., Nature Chemical Biology 5, 888-890 (2009); Yamagishi, Y. et al., ChemBioChem 10, 1469-1472 (2009); Sako, Y. et al., Journal of American Chemical Society 130, 7932-7934 (2008); Goto, Y. et al., ACS Chemical Biology 3, 120-129 (2008); Kawakami T. et al, Chemistry & Biology 15, 32-42 (2008), and International Publication No. 2008 / 117833, among others.

[0082] Examples of amino acids having the functional group (B-1) include propargylglycine, homopropagylglycine, 2-amino-6-heptynoid acid, 2-amino-7-octynoid acid, and 2-amino-8-nonynoid acid. 4-pentynoid or 5-hexynoid modified amino acids may also be used. Examples of 4-pentynoyl amino acids include N-(4-pentenoyl)-L-alanine, N-(4-pentenoyl)- L-phenylalanine, N-(4-pentenoyl)-L-tyrosine, N-(4-pentenoyl)-L-tryptop han, N-3-(4-pentynoylamido)benzoyl-L-phenylanine, N-3-(4-pentynoylam ido)benzoyl-L-tyrosine, N-3-(4-pentynoylamide)benzoyl-L-tryptophane, β Examples include -N-(4-pentenoyl)-L-diaminopropanoic acid, γ-N-(4-pentenoyl)-L-diaminobutyric acid, σ-N-(4-pentenoyl)-Lornitisine, ε-N-(4-pentenoyl)-L-lysine, and corresponding D-amino acid derivatives. Examples of 5-hexynoyl amino acids include amino acids in which the 4-pentynoyl group is replaced with a 5-hexynoyl group in the compounds exemplified as 4-pentynoyl amino acids.

[0083] Examples of amino acids having the functional group (B-2) include azidoalanine, 2-amino-4-azidobutanoic acid, azidoptonorvaline, azidonorleucine, 2-amino-7-azidoheptanoic acid, and 2-amino-8-azidooctanoic acid. Azidoacetyl-modified or 3-azidopentanoyl-modified amino acids can also be used. Examples of the azidoacetylated amino acids include N-azidoacetyl-L-alanine, N-azidoacetyl-L-phenylalanine, N-az Idoacetyl-L-tyrosine, N-azidoacetyl-L-tryptophan, N-3-(4-pentynoylamide)benzoyl-L-phe Examples include nylalanine, N-3-(4-pentynoylamide)benzoyl-L-tyrosine, N-3-(4-pentynoylamide)benzoyl-L-tryptophane, β-N-azidoacetyl-L-diaminopropanoic acid, γ-N-azidoacetyl-L-diaminobutyric acid, σ-N-azidoacetyl-L-ornithine, ε-N-azidoacetyl-L-lysine, and corresponding D-amino acid derivatives. Examples of 3-azidopentanoyl amino acids include amino acids in which the azidoacetyl group is replaced with a 3-azidopentanoyl group, as exemplified by the compounds used as azidoacetyl amino acids.

[0084] A cyclization method using an amino acid having the functional group (B-1) and an amino acid having the functional group (B-2) is described, for example, in Sako, Y. et al., Journal of American Chemical Society 130, 7932-7934 (2008), and International Publication No. 2008 / 117833, etc.

[0085] Examples of amino acids having the (C-1) functional group include N-(4-aminomethyl-benzol)-phenylalanine (AMBF) and 3-aminomethyltyrosine. Examples of amino acids having the (C-2) functional group include 5-hydroxytryptophan (WOH). Examples of cyclization methods using an amino acid having the (C-1) functional group and an amino acid having the (C-2) functional group include the method described in Yamagishi, Y. et al., Chem Bio Chem 10, 1469-1472 (2009) and International Publication No. 2008 / 117833.

[0086] Examples of amino acids having the functional group (D-1) include 2-amino-6-chloro-hexynoid acid, 2-amino-7-chloro-heptynoid acid, and 2-amino-8-chloro-octynoic acid. Examples of amino acids having the functional group (D-2) include cysteine, homocysteine, mercaptonorvaline, mercaptonorleucine, 2-amino-7-mercaptoheptanoic acid, and 2-amino-8-mercaptooctanoic acid. A cyclization method using an amino acid having the (D-1) functional group and an amino acid having the (D-2) functional group is, for example, the method described in International Publication No. 2012 / 074129.

[0087] Examples of amino acids in (E-1) include N-3-chloromethylbenzoyl-L-phenylalanine, N-3-chloromethylbenzoyl-L-tyrosine, N-3-chloromethylbenzoyl-L-tryptophane, and their corresponding D-amino acid derivatives. Examples of amino acids in (E-2) include cysteine, homocysteine, mercaptonorvaline, mercaptonorleucine, 2-amino-7-mercaptoheptanoic acid, and 2-amino-8-mercaptooctanoic acid. A cyclization method using an amino acid having the functional group (E-1) and an amino acid having the functional group (E-2) can be carried out by referring to, for example, the cyclization method of (A-1) and (A-2) or the cyclization method of (D-1) and (D-2).

[0088] As the ring-forming amino acids, a combination of an amino acid having the functional group of (A-1) and an amino acid having the functional group of (A-2) is preferred, a combination of N-acetyltryptophan with H substituted by a leaving group and cysteine ​​is more preferred, and a combination of N-haloacetyl-D-tyrosine or N-haloacetyl-D-tryptophan, preferably N-chloroacetyl-D-tyrosine or N-chloroacetyl-D-tryptophan and cysteine ​​(Cys) is even more preferred.

[0089] The cyclic peptide used in the present invention can be produced according to the RaPID method described above, but details of the production of the cyclic peptide described in Embodiment (12) above will be described in the [Examples] section.

[0090] As described in embodiments [5] to [7], etc., in the section [Means for Solving the Problems] above, one embodiment of the present invention includes a configuration in which all or part of a cyclic peptide sequence is incorporated into an amino acid sequence such as a scaffold protein having at least one loop structure. Such incorporation of a cyclic peptide sequence can be carried out using conventional genetic engineering techniques, but the LassoGraft method is preferred. The LassoGraft method is described in detail in Japanese Patent No. 6598344, and those skilled in the art can implement the method by referring to it, but the main points are described below.

[0091] Taking the embodiments [5] to [7] described above as examples, all or part of a cyclic peptide sequence is incorporated into the loop structure of an anticardiac cell type surface antigen antibody or scaffold protein (hereinafter also abbreviated as "target protein") having at least one loop structure, and it is preferable that this "incorporation" is by covalent bonding. Here, "incorporation" means that all or part of the amino acid sequence constituting the loop structure is replaced with all or part of the cyclic peptide sequence, or that all or part of the cyclic peptide sequence is inserted into the amino acid sequence constituting the loop structure. The site in the loop structure to which the cyclic peptide is incorporated is not particularly limited, and all or part of the cyclic peptide sequence (hereinafter also abbreviated as "cyclic peptide sequence") may be inserted into any site in the loop structure for generality. Any site in the loop structure means a site selected from within the loop structure for the two amino acid residues to be incorporated into the cyclic peptide. Preferably, the two amino acid residues within the loop structure are those that have a Cα interatomic distance of 4–7 Å within the loop structure, and both are at least partially exposed on the protein surface. Having a Cα interatomic distance of 4–7 Å between the two amino acid residues makes it easier for the cyclic peptide incorporated into the target protein to maintain the structure of the original cyclic peptide in the modified peptide. The Cα interatomic distance between the two amino acid residues can be confirmed from the structural data of the target protein whose three-dimensional structure is known. Even if the three-dimensional structure of the target protein is not known, the distance between the two amino acid residues can be estimated if a model structure constructed from the structural information of its homologous protein is available.

[0092] The two amino acid residues selected for the incorporation of a cyclic peptide within the loop structure may be adjacent amino acid residues within the loop structure, but it is preferable that the two amino acid residues are not adjacent. Two amino acid residues not being adjacent is equivalent to the two amino acid residues being discontinuous in the primary amino acid sequence of the protein's loop structure. For example, when selecting two amino acid residues such that the Cα interatomic distance between them is 4 to 7 Å in the protein's crystal structure, there may be 1 to 15 amino acid residues between the two amino acid residues constituting the loop structure in the target protein. The presence of 1 to 15 amino acid residues between two amino acid residues is equivalent to the two amino acid residues being separated by 1 to 15 amino acid residues in the primary amino acid sequence of the protein's loop structure. The 1 to 15 amino acid residue portion between the two amino acid residues is preferably the amino acid residues that form the loop structure.

[0093] In a method for presenting a cyclic peptide on a protein having a loop structure, the cyclic peptide is presented on the protein having the loop structure, but preferably, the structure of the protein and the structure of the cyclic peptide are maintained by replacing the loop structure with the cyclic peptide. By selecting two amino acid residues to bind to the cyclic peptide from the loop structure such that the Cα interatomic distance between the two amino acid residues is 4 to 7 Å, the original structure of the protein can be maintained, and the activity of the cyclic peptide can be maintained.

[0094] In embodiments of the present invention, when "replacing the chemical crosslink structure of a cyclic peptide with two amino acid residues constituting a loop structure," the chemical crosslink structure of the cyclic peptide may be replaced with two amino acid residues constituting a loop structure via a linker sequence. When the replacement is via a linker sequence, in the modified protein into which the cyclic peptide is incorporated, at the N-terminal end, the amino acid sequence derived from the loop structure is incorporated in the order of amino acid sequence derived from the loop structure, linker sequence, and amino acid sequence derived from the cyclic peptide, and at the C-terminal end, the amino acid sequence derived from the cyclic peptide is incorporated in the order of amino acid sequence derived from the cyclic peptide, linker sequence, and amino acid sequence derived from the loop structure. Linker sequences may be present at both the N-terminal and C-terminal ends, or at either the N-terminal or C-terminal end. The linker sequence is not particularly limited, but examples include sequences consisting of one or more Ser, Gly, and Cys.

[0095] Embodiments of the present invention include incorporating a cyclic peptide into a target protein having a loop structure, and such incorporation can be carried out using conventional genetic engineering techniques. Specifically, the modified protein with the incorporated peptide can be produced by the following procedure: 1) Select an amino acid sequence for incorporation into the target protein from the amino acid sequence of a cyclic peptide having a chemical crosslinking structure for forming an intramolecular cyclic structure, and select a base sequence corresponding to the amino acid sequence. If necessary, make adjustments to incorporate a linker sequence. 2) Select a base sequence corresponding to two amino acid residues constituting the loop structure of the target protein having a loop structure, delete the bases present between the selected base sequences as necessary, and prepare a nucleic acid having a base sequence in which the base sequence corresponding to the amino acid sequence of the selected cyclic peptide (including the linker sequence if necessary) is inserted. 3) Translate the nucleic acid to obtain the target protein with the incorporated cyclic peptide.

[0096] The methods for selecting base sequences corresponding to two amino acid residues constituting the loop structure of a target protein having a loop structure, and deleting the bases present between the selected base sequences as necessary, or for preparing nucleic acids having a base sequence incorporating a base sequence corresponding to the amino acid sequence of a selected cyclic peptide, are not particularly limited and can be carried out by conventionally known methods. Furthermore, since the methods for translating the prepared nucleic acids are already well known in the art to which the present invention belongs, the nucleic acids can be translated by appropriately applying those well known methods.

[0097] The various embodiments of the "molecule" described herein have been described in detail above, and more specific embodiments are listed below. (1) In some embodiments, module (A) is a protein that presents an AAV-binding cyclic peptide, and the protein may be a fibronectin type III module (FN3) fragment, an antibody Fc region fragment, streptavidin, serum albumin, ubiquitin, etc. The method of transplanting the cyclic peptide onto these proteins may be a linking reaction using a chemical crosslinking agent, a crosslinking reaction using a Cys residue, a non-covalent linking reaction using a biotin-avidin interaction, or a transplantation method in which a recombinant protein in which the internal sequence of the AAV-binding cyclic peptide is incorporated into a loop is genetically engineered using the LassoGraft method. (2) In some embodiments, module (A) may be an artificial protein that has acquired AAV binding ability by incorporating the internal sequence of an AAV-binding cyclic peptide into one or two superficial loops of an FN3 fragment using the LassoGraft method. (3) In some embodiments, module (A) may be an artificial protein that has acquired AAV binding ability by incorporating the internal sequence of an AAV-binding cyclic peptide into one or two superficial loops of an antibody Fc fragment using the LassoGraft method. (4) In some embodiments, module (A) may be an artificial protein that has acquired AAV binding ability by incorporating the internal sequence of an AAV-binding cyclic peptide into one or two superficial loops of serum albumin using the LassoGraft method. (5) In some embodiments, module (A) may be an artificial protein that has acquired AAV binding ability by incorporating the internal sequence of an AAV-binding cyclic peptide into one of the superficial loops of ubiquitin using the LassoGraft method, and an artificial protein formed by linking multiple such modules. (6) In some embodiments, module (B) may be an artificial protein that has acquired cardiac cell species surface antigen binding ability by incorporating the internal sequence of a cardiac cell species surface antigen-binding cyclic peptide into one or two of the superficial loops of an FN3 fragment using the LassoGraft method.(7) In some embodiments, module (B) may be an artificial protein that has acquired cardiac cell surface antigen binding ability by incorporating the internal sequence of a cardiac cell surface antigen-binding cyclic peptide into one or two superficial loops of an antibody Fc fragment using the LassoGraft method. (8) In some embodiments, module (B) may be an artificial protein that has acquired cardiac cell surface antigen binding ability by incorporating the internal sequence of a cardiac cell surface antigen-binding cyclic peptide into one or two superficial loops of serum albumin using the LassoGraft method. (9) In some embodiments, module (B) may be an artificial protein that has acquired cardiac cell surface antigen binding ability by incorporating the internal sequence of a cardiac cell surface antigen-binding cyclic peptide into one of the superficial loops of ubiquitin using the LassoGraft method, and an artificial protein formed by linking multiple such artificial proteins. (10) In some embodiments, module (B) may be a full-length protein, Fab fragment, or scFv fragment of an IgG-type cardiac cell-type surface antigen antibody. (11) In some embodiments, module (B) may be an anti-cardiac cell-type surface antigen nanobody. (12) In some embodiments, the “molecule” described herein may be a homodimer protein obtained by fusing module (A), which has acquired AAV binding ability by incorporating the internal sequence of an AAV-binding cyclic peptide into one or two superficial loops of an FN3 fragment using the LassoGraft method, and module (B), which has acquired cardiac cell-type surface antigen binding ability by incorporating the internal sequence of a cardiac cell-type surface antigen-binding cyclic peptide into one or two superficial loops of an antibody Fc fragment, via the hinge sequence of the antibody.(13) In some embodiments, the “molecule” described herein may be a homodimer protein obtained by fusing a module (A) that has acquired AAV binding ability by incorporating the internal sequence of an AAV-binding cyclic peptide into one or two superficial loops of an antibody Fc fragment by LassoGraft, and a module (B) that has acquired cardiac cell species surface antigen binding ability by incorporating the internal sequence of a cardiac cell species surface antigen-binding cyclic peptide into one or two superficial loops of an FN3 fragment by LassoGraft, via the hinge sequence of an antibody. (14) In some embodiments, the “molecule” described herein may be a homodimer protein obtained by fusing, via the hinge sequence of an antibody, one or more modules (A) which have acquired AAV binding ability by linking one or more artificial proteins that have acquired AAV binding ability by incorporating the internal sequence of an AAV-binding cyclic peptide into one of the superficial loops of ubiquitin using the LassoGraft method, and one or two modules (B) which have acquired cardiac cell species surface antigen binding ability by incorporating the internal sequence of a cardiac cell species surface antigen-binding cyclic peptide into one or two of the superficial loops of an antibody Fc fragment. (15) In some embodiments, the “molecule” described herein may be a homodimer protein obtained by fusing, via the hinge sequence of an antibody, a module (A) which has acquired AAV binding ability by incorporating the internal sequence of an AAV-binding cyclic peptide into one or two superficial loops of an antibody Fc fragment using the LassoGraft method, and a module (B) which has acquired target receptor binding ability by linking one or more artificial proteins by incorporating the internal sequence of a cardiac cell species surface antigen-binding cyclic peptide into one of the superficial loops of ubiquitin using the LassoGraft method. (16) In some embodiments, the “molecule” described herein may be an IgG antibody-like protein which has acquired AAV binding ability by incorporating the internal sequence of an AAV-binding cyclic peptide into one or two superficial loops of an antibody against a cardiac cell species surface antigen using the LassoGraft method.(17) In some embodiments, the “molecule” described herein may be a nanobody-Fc fusion protein obtained by fusing a module (B), which is a nanobody for cardiac cell type surface antigens, and a module (A), which has acquired AAV binding ability by incorporating the internal sequence of an AAV-binding cyclic peptide into one or two superficial loops of an antibody Fc fragment using the LassoGraft method, via the hinge sequence of an antibody. (18) In some embodiments, the “molecule” described herein may be an artificial protein that has acquired AAV binding ability by incorporating the internal sequence of an AAV-binding cyclic peptide into one or two superficial loops of serum albumin using the LassoGraft method, and has also acquired cardiac cell type surface antigen binding ability by incorporating the internal sequence of a cardiac cell type surface antigen-binding cyclic peptide into one or two other superficial loops using the LassoGraft method. (19) In some embodiments, the “molecule” described herein may be a complex obtained by reacting module (A), which is a biotinylated AAV-binding cyclic peptide, and module (B), which is a biotinylated cardiac cell-type surface antigen-binding cyclic peptide, with streptavidin having four equivalent biotin-binding sites in a ratio of 3:1, 2:2, or 1:3. (20) In some embodiments, the “molecule” described herein may be a complex obtained by reacting module (A), which is a biotinylated AAV-binding cyclic peptide, and module (B), which is a biotinylated scFv fragment of an antibody against a cardiac cell-type surface antigen or an anti-cardiac cell-type surface antigen nanobody, with streptavidin having four equivalent biotin-binding sites in a ratio of 3:1, 2:2, or 1:3. (21) In some embodiments, the “molecule” described herein may be a protein obtained by fusing a module (A) that has acquired AAV binding ability by incorporating the internal sequence of an AAV-binding cyclic peptide into one or two superficial loops of Fn10 by the LassoGraft method to the C-terminus or N-terminus of an anticardiac constituent cell type surface antigen antibody as module (B).Module (A) may be fused to either the heavy chain or the light chain of the anticardiac cell type surface antigen antibody.

[0098] [Regarding the use of the above-mentioned "molecule" as a gene transfer agent for cardiac cell types] The above-mentioned "molecule" described herein acts as an "engager" that brings AAV closer to the cell when a complex with recombinant AAV containing a natural AAV capsid is applied to the cell as a vector, thereby achieving a dramatically higher gene transfer efficiency in a cardiac cell type surface antigen-dependent manner than when natural AAV alone is used as a vector.

[0099] (Preparation of the complex of the engager and recombinant AAV) In one embodiment, the engager is added to an AAV containing a serotype capsid that has been genetically recombinant for the purpose of introducing the target gene ("recombinant AAV") to form a complex, which is then used as a "recombinant viral vector". The complex can be prepared, for example, by adding a solution containing the engager to a solution containing recombinant AAV in a test tube and then incubating it. The mixing ratio of the engager and recombinant AAV in the preparation of the complex is determined appropriately according to the characteristics of the engager and is not particularly limited, but for example, the complex can be prepared by mixing recombinant AAV:engager in a ratio of 1:0.1 to 1:10 when converted to a ratio of (moles of capsid subunits):(moles of AAV capsid-binding peptide in module A).

[0100] (Recombinant Viral Vectors) The recombinant viral vectors described herein comprise a naturally occurring serotype AAV viral capsid, the viral capsid encapsulating the target nucleotide. In some embodiments, the target nucleotide is under the control of a promoter selected from the group consisting of a viral promoter, a bacterial promoter, a mammalian promoter, a bird promoter, a fish promoter, an insect promoter, and any combination thereof. In some embodiments, the target nucleotide is under the control of a non-human promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the promoter is an EF1α promoter. In some embodiments, the promoter is a CAG promoter.

[0101] The target nucleotide can be one or more genes, which may encode a detectable marker, such as a reporter or a therapeutic polypeptide. In some embodiments, the target nucleotide is a reporter gene. In some embodiments, the target nucleotide is a reporter gene encoding a detectable marker selected from the group consisting of fluorescent proteins, luciferases, β-galactosidases, etc. In other embodiments, the target nucleotide is selected from the group consisting of a suicide gene, a nucleotide encoding an antibody or a fragment thereof, a nucleotide encoding the CRISPR / Cas system or a part(s) thereof, a nucleotide encoding antisense RNA, a nucleotide encoding siRNA, a secretory enzyme, a gene encoding a therapeutic protein, etc. In some embodiments, the target nucleotide encodes a cyclic peptide-embedded graft protein designed by the LassoGraft method.

[0102] The compositions described herein generally comprise the “molecule” described herein and a naturally occurring serotype viral capsid, the capsid encapsulating the target nucleotide. In some embodiments, the compositions described herein comprise (1) a viral vector comprising a complex of the “molecule” described herein and a viral capsid, and (2) a pharmaceutically acceptable carrier.

[0103] Methods for preparing and using compositions comprising the above-mentioned "molecule" as an engager (hereinafter referred to as "engager") and a viral capsid are also described herein. In some embodiments, methods including retargeting adeno-associated virus and delivering diagnostic / therapeutic cargo to target cells include contacting the engager-AAV capsid complex described herein with cardiac cell types (extracorporeally or intracorporeally, for example, in humans), wherein the engager-AAV capsid complex prevents AAV from adhering to cells other than cardiac cell types by covering the capsid surface of AAV via module (A) in the engager, and at the same time specifically binds to proteins expressed on the surface of cardiac cell types via module (B), thereby achieving highly efficient infection and transduction of cardiac cell types. Such methods may include steps such as generating a viral vector with a natural serotype capsid, for example, purifying a recombinant AAV vector containing the target nucleotide recombinantly expressed using packaging cells, chemically synthesizing an engager or recombinantly expressing and purifying it using a plasmid encoding it, and obtaining a complex by mixing these two in an appropriate ratio and incubating them.

[0104] (Use of Recombinant Viral Vectors) The recombinant viral vectors described herein can be used for gene therapy applications (in vivo and in vitro) for heart diseases, or as research tools, depending on the characteristics of the exogenous gene introduced into the AAV. Here, "heart diseases" are not particularly limited as long as they can be the target of gene therapy, but examples include heart failure, cardiomyopathy (dilated, hypertrophic, restrictive), arrhythmias, congenital heart disease, coronary artery disease, aortic disease, valvular heart disease, pulmonary vascular disease, and hypertension. When used for therapeutic purposes, the recombinant viral vectors can be used in the form of a pharmaceutical composition, either in a standalone form or in the form of a pharmaceutical composition, formulated in a manner known in the art using one or more pharmaceutically acceptable carriers and / or excipients (hereinafter collectively referred to as "pharmaceutical compositions"). Pharmaceutical compositions can be formulated for various modes of administration, including systemic, topical, or localized administration. Pharmaceutical compositions can be administered to subjects (humans or animals) that require them, for example, by injection, inhalation, or by oral, intraoral, parenteral, or rectal administration. More specifically, the pharmaceutical composition may be administered by any means known in the art, for example, intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intrainjured, intracranially, intraventricularly, intrathecally, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreously, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctivally, intravesicularly, mucous membrane, intrapericardially, intraumbilical cord, or intraocularly, by oral, topically, inhalation, injection, infusion, continuous infusion, etc. The route of administration may be appropriately determined depending on the purpose of administration.

[0105] Systemic administration includes intramuscular, intravenous, intraperitoneal, and subcutaneous administration, but administration by injection is preferred. For injection, the pharmaceutical composition may be formulated in a liquid solution, preferably with a physiologically compatible buffer such as Hank's solution or Ringer's solution. In addition, the pharmaceutical composition may be formulated in solid form and redissolved or suspended immediately before use for injection administration. A lyophilized form of the pharmaceutical composition is also preferred. The pharmaceutical composition may be formulated for parenteral administration by injection, e.g., bolus injection or continuous infusion. Formulations for injection may be provided in the form of unit formulations, such as ampoules, with optionally added preservatives. The pharmaceutical composition may be formulated as a suspension, solution, or emulsion in an oily or aqueous medium, and may contain other agents, including suspending agents, stabilizers, and / or dispersants.

[0106] For oral administration, pharmaceutical compositions may be prepared by means of conventional art, together with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose, crystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate), and may take the form of tablets or capsules, for example. Tablets may also be coated by methods well known in the art. Liquid preparations for oral administration may take the form of solutions, syrups, or suspensions, or may be presented as dried products for compositions containing water or other suitable media before use. Such liquid preparations may be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifiers (e.g., lecithin or acacia), non-aqueous media (e.g., iond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoic acid or sorbic acid). The preparations may also optionally contain buffer salts, flavorings, colorants, and sweeteners.

[0107] Systemic administration is also possible via transmucosal or transdermal means.

[0108] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions or dispersions, formulations containing sesame oil, peanut oil, or aqueous propylene glycol, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.

[0109] When administering the above-mentioned pharmaceutical composition to a target, a person skilled in the art can determine the appropriate dosage and duration of administration for each individual target by considering various factors such as the purpose of gene transfer, dosage form, route of administration, and the disease state of the target.

[0110] When the recombinant viral vector described above is administered to a target for therapeutic purposes, it can be used in combination with other drugs (combination agents). The necessity of combination therapy, the combination agents, and their dosage and administration can be appropriately determined by a person skilled in the art, and combination therapy can be implemented.

[0111] The embodiments of the present invention will be described below in detail based on examples, but the present invention is not limited thereto. Those skilled in the art can modify the embodiments of the present invention in various ways without departing from the meaning of the present invention, and such modifications are also included within the scope of the present invention.

[0112] material and method

[0113] [Pluripotent stem cell-derived cardiomyocytes] Pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, spermatogonial stem cells ("GS cells"), embryonic germ cells ("EG cells"), induced pluripotent stem (iPS) cells, and embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer. Preferred pluripotent stem cells are ES cells, iPS cells, and ntES cells.

[0114] ES cells are stem cells that are established from the inner cell mass of early embryos (e.g., blastocysts) of mammals such as humans and mice, and possess pluripotency and the ability to proliferate through self-renewal. ES cells are embryo-derived stem cells that originate from the inner cell mass of the blastocyst, which is the 8-cell stage of a fertilized egg after the morula stage. They possess the ability to differentiate into any cell that makes up an adult, known as pluripotency, and the ability to proliferate through self-renewal. ES cells were discovered in mice in 1981 (M.J. Evans and M.H. Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in primates such as humans and monkeys (J.A. Thomson et al. (1998), Science 282:1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; J.A. Thomson et al. (1996), Biol. Reprod., 55:254-259; J.A. Thomson and V. S. Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Furthermore, cell maintenance by subculturing can be carried out using a culture medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF).For methods of establishing and maintaining human and monkey ES cells, see, for example, USP 5, 843, 780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. USA. 92:7844-7848; Thomson JA, et al. (1998), Science. 282:1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. This is described in USA, 103:9554–9559; H. Suemori et al. (2001), Dev. Dyn., 222:273–279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580–1585; Klimanskaya I, et al. (2006), Nature. 444:481–485, etc. For ES cell production, a DMEM / F-12 culture medium supplemented with, for example, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 20% KSR, and 4 ng / ml bFGF was used, and the culture was maintained at 37°C and 2% CO2. ２ Human ES cells can be maintained in a humid atmosphere of 98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26:215-224). Furthermore, ES cells need to be passaged every 3-4 days, and during this passage, for example, 1 mM CaCl ２This can be done using 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 20% ​​KSR. ES cell selection can generally be performed by Real-Time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, and Nanog as indicators. In particular, for the selection of human ES cells, the expression of gene markers such as OCT-3 / 4, NANOG, and ECAD can be used as indicators (E. Kroon et al. (2008), Nat. Biotechnol., 26:443-452). For ES cells, various mouse ES cell lines established by inGenius Targeting Laboratory, RIKEN (the Institute of Physical and Chemical Research), etc., are available, and for human ES cells, various human ES cell lines established by the NIH, RIKEN, Kyoto University, and Cellartis are available. For example, as human ES cell lines, the NIH's CHB-1 to CHB-12, RUES1, RUES2, HUES1 to HUES28, etc., WisCell Research's H1 and H9, RIKEN's KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, SSES3, etc. can be used. Alternatively, clinical-grade cell lines and research and clinical cell lines prepared using those cell lines may be used.

[0115] Induced pluripotent stem cells (iPSCs) are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells." These include iPSCs established by Yamanaka et al. by introducing four factors—Oct3 / 4, Sox2, Klf4, and c-Myc—into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126:663-676), as well as human cell-derived iPSCs established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131:861-872), and Nanog-iPS cells established by selecting cells based on Nanog expression after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka). iPS cells produced by methods that do not include c-Myc (Nakagawa M, Yamanaka S, et al. Nature Biotechnology, (2008) 26, 101-106), and iPS cells established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May; 8(5): 409-12, Okita K et al. Stem Cells. 31(3): 458-66) can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, as created by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318:1917-1920.), induced pluripotent stem cells created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451:141-146), and induced pluripotent stem cells created by Sakurada et al. (Japanese Patent Publication No. 2008-307007) can also be used.In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA., et al., Nature) Any induced pluripotent stem cells known in the art as described in Biotechnology, (2008) 26, No. 7, 795-797, or in patents (e.g., JP 2008-307007, JP 2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852) can be used. Various iPSC lines established by the NIH, RIKEN, Kyoto University, etc. can be used as induced pluripotent cell lines. For example, human iPSC strains include RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 strains, and Kyoto University's 253G1, 201B7, 409B2, 454E2, 606A1, 610B1, and 648A1 strains. Alternatively, clinical-grade cell lines provided by Kyoto University or Cellular Dynamics International, etc., as well as research and clinical cell lines created using those cell lines, may be used.

[0116] In the present invention, "cardiomyocyte" means a cell that expresses at least one marker gene selected from the group consisting of cardiac troponin (cTNT), αMHC (α myosin heavy chain, MYH6), βMHC (MYH7), and troponin I. For cTNT, NCBI access number NM_000364 is an example in the case of humans, and NM_001130176 is an example in the case of mice. For αMHC, NCBI access number NM_002471 is an example in the case of humans, and NM_001164171 is an example in the case of mice. For βMHC, NCBI access number NM_000257 is an example in the case of humans, and NM_080728 is an example in the case of mice. It is known that as cardiomyocytes mature, an isoform switch occurs in which the expression of troponin I1 (TNNI1) decreases and the expression of troponin I3 (TNNI3) increases (Fikru B. Bedada, (2014) 3(4):594-605). "Epicardial cells" refer to cells that express at least one marker gene selected from at least WT1 (Wilms Tumor 1) and ZO-1 (Zonula occludens-1). In addition, these WT1, ZO-1 positive epicardial cells, when exposed to an epithelial-mesenchymal transition (EMT) cocktail containing TGFβ or TGFβ and FGF components, become a cell population that optionally expresses EMT markers such as SNAI1 or SNAI2, mesenchymal markers such as vimentin and / or CD90, and / or smooth muscle markers such as SMA, or smooth muscle genes, and optionally CNN1, MYH11, TAGLN, and SMTN, while the latter becomes a fibroblast cell population that expresses TCF21. "Endocardial cells" means cells that express at least one marker gene selected from at least NFATC1 and CD31.

[0117] As a method for inducing differentiation from pluripotent stem cells into immature cardiomyocytes, cardiomyocytes can be produced from pluripotent stem cells using a method reported by, for example, Laf Lamme MA et al. (Laf Lamme MA & Murry CE, Nature 2011, Review). Other examples, though not specifically defined, include methods for producing cardiomyocytes by forming cell aggregates (embryoid bodies) from induced pluripotent stem cells through suspension culture, methods for producing cardiomyocytes in the presence of a substance that suppresses Bone Morphogenic Protein (BMP) signaling (WO2005 / 033298), methods for producing cardiomyocytes by sequentially adding Activin A and BMP (WO2007 / 002136), methods for producing cardiomyocytes in the presence of a substance that promotes activation of the canonical Wnt signaling pathway (WO2007 / 126077), and methods for isolating FLk / KDR-positive cells from induced pluripotent stem cells and producing cardiomyocytes in the presence of cyclosporine A (WO2009 / 118928).Furthermore, methods for inducing differentiation of cardiomyocytes using cytokines in embryoid body formation (Yang L, et al., Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population., Nature., 2008 May 22; 453 (7194): 524-8), and methods for inducing differentiation of cardiomyocytes without using cytokines in adherent culture (Lian X, et al., Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of Canonical Wnt signaling., Proc Natl Acad Sci U.S.A., 2012 July 3;109(27):E1848-57, a method for inducing differentiation of cardiomyocytes without the use of cytokines, using a combination of adherent culture and suspension culture (Minami I, et al., A small molecule that promotes cardiomyocyte differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions., Cell Rep., 2012 Nov. Other methods have also been proposed, such as 29;2(5):1448-60). As a method for inducing differentiation from pluripotent stem cells into epicardial cells, they can be produced by methods such as those reported by Witty AC et al. (Nature Biotechnology 2014). As a method for inducing differentiation from pluripotent stem cells into endocardial cells, they can be produced by methods such as those reported by Neri T et al. and Mikryukov AA et al. (Nature Communications 2019, Cell Stem Cell 2020).

[0118] Any known culture medium can be used without particular limitation as a culture medium for obtaining cardiomyocytes from pluripotent stem cells. For example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies), StemPro34 (Invitrogen), StemFit AK02 medium with solution C removed (500 mL total of 400 mL of solution A and 100 mL of solution B from AJINOMOTO AK02), Essential 6 medium (Thermo Fischer Scientific) and mixed media thereof are used. These media can be supplemented with additives known to the body, depending on the cells and culture conditions. For example, the medium may contain serum or may be serum-free. Furthermore, if necessary, the medium may contain one or more serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 1-thiolglycerol, or one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts. Among these, preferred media are StemPro34 or StemFit AK02 containing transferrin, 1-thiolglycerol, L-glutamine, and ascorbic acid, respectively, with solution C removed, or RPMI1640 containing B27 supplement. The first additive used to induce differentiation into cardiomyocytes using the above media may be a combination of activin A, BMP4, and bFGF, or CHIR99021.In the case of a combination of activin A, BMP4, and bFGF, the concentrations used for these additives are preferably 1 ng / ml to 100 ng / ml for activin A, 1 ng / ml to 100 ng / ml for BMP4, and 1 ng / ml to 100 ng / ml for bFGF, and more preferably 6 ng / ml for activin A, 10 ng / ml for BMP4, and 5 ng / ml for bFGF. In the case of CHIR99021, the concentration used is preferably 100 nM to 100 μM, and more preferably 4 to 6 μM. After adding the above additives, cardiomyocytes can be induced by adding vascular endothelial growth factor (VEGF) and a Wnt inhibitor to the culture medium. The concentration of VEGF used is preferably 1 to 100 ng / ml, and more preferably 10 ng / ml. Examples of Wnt inhibitors include DKK1 protein (e.g., NCBI accession number: NM_012242 in humans), sclerostin (e.g., NCBI accession number: NM_025237 in humans), IWR-1 (Merck Millipore), IWP-2 (Sigma-Aldrich), IWP-3 (Sigma-Aldrich), IWP-4 (Sigma-Aldrich), PNU-74654 (Sigma-Aldrich), XAV939 (Sigma-Aldrich), and their derivatives, among which IWP-3, IWP-4, and IWR-1 are preferred. The concentration of the Wnt inhibitor used is not particularly limited as long as it inhibits Wnt, but is preferably 1 nM to 50 μM, and particularly preferably 1 to 2 μM. The culture period for obtaining cardiomyocytes from pluripotent stem cells is, for example, 5 to 365 days, preferably 5 to 100 days, more preferably 5 to 60 days, even more preferably 5 to 40 days, and even more preferably 5 to 30 days.

[0119] [Production and Purification of AAV Virus Vectors] AAV virus vectors were produced using the AAVpro Helper Free System manufactured by Takara Bio Inc. When preparing the AAV9 capsid, the plasmid pAAV2 / 9n (Addgene #112865) carrying the Rep gene derived from AAV2 and the Cap gene derived from AAV9 in tandem was used. As the reporter gene, pAAV-CMV-Fluc, in which Firefly luciferase (Fluc) was cloned into the pAAV-CMV vector, was used. Virus was produced by triple transfection of pHelper, pAAV2 / 9n, and pAAV-CMV-Fluc into HEK293T cells, which are packaging cells, by a conventional method. The transfected packaging cells were cultured in 5% CO ２ at 37°C for 2 days, then harvested, and resuspended in Phosphate-buffered saline (PBS, 20 mM phosphate, 150 mM NaCl, pH 7.5) containing 1 mM MgCl ２ , 2.5 mM KCl, and 0.5% Triton-X100. The operations of freezing the cell suspension at -80°C for 30 minutes, thawing in a 37°C warm bath, and centrifuging at 4000 x g for 10 minutes at 4°C were repeated 3 times, and finally centrifugation was performed at 4000 x g for 10 minutes at 4°C, after which the supernatant was collected. This was used as the virus extract. Virus purification was performed by affinity chromatography using POROS TM CaptureSelect AAVX resin (cat. no. A36739) manufactured by ThermoFisher. Briefly, the virus extract passed through a 0.45 μm filter was applied to POROS TMThe samples were passed through a CaptureSelect AAVX resin column, and after washing for unbound material, the virus was eluted using elution buffer (40% propylene glycol, 0.1 M Na-citrate pH 3, 0.3 M NaCl). The eluted fraction was neutralized with 1 / 12 volume of 2 M HEPES pH 8. The obtained purified virus solution was replaced with PBS containing 0.001% Pluronic F68 polyole (MP Biomedical, Cat. No. 2750049), concentrated by centrifugation to the desired volume using an Amicon Ultra-0.5 centrifugal filter unit with an Ultracel-100 membrane (100 K Da MWCO) filter cartridge, and stored at 4°C or -80°C until use. The viral titer (viral genome vg / mL per milliliter) was determined by qPCR using a standard curve for known viral concentrations.

[0120] [Preparation of Cyclic Peptides] Based on International Publication No. 2011 / 049157 and Japanese Patent Publication No. 2013-46637, etc., the RaPID system was performed on purified AAV9 capsid to identify three classes of cyclic peptides that have high affinity for and specifically bind to AAV9 capsid. The identified cyclic peptides were chemically synthesized using methods well known in the art and used in the preparation of additives and other experiments. Examples of methods for producing cyclic peptides include chemical synthesis methods such as liquid-phase methods, solid-phase methods, and hybrid methods combining liquid-phase and solid-phase methods, as well as genetic engineering methods and translational synthesis methods using cell-free translation systems. Representative examples of the AAV9 capsid-binding cyclic peptides of classes 1 to 3 identified above are shown below.

[0121] Example 1: Class 1 Cyclic Peptides The active structures of representative Class 1 cyclic peptides (Examples 1-1 to 1-15) are cyclic peptides represented by the following formula (Ia). For particularly representative examples, the cyclic peptide name (L number) is also indicated.

[0122]

[0123] (In the formula, Variable region 1 represents the amino acid sequence of Variable region 1 in the cyclic peptide of each example shown in Table 15 below, and S represents S derived from the thiol group of Cys.)

[0124]

[0125] Example 2: Class 2 Cyclic Peptides The active structures of representative Class 2 cyclic peptides (Examples 2-1 to 2-15) are cyclic peptides represented by the following formula (Ib). For particularly representative examples, the cyclic peptide name (L number) is also indicated.

[0126]

[0127] (In the formula, Variable region 1 represents the amino acid sequence of Variable region 1 in the cyclic peptide of each example shown in Table 16 below, Variable region 2 represents the amino acid sequence of Variable region 2 in the cyclic peptide of each example shown in Table 17 below, and S represents S derived from the thiol group of Cys.)

[0128]

[0129]

[0130] Example 3: Class 3 Cyclic Peptides The active structures of representative Class 3 cyclic peptides (Examples 3-1 to 3-8) are cyclic peptides represented by the following formula (Ic). For particularly representative examples, the cyclic peptide name (L number) is also indicated.

[0131]

[0132] (In the formula, Variable region 1 represents the amino acid sequence of Variable region 1 in the cyclic peptide of each example shown in Table 18 below, and S represents S derived from the thiol group of Cys.)

[0133]

[0134] [Various forms as an engager and their preparation] The core of the present invention is an engager molecule that attracts natural AAV capsids to cells expressing surface antigens of desired cardiac cell types, namely, a "molecule" comprising an AAV capsid-binding module (A) and a cardiac cell type surface antigen-binding module (B), which can simultaneously bind to AAV capsids and cardiac cell type surface antigens, and which can take on an extremely diverse range of engager forms. Representative embodiments include (a) heterologous cyclic peptide conjugates (referred to as type I engagers), (b) cyclic peptide-anticardiac cell type surface antigen antibody conjugates (referred to as type II engagers), (c) conjugates of two scaffold proteins to which cyclic peptides have been transplanted (referred to as type III engagers), (d) a single scaffold protein to which multiple cyclic peptides have been transplanted (referred to as type IV engagers), (e) an anticardiac cell type surface antigen antibody to which a cyclic peptide has been transplanted (referred to as type V engagers), and (f) a conjugate of a scaffold protein to which a cyclic peptide has been transplanted and an anticardiac cell type surface antigen antibody (referred to as type VI engagers). These embodiments are illustrated in Figure 1. Hereinafter, various embodiments using the above-mentioned "molecule" as an engager will be explained using examples. However, these are examples for the concrete understanding of the various embodiments, and the embodiments are not limited to these.

[0135] <Preparing the Engager>

[0136] Example 6: Preparation of a linked structure (Type III Engager) of two scaffold proteins implanted with cyclic peptides. A method for producing scaffold proteins implanted with AAV capsid-binding peptides and target receptor-binding peptides using the LassoGraft method is described in detail in Japanese Patent No. 6598344. Furthermore, by genetically fusing the two, molecules that bind to both AAV capsids and target receptors can be prepared. As one example, a molecule was prepared here by linking a module in which an AAV capsid-binding peptide was implanted inside the 10th Type III repeat domain of human fibronectin (hereinafter referred to as "Fn10") with a module in which a target receptor-binding peptide was implanted inside the Fc region derived from human IgG (hereinafter referred to as "Fc"), as follows. A construct for an Fn10:Fc fusion protein was created by concatenating DNA encoding the signal sequence of human prolactin, DNA encoding the region of human fibronectin from amino acid residue numbers 1418 to 1509, and DNA encoding the Fc region of human IgG1. This construct was then incorporated into the expression vector pcDNA3.1 (Thermo Fisher Scientific). Based on the Fn10:Fc expression vector, an expression vector was created in which one amino acid residue in the loop within the fibronectin region, for example, the loop region between Pro1440 and Val1442, was replaced with a sequence derived from the AAV9 capsid-binding cyclic peptide shown in Table 19. Furthermore, one amino acid residue in the loop within the Fc region, for example, the loop region between Glun419 and Asn421, was replaced with a sequence derived from the integrin a6b1-binding cyclic peptide IB8 (SGGYLVKRSGWLYTVDGGS) (SEQ ID NO: 55). Furthermore, an expression vector was constructed in which one amino acid residue in the loop within the Fc region, for example, the loop region between Ser383 and Gly385, was replaced with a sequence derived from a target receptor-binding cyclic peptide shown in Table 20. The transplanted sequence portion was amplified using extension PCR to obtain constructs of various target type III engagers.The target protein was transiently expressed using the Expi293 Expression System (Thermo Fisher Scientific) with this expression construct, and the culture supernatant was purified by affinity chromatography using Protein A-Sepharose (Cytiva). The purified type III engager protein was dialyzed to PBS and stored at -80°C until use. Cyclic peptide-grafted proteins were denoted as Fn10 (cyclic peptide name):Fc (cyclic peptide name), such as Fn10 (L19):Fc (IB8). However, proteins without peptide grafting on Fc were used as a control for type III engagers.

[0137]

[0138]

[0139] Example 8: Preparation of anti-target receptor antibodies (Type V engagers) with transplanted cyclic peptides. The technique of conferring new binding specificity to any antibody by applying the LassoGraft method is described in detail in Japanese Patent No. 7303527. Here, as the base anti-receptor antibodies, antibodies having a constant region of human IgG1 / kappa and a variable region of anti-human HER2 antibody Trastuzumab, anti-human SIRPα antibody Anzurstobart, or anti-mouse transferrin receptor antibody 8D3 were used. 8D3 was used as a control because it does not react with the human transferrin receptor. The variable region amino acid sequences for Trastuzumab, Anzurstobart, and 8D3 were taken from those described in DRUGBANK (https: / / go.drugbank.com / drugs / DB00072), Chan et al., Cancer Res. Commun. 2024, and Boado et al., Biotech Bioeng. 2008, respectively. Based on the antibody heavy chain expression vector, expression vectors were prepared by replacing, for example, one amino acid residue in the loop region between Tyr296 and Ser298 and one amino acid residue in the loop region between Ser383 and Gly385 with sequences derived from the AAV9 capsid-binding cyclic peptide shown in Table 19. The transplanted sequence was amplified using extension PCR to obtain heavy chain constructs for various target V-type engagers. These expression constructs, along with the corresponding light chain expression vectors, were used to transiently express the target bispecific antibodies using the Expi293 Expression System (Thermo Fisher Scientific). The culture supernatant was purified by affinity chromatography using Protein A-Sepharose (Cytiva). The purified V-type engager proteins were dialyzed to PBS and stored at -80°C until use. The cyclic peptide transplantation antibodies were each Trastuzumab-IgG (L19). ２ Like this: (Antibody name) - IgG (Cyclic peptide name) （価数） This was written as follows.

[0140] Example 9: Preparation of a conjugate (Type VI engager) of a scaffold protein implanted with a cyclic peptide and an anti-target receptor antibody. A method for producing a scaffold protein implanted with an AAV capsid-binding peptide using the LassoGraft method is described in detail in Japanese Patent No. 6598344. Furthermore, by genetically fusing this with any antibody, a molecule that binds to both the AAV capsid and the target receptor can be prepared. As one example, a molecule was prepared in which a module in which an AAV capsid-binding peptide was implanted inside Fn10 was fused to the C-terminus of an antibody against the target receptor as follows. Here, as the base anti-receptor antibody, an antibody having a constant region of human IgG1 / kappa and a variable region of anti-human HER2 antibody Trastuzumab, anti-human SIRPα antibody Anzurstobart, or anti-mouse transferrin receptor antibody 8D3 was used. 8D3 was used as a control because it does not respond to the human transferrin receptor. The variable region amino acid sequences for Trastuzumab, Anzurstobart, and 8D3 were taken from those described in DRUGBANK (https: / / go.drugbank.com / drugs / DB00072), Chan et al., Cancer Res. Commun. 2024, and Boado et al., Biotech Bioeng. 2008, respectively. By ligating an Fn10 module to the C-terminus of the antibody heavy chain and replacing two amino acid residues in the loop within the fibronectin region, such as the loop region between Arg1493 and Ser1496, with a sequence derived from the AAV9 capsid-binding cyclic peptide shown in Table 19, we obtained the heavy chain constructs for various target type VI engagers. Using these expression constructs, the target antibody-Fn10 fusions were transiently expressed together with the corresponding light chain expression vectors using the Expi293 Expression System (Thermo Fisher Scientific), and the culture supernatant was purified by affinity chromatography using Protein A-Sepharose (Cytiva). The purified type VI engager proteins were dialyzed to PBS and stored at -80°C until use.The antibody-Fn10 fusions were denoted as follows: (antibody name):Fn10(cyclic peptide name), such as Trastuzumab:Fn10(L17).

[0141] [Evaluation of cardiac cell-targeting engagers]

[0142] Example A

[0143] 1. Evaluated Engagers (1) AAV9+BiVE Type7 control (2) AAV9+BiVE Type7 Trustuzumab. Fn(L17) (3) AAV9+BiVE Type7 Anzurstobart_Fn(L17) (4) AAV9+BiVE Type1 control (5) AAV9+BiVE Type1 IB8 *) (1) to (3): Type VI Engagers *) (4) and (5): Type III Engagers

[0144] 2. Preparation of Engagers (1) To investigate the acquisition of receptor-specific gene transfer ability by the AAV9+BiVE Type7 control VI engager, an antibody-Fn10 fusion (8D3:Fn10(L17)) was prepared by transplanting the AAV9 capsid-binding peptide L17 (Table 19, Example No. 6-3) to the C-terminus of the target antigen antibody IgG (anti-mouse transferrin receptor (TfR) antibody 8D3), and this was complexed by adding 0.01 equivalents, 0.1 equivalents, 1 equivalent, or 10 equivalents per capsid subunit to purified AAV9. (2) AV9+BiVE Type7 Trustuzumab. To investigate the acquisition of gene transfer ability by the Fn(L17) VI engager, an antibody-Fn10 fusion product (referred to as Trastuzumab:Fn10(L17)) was prepared by transplanting the AAV9 capsid-binding peptide L17 (Table 19, Example No. 6-3) to the C-terminus of the target antigen antibody IgG (anti-human HER2 antibody Trastuzumab), and this was complexed by adding 0.01 equivalents, 0.1 equivalents, 1 equivalent, or 10 equivalents per capsid subunit to purified AAV9. (3) AAV9 + BiVE Type 7 Anzurstobart_Fn(L17) The above engager was prepared using L17 as the sequence derived from the cyclic peptide and Anzurstobart as the target antigen antibody, in accordance with the method described in Example 9. (4) AAV9+BiVE type1 control The above engager was prepared using L19 as the sequence derived from the cyclic peptide, without transplanting the peptide into Fc, according to the method described in Example 6. (5) AAV9+BiVE type1 IB8 The above engager was prepared using L19 as the sequence derived from the cyclic peptide and IB8 as the target antigen-binding peptide, according to the method described in Example 6.

[0145] 3. Evaluation of Engagers The cell population used in the present invention may be any cell population, for example, a cell population differentiated from pluripotent stem cells, or a population of cells extracted from a living organism. The timing of introducing AAV9 into the cell population that has undergone the process of differentiation from pluripotent stem cells is not particularly limited as long as it is the timing at which cell selection is desired. The infection efficiency can be evaluated using the amount of translation of the marker protein translated from AAV9 as an indicator. That is, if the amount of translation of the marker protein translated from AAV9 increases, it can be determined that the infection efficiency has increased. Specifically, the evaluation process can be carried out by detecting a signal from the marker protein, which is a translation product from the marker gene, using a predetermined detection device. Examples of detection devices include, but are not limited to, plate readers, flow cytometers, imaging cytometers, fluorescence microscopes, emission microscopes, and CCD cameras. Human iPS cell-derived cardiomyocytes were infected with a complex of three types of target surface antigen-targeting engagers ((2), (3), and (5) above) and their control engagers ((1) and (4) above) and AAV9 possessing a luciferase gene at MOI = 25,000, and the cells were incubated at 37°C and 5% CO2. ２ After culturing under the specified conditions for two days, luciferase expression levels were measured using the Luciferase Assay System (Promega, Cat. No. E1501). Two days later, the activity of the reporter gene, luciferase, was measured and expressed as a percentage relative to the value without the enhancer. The evaluation results are shown in Figure 2.

[0146] Example B

[0147] 1. Evaluated Engagers (1) AAV+Type3 control (2) AAV+Type3 Trustuzumab (3) AAV+Type3 Anzurstobart *) (1) to (3): V-type Engagers

[0148] 2. Preparation of Engagers (1) AAV+Type3 control The above engager was prepared using L19 as the sequence derived from the cyclic peptide and 8D3 as the antibody, in accordance with the method described in Example 8. (2) AAV+Type3 Trastuzumab The above engager was prepared using L19 as the sequence derived from the cyclic peptide and Trastuzumab as the antibody, in accordance with the method described in Example 8.

[0149] (3) AAV+Type3 Anzurstopart The above-mentioned engager was prepared using L19 as the sequence derived from the cyclic peptide and Anzurstopart as the antibody, in accordance with the method described in Example 8. 3. Evaluation of Engagers Except for using two types of engagers that target surface antigens ((2) and (3) above) and their control engager ((1) above), the evaluation was carried out according to the method in Example A. The evaluation results are shown in Table 21. A graph of the evaluation results is shown in Figure 3.

[0150]

[0151] Example C

[0152] 1. Evaluated Engagers (1) AAV+Type3 Trustuzumab (2) AAV+Type3 Anzurstobart

[0153] 2. Preparation of Engagers The above-mentioned engagers (1) and (2) were prepared in accordance with Example B.

[0154] 3. Evaluation of Engagers (1) Evaluation using human iPS-derived cardiomyocytes was performed in accordance with the method in Example A. (2) In the evaluation using human fibroblasts, human fibroblasts were also infected with MOI = 25,000 and incubated at 37°C and 5% CO2. ２After culturing under the specified conditions for two days, luciferase expression levels were measured using the Luciferase Assay System (Promega, Cat. No. E1501). Two days later, the activity of the reporter gene, luciferase, was measured and expressed as a percentage relative to the value without the enhancer. The evaluation results are shown in Figure 4.

[0155] Example D

[0156] 1. Evaluated Engagers (1) AAV9 + BiVE Type3 control (H460) (2) AAV9 + BiVE Type3 ErbB2 (H2Mab300) (3) AAV9 + BiVE Type3 SIRPa (mab3) (4) AAV9 + BiVE Type3 ITGA7 (M36) *) (1) to (4): V-type Engagers

[0157] 2. Preparation of Engagers In order to investigate the efficiency of cardiac infection by type V engagers, the following type V engagers were prepared. Note that each antibody is a known antibody described in the literature, etc., and can be readily obtained by those skilled in the art. (1) AAV9 + BiVE Type 3 control (H460) The above engager was prepared according to the method described in Example 8, using L19 as the sequence derived from a cyclic peptide and H460 as the antibody that does not have specificity for cardiac surface antigens. (2) AAV9 + BiVE Type 3 ErbB2 (H2Mab300) The above engager was prepared according to the method described in Example 8, using L19 as the sequence derived from a cyclic peptide and ErbB2 (H2Mab300) as the antibody that has specificity for cardiac surface antigens. (3) AAV9+BiVE Type3 SIRPa(mab3) The above engager was prepared according to the method described in Example 8, using L19 as the sequence derived from the cyclic peptide and SIRPa(mab3) as the antibody having specificity to cardiac surface antigens. (4) AAV9+BiVE Type3 ITGA7(M36) The above engager was prepared according to the method described in Example 8, using L19 as the sequence derived from the cyclic peptide and ITGA7(M36) as the antibody having specificity to cardiac surface antigens.

[0158] 3. Evaluation of Engagers (a) Preparation of Samples Used in the Test The engager solutions (1 μM) (1) to (4) prepared in 2 above were each subjected to a conventional method to introduce CAG-Fluc into wild-type AAV9 (5 x 10) ９ A solution was prepared by mixing with (vg / μl). Two mixing ratios were used: 1:1 and 1:3, resulting in a total of eight test samples. Table 22 shows the details of the mixing ratios.

[0159]

[0160] (b) Verification of organ infection status after administration to mice The organ infection status of AAV9 was examined using a luciferase assay with 8-12 week old C57BL / 6J mice (4 mice per group). Eight test samples prepared in (a) above were administered intraperitoneally to the mice, and one week later, D-luciferin (150 mg / kg) was administered intraperitoneally. Organs were removed 10 minutes later, perfused with PBS, and then immersed in D-luciferin, and all organs were imaged. An In Vivo Imaging System was used for evaluation. The evaluation results (images taken) for the heart and liver are shown in Figure 5.

[0161] - Effects on the heart: When the mixing ratio was 1:1, administration of any of the Type 3 engagers (2) to (4) resulted in an increased efficiency of AAV9 infection in the heart compared to administration of AAV9 alone or Control (1). When the mixing ratio was 1:3, administration of any of the Type 3 engagers (2) to (4) also resulted in an increased efficiency of AAV9 infection in the heart compared to administration of Control (1). (2) Effects on the liver: In the liver, which does not express cardiac constituent cell type surface antigens, when the mixing ratio was 1:1, administration of Control (1) and Type 3 engagers (2) to (4) suppressed the efficiency of AAV9 infection in the liver to 1 / 5 to 1 / 10 compared to administration of AAV9 alone. Furthermore, in a mixing ratio of 1:3, administration of Control (1) and Type 3 Engagers (2) to (4) almost completely suppressed AAV9 infection of the liver.

[0162] Example E

[0163] 1. Evaluated Engagers (1) AAV9+BiVE Type7 control (HR460) (2) AAV9+BiVE Type7 ErbB2 (H2Mab300)_Fn (L17) (3) AAV9+BiVE Type7 SIRPa (mab3)_Fn (L17) (4) AAV9+BiVE Type7 ITGA7 (M36)_Fn (L17) *) (1) to (4): VI type Engagers

[0164] 2. Preparation of Engagers To investigate the efficiency of cardiac infection by type VI engagers, the following type VI engagers were prepared: (1) AAV9+BiVE Type7 control (HR460) The above engager was prepared according to the method described in Example 9, using L17 as the sequence derived from a cyclic peptide and H460 as the antibody that does not have specificity for cardiac surface antigens. (2) AAV9+BiVE Type7 ErbB2 (H2Mab300)_Fn (L17) The above engager was prepared according to the method described in Example 9, using L17 as the sequence derived from a cyclic peptide and ErbB2 (H2Mab300) as the antibody that has specificity for cardiac surface antigens. (3) AAV9+BiVE Type7 SIRPa(mab3)_Fn(L17) The above engager was prepared according to the method described in Example 9, using L17 as the sequence derived from the cyclic peptide and SIRPa(mab3) as the antibody having specificity to cardiac surface antigens. (4) AAV9+BiVE Type7 ITGA7(M36)_Fn(L17) The above engager was prepared according to the method described in Example 9, using L17 as the sequence derived from the cyclic peptide and ITGA7(M36) as the antibody having specificity to cardiac surface antigens.

[0165] 3. Evaluation of Engagers (a) Preparation of Samples Used in the Test Following the preparation method described in 3. (a) of Example D above, samples were prepared for each of the engagers (1) to (4) above in two mixing ratios (1:1 and 1:3), resulting in a total of eight test samples. (b) Verification of Organ Infection Status After Administration to Mice Following the test method described in 3. (b) of Example D above, the infection status of organs was verified after administration of the eight test samples prepared in (a) above to mice. The evaluation results for the heart and liver are shown in Figure 6.

[0166] (1) Effects on the heart: When the mixing ratio was 1:1, administration of any of the Type 7 engagers (2) to (4) showed an increased efficiency of AAV9 infection in the heart compared to administration of AAV9 alone or Control (1), and the degree of this increase tended to be higher than that of administration of any of the Type 3 engagers. This high increase in the efficiency of AAV9 infection in the heart was maintained even when the mixing ratio was 1:3. (2) Effects on the liver: When the mixing ratio was 1:1, administration of any of the Type 7 engagers (2) to (4) did not show any effect in suppressing AAV9 infection in the liver. However, when the mixing ratio was 1:3, administration of any of the Type 7 engagers showed an effect in suppressing AAV9 infection in the liver compared to the case of a mixing ratio of 1:1.

[0167] As demonstrated above, the engager, provided as one embodiment of the present invention, consisting of a bivalent molecule capable of binding to both natural AAV capsid and cardiac cell surface antigens, demonstrated in all embodiments that, while using natural (unmodified) AAV capsid, exhibited excellent AAV9 infection effects on cells expressing the target cardiac cell surface antigen. On the other hand, it was confirmed that it actually suppressed AAV9 infection in cells that did not express the cardiac cell surface antigen. Therefore, it has become clear that genes can be efficiently introduced into cardiac cell species using the engager utilizing the "molecule" described herein.

[0168] The present invention relates to a method for conferring cardiac cell type surface antigen specificity to AAV, and as one embodiment of the invention, provides a gene delivery agent for cardiac cell types containing a bivalent molecule that can bind to both natural AAV capsids and cardiac cell type surface antigens. This delivery agent is useful for gene therapy for heart diseases, etc. This application is based on Japanese Patent Application No. 2025-067747 (filing date: April 16, 2025), the contents of which are entirely contained herein.

Claims

1. A gene transfer agent for cardiac cell species comprising a molecule ("molecule") that can bind to the capsid of AAV (adeno-associated virus) for carrying foreign genes ("AAV capsid-binding") and a module (B) that can bind to the surface antigen of cardiac cell species ("cardiac cell species surface antigen-binding"), wherein the molecule can simultaneously bind to the capsid of AAV and the surface antigen of cardiac cell species, and module (A) is 1) an AAV capsid-binding peptide, or 2) a scaffold protein having a structure into which the AAV capsid-binding peptide has been transplanted, and module (B) is 1) an anti-cardiac cell species surface antigen antibody, 2) a cardiac cell species surface antigen-binding peptide, or 3) a scaffold protein having a structure into which the cardiac cell species surface antigen-binding peptide has been transplanted.

2. The gene transfer agent according to claim 1, wherein the molecule is an anticardiac cell species surface antigen antibody having a structure in which an AAV capsid-binding peptide is transplanted.

3. The gene transfer agent according to claim 1, wherein the molecule is a protein comprising a scaffold protein having a structure in which an AAV capsid-binding peptide is transplanted, and an anti-cardiac constituent cell type surface antigen antibody linked to it.

4. The gene transfer agent according to claim 1, wherein the molecule is a protein formed by linking a scaffold protein having a structure into which an AAV capsid-binding peptide has been transplanted, and a scaffold protein having a structure into which a cardiac cell type surface antigen-binding peptide has been transplanted.

5. The gene transfer agent according to claim 1, wherein the molecule is a scaffold protein having a structure in which an AAV capsid-binding peptide and a cardiac cell type surface antigen-binding peptide are transplanted.

6. The gene transfer agent according to claim 1, wherein the molecule is an anticardiac cell surface antigen antibody having at least one loop structure, wherein all or part of the amino acid sequence constituting at least one loop structure is replaced with all or part of an AAV capsid-binding peptide sequence, or all or part of an AAV capsid-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure, and the anticardiac cell surface antigen antibody has an amino acid sequence into which all or part of an AAV capsid-binding peptide sequence is incorporated.

7. The gene transfer agent according to claim 1, wherein the molecule is a scaffold protein having at least one loop structure, wherein all or part of the amino acid sequence constituting at least one loop structure is replaced with all or part of an AAV capsid-binding peptide sequence, or all or part of an AAV capsid-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure, and the AAV capsid-binding scaffold protein having an amino acid sequence into which all or part of an AAV capsid-binding peptide sequence is incorporated, and an anti-cardiac constituent cell type surface antigen antibody is linked to it.

8. The molecule is a protein formed by linking two components: an AAV capsid-binding scaffold protein having an amino acid sequence incorporating an AAV capsid-binding peptide sequence, wherein in a scaffold protein having at least one loop structure, all or part of the amino acid sequence constituting at least one loop structure is replaced with all or part of an AAV capsid-binding peptide sequence, or in which all or part of an AAV capsid-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure; and a cardiac cell species surface antigen-binding scaffold protein having an amino acid sequence incorporating all or part of an AAV capsid-binding peptide sequence, wherein in a scaffold protein having at least one loop structure, all or part of the amino acid sequence constituting at least one loop structure is replaced with all or part of a cardiac cell species surface antigen-binding peptide sequence, or in which all or part of a cardiac cell species surface antigen-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure; The gene transfer agent according to claim 1.

9. The gene transfer agent according to claim 1, wherein the molecule is a scaffold protein having two or more loop structures, wherein all or part of the amino acid sequence constituting at least one loop structure is replaced with all or part of an AAV capsid-binding peptide sequence, or all or part of an AAV capsid-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure, and all or part of the amino acid sequence constituting at least one loop structure different from the above loop structure is replaced with all or part of a cardiac cell species surface antigen-binding peptide sequence, or all or part of a cardiac cell species surface antigen-binding peptide sequence is inserted into the amino acid sequence constituting at least one loop structure different from the above loop structure, and the protein has an amino acid sequence into which all or part of the AAV capsid-binding peptide sequence and the cardiac cell species surface antigen-binding peptide sequence are incorporated.

10. The gene transfer agent according to claim 1, wherein the molecule is a protein formed by linking a biotinylated AAV capsid-binding peptide and a biotinylated cardiac cell type surface antigen-binding peptide via a protein having two or more biotin-binding sites.

11. The gene transfer agent according to claim 1, wherein the molecule is a protein formed by linking a biotinylated AAV capsid-binding peptide and a biotinylated anticardiac cell type surface antigen antibody via a protein having two or more biotin-binding sites.

12. A gene transdermal agent according to any one of claims 1 to 11, wherein the cardiac cell type surface antigen is one or more antigens selected from CD29, CD46, CD47, CD49f, CD58, CD59, CD98, CD147, CD321, CD340, SSEA-4, CD172a, ITGA7 (integrin α7), and VCAM1, which are expressed on cardiac cell types.

13. The gene transfer agent according to claim 12, wherein the cardiac cell type surface antigen is one or more antigens selected from CD49f, CD340, and CD172a that are expressed on cardiac cell types.

14. The gene transfer agent according to any one of claims 1 to 11, wherein the cardiac cell type surface antigen-binding peptide is IB8.

15. The gene transfer agent according to any one of claims 1 to 11, wherein the anticardiac constituent cell type surface antigen antibody is trastuzumab, anzurstwald, or pertuzumab.

16. The AAV capsid-binding peptide is a core motif sequence (I) represented by: (1) the amino acid sequence represented by Tyr-Thr-Tyr-Arg-Thr-Thr-Ile-Pro-His-Glu-Tyr-Leu-Ala (SEQ ID NO: 1), or (2) an amino acid sequence that has 80% or more homology to the amino acid sequence represented by SEQ ID NO: 1 and is AAV9 capsid-binding; or, a core motif sequence (II) represented by: Formula: Xaa1-Ser-Lys-Ser-Xaa2-Xaa3-Leu-Ala-Gln-Xaa4 (wherein Xaa1 represents Phe or Tyr, Xaa2 represents Leu, Val, Ile or Phe, Xaa3 represents any amino acid, and The gene transfer agent according to any one of claims 1 to 11, wherein Xaa4 is an amino acid sequence represented by (Gln, Asp, Glu, or Asn) or a sequence containing such a sequence.

17. The gene transfer agent according to claim 16, wherein the core motif sequence (II) is (1) an amino acid sequence represented by Tyr-Ser-Lys-Ser-Val-Cys-Leu-Ala-Gln-Asp (SEQ ID NO: 2), or an amino acid sequence having 70% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and being AAV9 capsid-binding, or (2) an amino acid sequence represented by Phe-Ser-Lys-Ser-Leu-His-Leu-Ala-Gln-Glu (SEQ ID NO: 3), or an amino acid sequence having 70% or more homology with the amino acid sequence represented by SEQ ID NO: 3 and being AAV9 capsid-binding.

18. The gene transfer agent according to any one of claims 1 to 11, wherein the AAV capsid-binding peptide is the whole sequence or a partial sequence constituting a cyclic peptide represented by the following formula (Ia), formula (Ib), or formula (Ic). (In the formula, Variable region 1 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 1 below, and S represents S derived from the thiol group of Cys.) (In the formula, Variable region 1 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 2 below, Variable region 2 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 3 below, and S represents S derived from the thiol group of Cys.) (In the formula, Variable region 1 represents an AAV9-binding amino acid sequence selected from the amino acid sequences listed in Table 4 below, and S represents S derived from the thiol group of Cys.) 19. A gene transfer agent according to any one of claims 1 to 11, which reduces the nonspecific infectivity of AAV by binding to AAV via module (A), while simultaneously increasing the specific infectivity to cardiac constituent cell type surface antigens that are targets of module (B).

20. A gene transfer agent according to any one of claims 1 to 11, further comprising AAV having a wild-type capsid.

21. The gene transfer agent according to any one of claims 1 to 11, wherein the molecule is mixed with AAV having a wild-type capsid to form a complex of the two.

22. The gene transfer agent according to claim 20 or 21, wherein an exogenous gene is loaded onto the AAV.

23. The gene transfer agent according to claim 22 for infecting cardiac constituent cell types with AAV.

24. The gene delivery agent according to claim 23 for introducing an exogenous gene into cardiac cell types.

25. The gene transfer agent according to any one of claims 1 to 11, wherein the AAV is AAV9.

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