Modified aav particles, method for producing same, related medicine, and production of same

Abstract

The present disclosure provides a method for producing recombinant adeno-associated virus particles having a ligand on surfaces thereof, virus-like particles, host cells, and virus virions (VP) having low hepatotoxicity, the method comprising: (A) a step for introducing, into host cells via gene delivery, a nucleic acid molecule containing a nucleotide sequence that encodes a desired protein, and one or more types of nucleic acid molecules containing a nucleotide sequence that allows the expression of VP1, VP2, VP3, and VP3 modified with the ligand upon gene delivery; and (B) a step for subjecting the host cells to conditions that allow the generation of the recombinant adeno-associated virus particles.

Description

Modified AAV particles, their production method, related medicines and their production

[0001] The present disclosure relates to improved rAAV particles and methods for producing same.

[0002] Adeno-associated viruses (AAVs) are linear, single-stranded DNA viruses that belong to the Parvoviridae family and are among the smallest naturally occurring viruses. Their viral genome is approximately 4.7 kb. They are non-enveloped and form icosahedral particles with a diameter of approximately 22 nm.

[0003] When wild-type AAV infects human host cells alone, the viral genome is site-specifically integrated into chromosome 19 via inverted terminal repeats (ITRs) present at both ends of the viral genome. Genes of the viral genome integrated into the host cell genome are rarely expressed, but when the cells are infected with a helper virus, AAV is excised from the host genome and replication of the infectious virus begins. When the helper virus is an adenovirus, the genes responsible for the helper function are E1A, E1B, E2A, VA1, and E4. Here, when the host cells are HEK293 cells, which are human fetal kidney tissue-derived cells transformed with adenovirus E1A and E1B, the E1A and E1B genes are naturally expressed in the host cells.

[0004] The AAV genome also contains two genes, Rep and Cap. The REP proteins (Rep78, Rep68, Rep52, and Rep40) produced by the Rep gene are essential for capsid formation and mediate the integration of the viral genome into chromosomes. The Cap gene is responsible for the production of three capsid proteins (VP1, VP2, and VP3).

[0005] In the wild-type AAV genome, ITRs are present at both ends, and the Rep gene and Cap gene are present between the ITRs. Recombinant AAV particles (rAAV particles) are AAV particles in which the region containing the Rep gene and Cap gene has been replaced with a gene encoding a foreign protein, and the resulting particle is encapsulated in a capsid.

[0006] Three types of plasmids are generally used to produce rAAV particles: a plasmid containing a gene encoding a foreign protein flanked by ITRs (Patent Document 1), a plasmid containing a gene encoding an REP protein and a gene encoding a CAP protein (Patent Document 2), and a plasmid containing genes encoding adenovirus-derived E2A, E4, and VA1 RNAs (Patent Document 3). To produce rAAV particles encapsulating a gene of interest in transfected cells, these three types of plasmids are introduced into host cells such as HEK293 cells. rAAV particles are synthesized in host cells into which the three types of plasmids have been introduced.

[0007] AAV has many variants with different serotypes, called serotypes, and 11 of these serotypes are known to infect human cells. Each AAV serotype has a specific organ tropism. For example, AAV9 is known to infect the central nervous system (CNS), heart, lung, liver, and skeletal muscle.

[0008] Although AAV infects various human organs, it is believed to be non-pathogenic. Therefore, attempts have been made to use AAV as a material for producing recombinant viruses for gene therapy. Since each AAV serotype has its own tropism for the infected organ, attempts have been made to use different serotypes depending on the organ to which a gene is to be introduced by gene therapy. Since AAV9 has tropism for the central nervous system (CNS), heart, lung, liver, and skeletal muscle, attempts have also been made to use AAV9 as a material for producing recombinant viruses for introducing genes into these organs and tissues.

[0009] In gene therapy using AAV, a recombinant AAV genome (rAAV genome) in which a part of the wild-type AAV genome has been replaced with a foreign gene is administered to a patient in the form of a recombinant AAV particle (rAAV particle) encapsulated in capsid protein. The rAAV particle is produced by carrying out in vitro the process in which the wild-type AAV genome is encapsulated in capsid protein to form wild-type AAV.

[0010] By using different serotypes depending on the target organ in gene therapy, it is expected that more genes can be introduced into the organ. However, the organ tropism of the infectivity of each serotype is not high. As a means of increasing the infection tropism of rAAV particles to a specific organ, various methods for modifying the capsid of rAAV particles have been considered. For example, various attempts have been made to modify the capsid of rAAV particles so that they have affinity for proteins that are specifically present on the surface of target cells, thereby allowing the rAAV particles to preferentially bind to the target cells and increasing the tropism of the rAAV particles.

[0011] Well-known methods for enhancing the organ tropism of rAAV particles include direct recombinant targeting and indirect recombinant targeting. Direct recombinant targeting, for example, involves using rAAV particles containing capsids modified to have affinity for proteins expressed on the surface of target cells. Indirect recombinant targeting involves producing rAAV particles containing capsids with scaffolds capable of binding to other molecules, and then binding a substance with affinity for proteins expressed on the surface of target cells via the scaffold to impart targeting to the rAAV particles. Many reports have been published on these direct and indirect recombinant targeting methods (Patent Documents 4 and 5).

[0012] International Publication No. WO 95 / 34670 International Publication No. WO 97 / 06272 International Publication No. WO 97 / 17458 International Publication No. WO 2019 / 006046 International Publication No. WO 2019 / 006043

[0013] Gigout L. et al. , Mol Ther. 11:856-65 (2005) Stachler MD. et al. , Mol Ther. 16:1467-73 (2008) Girod A. et al. , Nat Med. 5:1052-6 (1999) Grifman M. et al. , Mol Ther. 3:964-75 (2001) Nicklin SA. et al. , Mol Ther. 4:174-81 (2001) Jackson, CB. et al. , Mol Ther. 19:496-506 (2020)

[0014]

[0003] As a result of extensive research, the present inventors have found that rAAV particles containing capsids comprising VP1, VP2, and VP3, as well as fusion proteins of these with another protein (A), and in which the ratio of the total number of VP1, VP2, and VP3 molecules to the total number of fusion protein molecules in the capsid is specific, can be produced in particularly good yield as rAAV particles and have high infectivity for target cells. Furthermore, the present inventors have found that a similar effect can be achieved by appropriately incorporating VP3 modified with a ligand. That is, the present disclosure includes the following. [Production Method] [Item 1] A method for producing recombinant adeno-associated virus particles having a ligand on their surface, comprising: (A) a step of transfecting a host cell with a nucleic acid molecule comprising a VP nucleic acid sequence that, upon transfection, enables expression of VP1, VP2, VP3, and VP3 modified with the ligand, and a nucleic acid molecule comprising a nucleic acid sequence encoding a desired protein; and (B) a step of subjecting the host cell to conditions under which the recombinant adeno-associated virus particles are produced. [Item 1A] The method according to any one of the above items, wherein one or more types of nucleic acid molecule are present, and / or one or more types of ligand (which may be the same or different) are present. [Item 2] A method for producing recombinant adeno-associated virus particles having a ligand on their surface, comprising the steps of: (A) introducing into a host cell (1) a first nucleic acid sequence that, when introduced, enables expression of VP1, VP2, and VP3; (2) a second nucleic acid sequence that, when introduced, enables expression of ligand-modified VP3; and (3) a nucleic acid sequence encoding a desired protein; and (B) subjecting the host cell to conditions under which the recombinant adeno-associated virus particles are produced. [Item 2A] The method of any one of the above items, wherein the ligand is present in one or more forms (which may be the same or different types). [Item 3] The method of any one of the above items, wherein the host cell contains elements necessary for producing adeno-associated virus particles. [Item 4] The method of any one of the above items, wherein the necessary elements include a nucleic acid sequence encoding a Rep protein.[Item 5] The method of any one of the above items, wherein the nucleic acid sequence encoding the Rep protein and the VP nucleic acid sequence are located between at least two inverted terminal repeats (ITRs), either individually or together. [Item 6] The method of any one of the above items, wherein the necessary elements further comprise a nucleic acid sequence encoding a protein responsible for helper function. [Item 7] The method of any one of the above items, wherein the proteins responsible for helper function include at least one, two, three, four, or all five selected from the group consisting of E1A, E1B, E2A, VA1, and E4. [Item 8] The method of any one of the above items, wherein the proteins responsible for helper function, when there are two or more, are located between at least two inverted terminal repeats (ITRs), either individually or together. [Item 9] The method of any one of the above items, wherein the nucleic acid sequence encoding the desired protein is located between at least two inverted terminal repeats (ITRs). [Item 10] The method according to any one of the above items, wherein the desired protein includes a therapeutic protein, a protein for genome editing, a protein for testing, etc. [Item 11] The method according to any one of the above items, wherein the nucleic acid sequence encoding the desired protein is incorporated into the recombinant adeno-associated virus particle under conditions for producing the particle so that it is subsequently incorporated into the particle in an expressible state. [Item 12] The method according to any one of the above items, wherein the first nucleic acid sequence and the second nucleic acid sequence are introduced as separate nucleic acid molecules, and are introduced into the host cell such that the ratio of the number of molecules of the second nucleic acid sequence to the sum of the number of molecules of the first nucleic acid sequence to be introduced and the number of molecules of the second nucleic acid sequence to be introduced is 1 to 50%. [Item 13] The method according to any one of the preceding items, wherein the first nucleic acid sequence and the second nucleic acid sequence are introduced as separate nucleic acid molecules, and are introduced into the host cell so that the ratio of the number of molecules of the second nucleic acid sequence to the sum of the number of molecules of the first nucleic acid sequence to be introduced and the number of molecules of the second nucleic acid sequence to be introduced is 3 to 30%.[Item 14] The method of any one of the above items, wherein the first nucleic acid sequence and the second nucleic acid sequence are introduced as separate nucleic acid molecules and are transfected into the host cell so that the ratio of the number of molecules of the second nucleic acid sequence to the sum of the number of molecules of the first nucleic acid sequence to be introduced is 5% to 20%. [Item 15] The method of any one of the above items, wherein the ratio of ligand-modified VP3 mRNA to the sum of the number of molecules of VP3 mRNA and the number of molecules of the ligand-modified VP3 mRNA in the host cell under conditions for producing the recombinant adeno-associated virus particles is 0.5% to 20%. [Item 16] The method of any one of the above items, wherein the ratio of ligand-modified VP3 mRNA to the sum of the number of molecules of VP3 mRNA and the number of molecules of the ligand-modified VP3 mRNA in the host cell under conditions for producing the recombinant adeno-associated virus particles is 1% to 15%. [Item 17] The method according to any one of the above items, wherein the ligand is a polypeptide having a length of 41 amino acids or more. [Item 18] The method according to any one of the above items, wherein the ligand is a polypeptide having a size of 4.5 kDa or more. [Item 19] The method according to any one of the above items, wherein the ligand is a VHH. [Item 20] The method according to any one of the above items, wherein the ligand has specific affinity for a protein present on the surface of vascular endothelial cells. [Item 21] The method according to any one of the above items, wherein the host cell does not express ligand-modified VP1 and / or ligand-modified VP2. [Item 22] The method according to any one of the above items, wherein any one, two, three, or all of the VP1, VP2, VP3, and VP3 modified with the ligand are mutated VPs in which one or more amino acid residues of Loop-4 and / or Loop-5 and / or Loop-8 of VP are absent and / or substituted with one or more other amino acid residues.[Item 23] The method of any one of the above items, wherein the amino acid residues in VP1 represent serotype 9 adeno-associated virus amino acid residues, or in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), the corresponding adeno-associated virus amino acid residues of other serotypes when aligned with serotype 9 adeno-associated virus amino acids. [Item 24] A host cell that produces recombinant adeno-associated virus particles having VP3 modified with a ligand on its surface. [Item 24A] The host cell of any one of the above items, wherein the ligand is present in one or more forms (which may be the same or different types). [Item 25] The cell of any one of the above items, wherein the host cell contains elements necessary for producing adeno-associated virus particles. [Item 26] The host cell of any one of the preceding items, wherein the necessary elements include a nucleic acid sequence encoding a Rep protein. [Item 27] ​​The host cell of any one of the preceding items, wherein the nucleic acid sequence encoding the Rep protein and the VP nucleic acid sequence, individually or together, are located between at least two inverted terminal repeats (ITRs). [Item 28] The host cell of any one of the preceding items, wherein the necessary elements further include a nucleic acid sequence encoding a protein responsible for helper function. [Item 29] The host cell of any one of the preceding items, wherein the proteins responsible for helper function include at least one, two, three, four, or all five selected from the group consisting of E1A, E1B, E2A, VA1, and E4. [Item 30] The host cell of any one of the preceding items, wherein the proteins responsible for helper function, when two or more, individually or together, are located between at least two inverted terminal repeats (ITRs). [Item 31] The host cell according to any one of the preceding items, wherein the nucleic acid sequence encoding the desired protein is located between at least two inverted terminal repeats (ITRs). [Item 32] The host cell according to any one of the preceding items, wherein the desired protein includes a therapeutic protein, a protein for genome editing, a protein for testing, etc.[Item 33] The host cell of any one of the preceding items, comprising: (1) a first nucleic acid sequence that, when expressed, enables the expression of VP1, VP2, and VP3; (2) a second nucleic acid sequence that, when expressed, enables the expression of VP3 modified with the ligand; and (3) a nucleic acid sequence encoding a desired protein. [Item 34] The host cell of any one of the preceding items, wherein the nucleic acid sequence encoding the desired protein is incorporated into the recombinant adeno-associated virus particle under conditions for the production of the particle so that it is subsequently incorporated into the particle in an expressible state. [Item 35] The host cell of any one of the preceding items, wherein the first nucleic acid sequence and the second nucleic acid sequence are exogenous and have been transfected so that the ratio of the number of molecules of the second nucleic acid sequence to the sum of the number of molecules of the first nucleic acid sequence and the number of molecules of the second nucleic acid sequence is 1% to 50%. [Item 36] The host cell of any one of the preceding items, wherein the first nucleic acid sequence and the second nucleic acid sequence are exogenous and have been transfected so that the proportion of the number of molecules of the second nucleic acid molecule in the sum of the number of molecules of the first nucleic acid sequence and the number of molecules of the second nucleic acid sequence is 3% to 30%. [Item 37] The host cell of any one of the preceding items, wherein the first nucleic acid sequence and the second nucleic acid sequence are exogenous and have been transfected so that the proportion of the number of molecules of the second nucleic acid molecule in the sum of the number of molecules of the first nucleic acid sequence and the number of molecules of the second nucleic acid sequence is 5% to 20%. [Item 38] The host cell of any one of items 2 to 2A2, wherein the proportion of ligand-modified VP3 mRNA in the sum of the number of molecules of VP3 mRNA and the number of molecules of ligand-modified VP3 mRNA is 0.5% to 20%. [Item 39] The host cell of any one of the above items, wherein the proportion of ligand-modified VP3 mRNA in the sum of the number of VP3 mRNA molecules and the number of ligand-modified VP3 mRNA molecules is 1% to 15%. [Item 40] The host cell of any one of the above items, wherein the ligand is a polypeptide having a length of 41 amino acids or more. [Item 41] The host cell of any one of the above items, wherein the ligand is a polypeptide having a size of 4.5 kDa or more. [Item 42] The host cell of any one of the above items, wherein the ligand is a VHH.[Item 43] The host cell of any one of the preceding items, wherein the ligand has specific affinity for a protein present on the surface of a vascular endothelial cell. [Item 44] The host cell of any one of the preceding items, which does not express ligand-modified VP1 and ligand-modified VP2. [Item 45] The host cell of any one of the preceding items, which is substantially free of ligand-modified VP1 and / or ligand-modified VP2. [Item 46] The host cell of any one of the preceding items, wherein the number of molecules of the ligand per recombinant adeno-associated virus particle is 1 to 50. [Item 47] The host cell of any one of the preceding items, wherein the number of molecules of the ligand per recombinant adeno-associated virus particle is 1 to 30. [Item 48] The host cell of any one of the preceding items, wherein the number of molecules of the ligand per recombinant adeno-associated virus particle is 1 to 20. [Item 49] The host cell according to any one of the preceding items, wherein the number of molecules of the ligand per recombinant adeno-associated virus particle is 1 to 16. [Item 50] The host cell according to any one of the preceding items, wherein any one, two, three, or all of VP1, VP2, VP3, and ligand-modified VP3 are each independently mutated VPs that do not have and / or have substituted with one or more amino acid residues in Loop-4 and / or Loop-5 and / or Loop-8 of VP. [Item 51] The host cell according to any one of the preceding items, wherein the amino acid residues in VP1 represent serotype 9 adeno-associated virus amino acid residues, or, in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), the corresponding adeno-associated virus amino acid residues of other serotypes when aligned with serotype 9 adeno-associated virus amino acids. [Item 52] A recombinant adeno-associated virus (rAAV) particle having VP3 modified with a ligand on its surface. [Item 53] A recombinant adeno-associated virus (rAAV) vector, preferably a therapeutic rAAV vector, having VP3 modified with a ligand on its surface.[Item 53A] The rAAV particle or rAAV vector according to any one of the preceding items, wherein the ligand is of one or more types (which may be the same or different types). [Item 54] The rAAV particle or rAAV vector according to any one of the preceding items, wherein the adeno-associated virus particle comprises elements necessary for constituting the particle. [Item 55] The rAAV particle or rAAV vector according to any one of the preceding items, wherein the adeno-associated virus particle comprises elements necessary for constituting the particle. Item 56: The rAAV particle or rAAV vector of any one of the preceding items, further comprising a nucleic acid sequence encoding a desired protein. [Item 57] The rAAV particle or rAAV vector of any one of the preceding items, wherein the ligand is a polypeptide having a length of 41 amino acids or more. [Item 58] The rAAV particle or rAAV vector of any one of the preceding items, wherein the ligand is a polypeptide having a size of 4.5 kDa or more. [Item 59] The rAAV particle or rAAV vector of any one of the preceding items, wherein the ligand is a VHH. [Item 60] The rAAV particle or rAAV vector of any one of the preceding items, wherein the ligand has specific affinity for a protein present on the surface of vascular endothelial cells. [Item 61] The rAAV particle or rAAV vector of any one of the preceding items, which is substantially free of ligand-modified VP1 and ligand-modified VP2. [Item 62] Item 63: The rAAV particle or rAAV vector of any one of the preceding items, configured so that the number of molecules of the ligand per rAAV particle is 1 to 30. Item 64: The rAAV particle or rAAV vector of any one of the preceding items, configured so that the number of molecules of the ligand per rAAV particle is 1 to 20. Item 65: The rAAV particle or rAAV vector of any one of the preceding items, configured so that the number of molecules of the ligand per recombinant adeno-associated virus particle is 1 to 16.[Item 66] The rAAV particle or rAAV vector according to any one of the preceding items, wherein any one, two, three, or all of VP1, VP2, VP3, and ligand-modified VP3 are mutated VPs in which one or more amino acid residues of Loop-4 and / or Loop-5 and / or Loop-8 of VP are absent and / or substituted with one or more other amino acid residues. [Item 67] The rAAV particle according to any one of the preceding items, wherein the amino acid residues in the VP1 represent serotype 9 adeno-associated virus amino acid residues, or, in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), represent the corresponding adeno-associated virus amino acid residues of other serotypes when aligned with serotype 9 adeno-associated virus amino acids. [Item 68] The rAAV particle or rAAV vector according to any one of the preceding items, wherein the ligand has a first linker on the N-terminus, or a second linker on the C-terminus, or a first linker on the N-terminus and a second linker on the C-terminus. [Item 69] The rAAV particle or rAAV vector of any one of the preceding items, wherein the ligand has specific affinity for another molecule. [Item 70] The rAAV particle or rAAV vector of any one of the preceding items, having two or more types of ligands on its surface. [Item 71] The rAAV particle or rAAV vector of any one of the preceding items, wherein the ligand comprises an anti-transferrin receptor VHH.[Item 72] The ligand comprises a first linker having an amino acid sequence set forth in any one or more of SEQ ID NOS: 79-91, an amino acid sequence set forth in any one of SEQ ID NOS: 5-13, or the following combinations: (1) CDR1 having an amino acid sequence set forth in SEQ ID NOS: 14, 15, 20, 21, 26, 27, 32, 33, 40, 41, 46, or 47; (2) CDR2 having an amino acid sequence set forth in SEQ ID NOS: 16, 17, 22, 23, 28, 29, 34, 35, 38, 39, 42, 43, 48, or 49; and (3) CDR3 having an amino acid sequence set forth in SEQ ID NOS: 18, 19, 24, 25, 30, 31, 36, 37, 44, 45, 50, or 51, or preferably (A1) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 14 or 15, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 16 or 17, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18 or 19; (A2) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 20 or 21, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 22 or 23, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 24 or 25; (A3) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26 or 27, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 28 or 29, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 30 or 31; (A4) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 32 or 33, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 34 or 35, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 36 or 37; (A5) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26 or 27, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 28 or 29, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 30 or 31; (A6) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26 or 27, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 38 or 39, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 30 or 31;(A7) the rAAV particle or rAAV vector according to any one of the preceding items, comprising: (A8) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40 or 41, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42 or 43, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44 or 45; (A9) an anti-transferrin receptor VHH comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 46 or 47, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 48 or 49, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50 or 51, and a second linker having the amino acid sequence set forth in any one or more of SEQ ID NOs: 79 to 91. [Item 73] A pharmaceutical composition comprising the rAAV particle or rAAV vector according to any one of the preceding items. [Item 73A] A method for preventing or treating a disease, disorder, or symptom prevented or treated by a desired protein encoded by a nucleic acid molecule contained in an rAAV particle or rAAV vector according to any one of the preceding items, comprising administering an effective amount of the rAAV particle or rAAV vector according to any one of the preceding items to a subject in need thereof. [Item 73B] The rAAV particle or rAAV vector according to any one of the preceding items for use as a medicament. [Item 73C] Use of the rAAV particle or rAAV vector according to any one of the preceding items for the manufacture of a medicament. [Item 74] A composition comprising rAAV particles having VP3 modified with a ligand on their surface. [Item 75] A composition comprising an rAAV vector having VP3 modified with a ligand on their surface. [Item 75A] The composition according to any one of the preceding items, wherein one or more ligands (which may be the same or different types) are present. [Item 76] The composition according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 1 to 50%. [Item 77] The composition according to any one of the preceding items, wherein the actual modification rate of the modification with the ligand is 3 to 30%.[Item 78] The composition according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 4 to 25%. [Item 79] The composition according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 4 to 10%. [Item 80] The composition according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 1 (number / particle) or more. [Item 81] The composition according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 1 to 50 (number / particle). [Item 82] The composition according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 1 to 22 (number / particle) or more. [Item 83] The composition according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 2 to 16 (number / particle) or more. [Item 84] The composition according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 2 to 13 (number / particle) or more. [Item 85] The composition according to any one of the preceding items, wherein the actual modification rate is the ratio of VLPs (preferably rAAV particles) having a ligand-modified VP3 on their surface to the total of VLPs (preferably rAAV particles) having the ligand-modified VP3 on their surface and VLPs (preferably rAAV particles) having unmodified VP3. [Item 86] A virus-like particle (VLP) comprising a capsid having a VP3 modified with a ligand on its surface. [Item 86A] The VLP according to any one of the preceding items, wherein one or more ligands (which may be the same or different types) are present. [Item 87] The VLP according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 1 to 50%. [Item 88] The VLP according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 3 to 30%. [Item 89] The VLP according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 4 to 25%. [Item 90] The VLP according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 4 to 10%. [Item 91] The VLP according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 1 (number / particle) or more. [Item 92] The VLP according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 1 to 50 (number / particle).[Item 93] The VLP according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 1 to 22 (number / particle) or more. [Item 94] The VLP according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 2 to 16 (number / particle) or more. [Item 95] The VLP according to any one of the preceding items, wherein the actual modification rate of modification with the ligand is 2 to 13 (number / particle) or more. [Item 96] A pharmaceutical composition comprising the VLP of any one of the preceding items, comprising a nucleic acid sequence encoding a therapeutic or prophylactic protein. [Item 96A] A method for preventing or treating a disease, disorder, or symptom prevented or treated by a desired protein encoded by a nucleic acid molecule contained in the composition or VLP, comprising administering an effective amount of the composition or VLP of any one of the preceding items to a subject in need thereof. [Item 96B] The composition or VLP of any one of the preceding items for use as a medicament. [Item 96C] Use of the composition or VLP according to any one of the preceding items for the manufacture of a medicine. [Item 97] A method for producing a recombinant adeno-associated virus particle, comprising the step of incorporating into the adeno-associated virus particle a mutated VP in which any one, two, or all of VP1, VP2, and VP3 lacks and / or has substituted with one or more other amino acid residues one or more amino acid residues in Loop-4 and / or Loop-5 and / or Loop-8 of the VP, respectively. [Item 98] The method for production according to any one of the preceding items, wherein the amino acid residues in VP1 represent serotype 9 adeno-associated virus amino acid residues, or in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), the corresponding adeno-associated virus amino acid residues of other serotypes when aligned with serotype 9 adeno-associated virus amino acids.[Item 99] A host cell having a group of genes necessary for producing recombinant adeno-associated virus particles, which produces recombinant adeno-associated virus particles in which any one, two, or all of VP1, VP2, and VP3 are mutated VPs in which one or more amino acid residues of Loop-4 and / or Loop-5 and / or Loop-8 of VP are absent and / or substituted with one or more other amino acid residues. [Item 100] The host cell according to any one of the preceding items, wherein the amino acid residues in VP1 represent serotype 9 adeno-associated virus amino acid residues, or, in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), the corresponding adeno-associated virus amino acid residues of other serotypes when aligned with serotype 9 adeno-associated virus amino acids. [Item 101] A recombinant adeno-associated virus (rAAV) particle comprising a mutated VP in which any one, two, or all of VP1, VP2, and VP3 are absent and / or substituted with one or more other amino acid residues in Loop-4 and / or Loop-5 and / or Loop-8 of the VP, respectively. [Item 102] The rAAV particle according to any one of the preceding items, wherein the amino acid residues in VP1 represent serotype 9 adeno-associated virus amino acid residues, or, in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), the corresponding adeno-associated virus amino acid residues of other serotypes when aligned with serotype 9 adeno-associated virus amino acids. [Item 103] A vector for producing a recombinant adeno-associated virus, which is configured to produce recombinant adeno-associated virus particles containing a mutated VP in which any one, two, or all of VP1, VP2, and VP3 are missing and / or have one or more amino acid residues in Loop-4 and / or Loop-5 and / or Loop-8 of the VP substituted with one or more other amino acid residues, preferably a nucleic acid molecule containing a nucleic acid sequence encoding the mutated VP.[Item 104] The vector for producing a recombinant adeno-associated virus according to any one of the preceding items, wherein the amino acid residues in VP1 represent serotype 9 adeno-associated virus amino acid residues, or, in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), represent the corresponding adeno-associated virus amino acid residues of other serotypes when aligned with serotype 9 adeno-associated virus amino acids. [Item 105] A mutated VP in which one or more amino acid residues of Loop-4 and / or Loop-5 and / or Loop-8 of VP are absent and / or substituted with one or more other amino acid residues. [Item 106] The mutated VP according to any one of the above items, wherein the amino acid residues in the VP1 represent serotype 9 adeno-associated virus amino acid residues, or in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), represent the corresponding adeno-associated virus amino acid residues of other serotypes when aligned with serotype 9 adeno-associated virus amino acids. [Item 107] A virus-like particle (VLP) comprising a mutated VP in which one or more amino acid residues in Loop-4 and / or Loop-5 and / or Loop-8 of the VP are absent and / or substituted with one or more other amino acid residues. [Item 108] The VLP according to any one of the preceding items, wherein the amino acid residues in VP1 represent serotype 9 adeno-associated virus amino acid residues, or, in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), the corresponding adeno-associated virus amino acid residues of other serotypes when aligned with serotype 9 adeno-associated virus amino acids. [Item 109] The mutated VP according to any one of the preceding items, wherein the VP is VP3. [Item 110] The mutated VP according to any one of the preceding items, wherein the mutation is contained in Loop-8.[Item 111] The mutated VP according to any one of the preceding items, which does not have any one of the amino acid residues at positions 589 and 590, 590 and 591, 591 and 592, and 594 and 595 of VP1, or any combination of amino acid residues corresponding thereto. [Item 112] The mutated VP according to any one of the preceding items, which does not have any one of the amino acid residues at positions 496, 497, 498, 499, 502, 504, 591, 592, 593, 594, and 595 of VP1, or any one or more amino acid residues corresponding thereto. [Item 113] The rAAV particle according to any one of the preceding items, which does not have one or more amino acid residues at positions 496, 497, 498, 499, 502, 504, 591, 592, 593, 594, and 595 of VP1, or positions corresponding thereto. [Item 114] The rAAV particle according to any one of the preceding items, which does not have any amino acid residues at any one of the combinations of positions 589 and 590, 590 and 591, 591 and 592, and 594 and 595 of VP1, or positions corresponding thereto. The present disclosure also provides the following. 1. 1. A method for producing recombinant adeno-associated virus particles (rAAV particles) whose capsid contains a fusion protein of a protein (CAP protein) encoded in the Cap region of the adeno-associated virus genome (AAV genome) and another protein (A), the method comprising introducing into a host cell a first nucleic acid molecule encoding the CAP protein and a second nucleic acid molecule encoding the fusion protein of the CAP protein and another protein (A). 2. The method according to item 1 above, wherein the first nucleic acid molecule is capable of expressing VP1, VP2, and VP3 when introduced into a host cell, and the second nucleic acid molecule is capable of expressing at least one of the fusion protein of VP1 and another protein (A), the fusion protein of VP2 and another protein (A), and the fusion protein of VP2 and another protein (A) when introduced into a host cell.3. The production method according to 1 or 2 above, wherein the other protein (A) comprises a first linker, a protein having a function, and a second linker in this order from the N-terminus, or a protein having a function and a second linker in this order from the N-terminus. The method for producing a fusion protein according to any one of items 1 to 3 above, which is selected from the group consisting of the following items (1) to (8): (1) The fusion protein encoded by the second nucleic acid molecule is such that the other protein (A) consists of 100 or more amino acid residues, the CAP protein is an AAV8 CAP protein, and the other protein (A) is added to a position corresponding to the C-terminal side of the 450 to 465, 584 to 602, 455, 457, 462, 501, 588, or 599 amino acid residues from the N-terminus of VP1, or the amino acid sequence corresponding to the 456 to 462 or 455 to 460 amino acid sequence from the N-terminus of VP1 is substituted for the amino acid sequence of the other protein (A); (2) In the fusion protein encoded by the second nucleic acid molecule, the other protein (A) consists of less than 100 amino acid residues, the CAP protein is an AAV8 CAP protein, and the other protein (A) is added to a position corresponding to the C-terminal side of the 450-465, 584-602, 707-717, or 455, 457, 462, 501, 588, 599, or 707 amino acid residues from the N-terminus of the VP1, or the amino acid sequence corresponding to the 456-462 or 455-460 amino acid sequence from the N-terminus of the VP1 is replaced with the amino acid sequence of the other protein (A); (3) The fusion protein encoded by the second nucleic acid molecule is such that the other protein (A) consists of 100 or more amino acid residues, the CAP protein is a CAP protein other than AAV8, and the other protein (A) is added at a position corresponding to the C-terminal side of amino acid residues corresponding to amino acid residues 450 to 465, 584 to 602, 455, 457, 462, 501, 588, or 599 from the N-terminus of VP1 of AAV8, or the amino acid sequence corresponding to the amino acid sequence of amino acids 456 to 462 or 455 to 460 from the N-terminus of VP1 of AAV8 is substituted with the amino acid sequence of the other protein (A);(4) The fusion protein encoded by the second nucleic acid molecule is one in which the other protein (A) consists of less than 100 amino acid residues, the CAP protein is a CAP protein other than AAV8, and the other protein (A) is added at a position corresponding to the C-terminal side of amino acid residues corresponding to amino acid residues at positions 450 to 465, 584 to 602, 707 to 717, or 455, 457, 462, 501, 588, 599, or 707 from the N-terminus of VP1 of AAV8, or in which the amino acid sequence corresponding to amino acid sequence at positions 456 to 462 or 455 to 460 from the N-terminus of VP1 of AAV8 is replaced with the amino acid sequence of the other protein (A); (5) The fusion protein encoded by the second nucleic acid molecule is one according to (1) or (2) above, further comprising deletion of one or more amino acid residues corresponding to amino acid residues at positions 582 to 604 from the N-terminus of VP1; (6) The fusion protein encoded by the second nucleic acid molecule is the one described in (1) or (2) above, further having one or more amino acid residues deleted corresponding to the 591st, 592nd, 593rd, or 593rd amino acid residue from the N-terminus of VP1; (7) The fusion protein encoded by the second nucleic acid molecule is the one described in (3) or (4) above, further having one or more amino acid residues deleted corresponding to the 582nd to 604th amino acid residues from the N-terminus of AAV8 VP1; (8) The fusion protein encoded by the second nucleic acid molecule is the one described in (3) or (4) above, further having one or more amino acid residues deleted corresponding to the 591st, 592nd, 593rd, or 593rd amino acid residue from the N-terminus of AAV8 VP1. 5. The method for production according to any one of items 1 to 4 above, which is selected from the group consisting of the following items (1) to (4): (1) the CAP protein encoded by the first nucleic acid molecule is an AAV8 CAP protein, in which one or more amino acid residues corresponding to the 582nd to 604th amino acid residues from the N-terminus of VP1 have been deleted; (2) the CAP protein encoded by the first nucleic acid molecule is an AAV8 CAP protein, in which one or more amino acid residues corresponding to the 591st, 592nd, 593rd, or 593rd amino acid residues from the N-terminus of VP1 have been deleted;(3) The CAP protein encoded by the first nucleic acid molecule is a CAP protein other than AAV8, in which one or more amino acid residues corresponding to amino acid residues 582 to 604 from the N-terminus of AAV8 VP1 have been deleted; (4) The CAP protein encoded by the first nucleic acid molecule is a CAP protein other than AAV8, in which one or more amino acid residues corresponding to amino acid residues 591, 592, 593, or 593 from the N-terminus of AAV8 have been deleted. 6. The production method according to any of 1 to 5 above, in which the first nucleic acid molecule and the second nucleic acid molecule are introduced into a host cell so that the ratio of the number of molecules thereof is 9.9:0.1 to 8.0:2.0 or 9.9:0.1 to 8.0:3.0. 7. The production method according to any of 1 to 6 above, in which the other protein (A) has a specific affinity for a protein present on the surface of vascular endothelial cells. 8. The method for production according to the above item 7, wherein the protein present on the surface of the vascular endothelial cell is selected from the group consisting of transferrin receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transporter 1, organic anion transporter, monocarboxylate transporter, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 8, and membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor. 9. The method for production according to any of the above items 1 to 6, wherein the other protein (A) comprises a single-chain antibody or a single-domain antibody having specific affinity for the transferrin receptor. 10. The method for production according to any of the above items 1 to 6, wherein the other protein (A) is at least one of a fusion protein of VP1, VP2, and VP3 that is capable of specifically binding to another molecule. 11. The method for production according to the above item 10, wherein the other protein (A) comprises an antibody having affinity for the other molecule. 12. 12. The method for producing according to claim 10, wherein the other molecule is an antibody having affinity for the other protein (A). 13. The method for producing according to claim 10, wherein the other molecule is a fusion protein of an antibody having affinity for the other protein (A) and the other protein (B).14. The production method according to the above 13, wherein the other protein (B) has specific affinity for a protein present on the surface of vascular endothelial cells. 15. The production method according to the above 14, wherein the protein present on the surface of vascular endothelial cells is selected from the group consisting of transferrin receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transporter 1, organic anion transporter, monocarboxylate transporter, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 8, and membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor. 16. The production method according to the above 13, wherein the other protein (B) is a single-chain antibody or a single-domain antibody having specific affinity for the transferrin receptor. 17. The production method according to any of the above 3 to 16, wherein the second linker consists of 10 to 50 amino acid residues. 18. 19. rAAV particles selected from the following (1) or (2): (1) a capsid comprising VP1, VP2, and VP3, and a fusion protein of any of these with another protein (A), wherein the other protein (A) consists of 100 or more amino acid residues, and wherein the ratio of the total number of molecules of the VP1, VP2, and VP3 in the capsid to the total number of molecules of the fusion protein is 9.9:0.1 to 8.0:2.0; (2) a capsid comprising VP1, VP2, and VP3, and a fusion protein of any of these with another protein (A), wherein the other protein (A) consists of less than 100 amino acid residues, and wherein the ratio of the total number of molecules of the VP1, VP2, and VP3 in the capsid to the total number of molecules of the fusion protein is 9.9:0.1 to 7.0:3.0. rAAV particles selected from the following (1) or (2): (1) a capsid comprising VP2 and VP3, and a fusion protein of these with another protein (A), the other protein (A) consisting of 100 or more amino acid residues, and the ratio of the total number of molecules of the VP2 and VP3 to the total number of molecules of the fusion protein in the capsid is 9.9:0.1 to 8.0:2.0;(2) A capsid comprising VP2 and VP3, and a fusion protein of these with another protein (A), wherein the other protein (A) consists of less than 100 amino acid residues, and the ratio of the total number of molecules of the VP2 and VP3 in the capsid to the total number of molecules of the fusion protein is 9.9:0.1 to 8.0:3.0. 20. rAAV particles according to 18 or 19 above, which are selected from the following (1) to (8): (1) the fusion protein, wherein the other protein (A) consists of 100 or more amino acid residues, the CAP protein is the CAP protein of AAV8, and the other protein (A) is added at a position corresponding to the C-terminal side of the 450 to 465, 584 to 602, 455, 457, 462, 501, 588, or 599 amino acid residues from the N-terminus of VP1, or the amino acid sequence corresponding to the 456 to 462 or 455 to 460 amino acid sequence from the N-terminus of VP1 is substituted with the amino acid sequence of the other protein (A); (2) The fusion protein is one in which the other protein (A) consists of less than 100 amino acid residues, the CAP protein is an AAV8 CAP protein, and the other protein (A) is added to a position corresponding to the C-terminal side of the 450-465, 584-602, 707-717, or 455, 457, 462, 501, 588, 599, or 707 amino acid residues from the N-terminus of the VP1, or in which the amino acid sequence corresponding to the 456-462 or 455-460 amino acid sequence from the N-terminus of the VP1 is replaced with the amino acid sequence of the other protein (A); (3) The fusion protein is one in which the other protein (A) consists of 100 or more amino acid residues, the CAP protein is a CAP protein other than AAV8, and the other protein (A) is added at a position corresponding to the C-terminal side of amino acid residues corresponding to amino acid residues 450 to 465, 584 to 602, 455, 457, 462, 501, 588, or 599 from the N-terminus of VP1 of AAV8, or in which the amino acid sequence corresponding to the amino acid sequence of amino acids 456 to 462 or 455 to 460 from the N-terminus of VP1 of AAV8 is replaced with the amino acid sequence of the other protein (A);(4) The fusion protein is one in which the other protein (A) consists of less than 100 amino acid residues, the CAP protein is a CAP protein other than AAV8, and the other protein (A) is added at a position corresponding to the C-terminal side of amino acid residues corresponding to amino acid residues at positions 450 to 465, 584 to 602, 707 to 717, or 455, 457, 462, 501, 588, 599, or 707 from the N-terminus of VP1 of AAV8, or one in which the amino acid sequence corresponding to amino acid sequence at positions 456 to 462 or 455 to 460 from the N-terminus of VP1 of AAV8 is replaced with the amino acid sequence of the other protein (A); (5) The fusion protein is one in which, in addition to the one described in (1) or (2) above, one or more amino acid residues corresponding to amino acid residues 582 to 604 from the N-terminus of VP1 are deleted; (6) The fusion protein is one according to (1) or (2) above, further comprising one or more amino acid residues deleted from the N-terminus of VP1, which correspond to the amino acid residues at positions 591, 592, 593, or 593; (7) The fusion protein is one according to (3) or (4) above, further comprising one or more amino acid residues deleted from the N-terminus of AAV8 VP1, which correspond to the amino acid residues at positions 582 to 604; (8) The fusion protein is one according to (3) or (4) above, further comprising one or more amino acid residues deleted from the N-terminus of VP1, which correspond to the amino acid residues at positions 591, 592, 593, or 593. 21. 21. The rAAV particle according to any one of items 18 to 20 above, which is selected from the following (1) or (2): (1) all CAP proteins lacking one or more amino acid residues corresponding to amino acid residues 582 to 604 from the N-terminus of VP1; (2) all CAP proteins lacking one or more amino acid residues corresponding to amino acid residues 591, 592, 593, or 593 from the N-terminus of VP1. 22. The rAAV particle according to any one of items 18 to 21 above, wherein the other protein (A) comprises a first linker, a functional protein, and a second linker in this order from the N-terminus, or a functional protein and a second linker in this order from the N-terminus.23. The rAAV particle according to any one of items 18 to 22 above, wherein the other protein (A) has specific affinity for a protein present on the surface of vascular endothelial cells. 24. The rAAV particle according to any one of items 18 to 22 above, wherein the other protein (A) comprises a single-chain antibody or a single-domain antibody having specific affinity for a transferrin receptor. 25. The rAAV particle according to any one of items 18 to 24 above, wherein at least one of the fusion proteins of the other protein (A) with VP1, VP2, and VP3 is capable of specifically binding to another molecule. 26. The rAAV particle according to item 25 above, wherein the other protein (A) comprises an antibody having affinity for the other molecule. 27. The rAAV particle according to item 25 above, wherein the other molecule is an antibody having affinity for the other protein (A). 28. 28. The rAAV particle according to claim 25, wherein the other molecule is a fusion protein of the other protein (B) and an antibody having affinity for the other protein (A). 29. The rAAV particle according to claim 28, wherein the other protein (B) has specific affinity for a protein present on the surface of vascular endothelial cells. 30. The rAAV particle according to claim 29, wherein the other protein (B) is a single-chain antibody or a single-domain antibody having specific affinity for a transferrin receptor. 31. A pharmaceutical composition comprising the rAAV particle according to any of claims 18 to 30. 32. rAAV particles selected from the group consisting of the following (1) to (4): (1) a CAP protein that is an AAV8 CAP protein from which one or more amino acid residues corresponding to the 582nd to 604th amino acid residues from the N-terminus of VP1 have been deleted; (2) the CAP protein is an AAV8 CAP protein from which one or more amino acid residues corresponding to the 591st, 592nd, 593rd, or 593rd amino acid residues from the N-terminus of VP1 have been deleted; (3) the CAP protein is a CAP protein other than that of AAV8 from which one or more amino acid residues corresponding to the 582nd to 604th amino acid residues from the N-terminus of AAV8 VP1 have been deleted; (4) the CAP protein is a CAP protein other than that of AAV8 from which one or more amino acid residues corresponding to the 591st, 592nd, 593rd, or 593rd amino acid residues from the N-terminus of AAV8 have been deleted.33. A pharmaceutical composition comprising the rAAV particles described in 32 above.

[0015] It is contemplated that one or more of the above features may be provided in combinations other than those explicitly stated, and further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0016] Note that features and significant actions and effects of the present disclosure other than those described above will become clear to those skilled in the art by referring to the following description of the preferred embodiments of the invention and the drawings.

[0017] According to the present disclosure, highly infectious rAAV particles that exhibit high tropism for specific organs can be produced in high yields. By packaging a nucleotide sequence encoding a physiologically active protein or a nucleic acid molecule containing the nucleotide sequence into such rAAV particles, rAAV particles that can express the protein in a desired organ can be produced. For example, by administering such rAAV particles to a patient with organ damage, the desired protein that is expected to have a therapeutic effect can be expressed in the organ, thereby alleviating the patient's symptoms.

[0018]

[0033] Figure 1 shows the yield of rAAV particles when rAAV particles were produced using a mixed vector prepared from pR2C8 (VHH456-462) and pR2C8 such that the ratio (molar ratio) of pR2C8 (VHH456-462) was 10%, 30%, and 50% of the total. The vertical axis shows the relative yield of each rAAV particle when the yield when rAAV was produced using only pR2C8 was set to 1.

[0034] Figure 1 shows the yield of rAAV particles when rAAV particles were produced using a mixed vector prepared from pR2C9 (VHH455 Hinge-Hinge) and pR2C9 such that the ratio (molar ratio) of pR2C9 (VHH455 Hinge-Hinge) was 0%, 5%, 10%, 30%, and 50% of the total. The vertical axis shows the relative yield of each rAAV particle, with the yield when rAAV was produced using only pR2C9 set to 100%. Figure 1 shows the results of immunohistochemical staining of the brains of hTfRKI mice 2 weeks after administration of rAAV particles produced using a mixed vector containing pR2C9(VHH455) and pR2C9, adjusted so that the proportion (molar ratio) of pR2C9(VHH455) was 5% and 10% of the total. (a) Immunohistochemical staining of an uninfected mouse; (b) a mouse administered with anti-TfR VHH(455)(CAG-GFP-WPRE) AAV9 (5%); and (c) a mouse administered with anti-TfR VHH(455)(CAG GFP-WPRE) AAV9 (10%).

[0033] Figure 1 shows the yield of each rAAV particle when rAAV particles were produced using a pR2C8 plasmid in which a VHH-encoding gene was introduced into the Cap region. The vertical axis shows the relative yield of each rAAV particle when the yield when rAAV particles were produced using only pR2C8 was set to 1. In the figure, mod3 indicates rAAV particles produced using only pR2C8. An asterisk indicates particles with significantly reduced infectivity. Figure 1 shows the yield of each rAAV particle when rAAV particles were produced using a pR2C8 plasmid in which a VHH-encoding gene was introduced into the Cap region so that linkers were positioned on both the N- and C-terminal sides of the VHH. The vertical axis shows the relative yield of each rAAV particle when the yield when rAAV particles were produced using only pR2C8 was set to 1.The table on the left side of each figure shows the combination of linkers on the N-terminus and C-terminus. Same as Figure 5. Same as Figure 5. Same as Figure 5. Figure showing the yield of each rAAV particle when rAAV particles were produced using pR2C8 plasmid in which a VHH-encoding gene was introduced into the Cap region so that a linker was positioned at each of the N-terminus and C-terminus of VHH. The vertical axis shows the rAAV genome amount (vg). (A) shows rAAV particles produced using pR2C8 alone, (B) pR2C8 and pR2C8(VHH456-462), (C) pR2C8 and pR2C8(VHH456-462)Hinge-Hinge, (D) pR2C8 and pR2C8(VHH501)GS1-Hinge, and (E) pR2C8 and pR2C8(VHH599)GS1-Hinge. Figures showing the results of an infectivity test of rAAV particles using mice. (a), (b), and (c) show the levels of GFP expression in the brain, spinal cord, and liver of mice infected with rAAV particles. Vertical bars indicate standard error. Figures showing the quantification of rAAV particles in the brain and liver of mice infected with rAAV particles. The vertical axis shows the quantitative value of the rAAV genome (vg / gDNA). (a) shows the brain, and (b) shows the quantitative value of the rAAV particles in the brain. The vertical bar shows the standard error. Figure showing the results of an infectivity test of rAAV particles using mice. (a) shows the quantitative result of the rAAV genome in the brain of a mouse infected with rAAV particles, and the vertical axis shows the quantitative value of the rAAV particles as the quantitative value of the rAAV genome (vg / gDNA). The vertical bar shows the standard error. (b) shows the result of measuring the expression level of GFP in the brain of a mouse infected with rAAV particles, and the vertical axis shows the GFP concentration (μg / g wet tissue weight). The vertical bar shows the standard error. Figure showing the results of immunohistochemical staining of the whole brain of a mouse infected with rAAV particles. (a) shows the results of immunohistochemical staining of uninfected mice, (b) shows the results of immunohistochemical staining of mice infected with AAV9-WT, (c) shows the results of immunohistochemical staining of mice infected with anti-TfR VHH(CMV-GFP-WPRE)-AAV9, (d) shows the results of immunohistochemical staining of mice infected with anti-TfR VHH(CAG-GFP-WPRE)-AAV9, (e) shows the results of immunohistochemical staining of the cerebrum of mice infected with rAAV particles.(a) shows the results of immunohistochemical staining of the cerebrum of a mouse infected with an uninfected mouse, (b) AAV9-WT, (c) anti-TfR VHH(CMV-GFP-WPRE)-AAV9, (d) anti-TfR VHH(CAG-GFP-WPRE)-AAV9, (e) anti-TfR VHH(CBh-GFP-WPRE)-AAV9, or (f) anti-TfR VHH(PGK-GFP-WPRE)-AAV9. (b) shows the results of immunohistochemical staining of the cerebellum of a mouse infected with rAAV particles. (a) shows the results of immunohistochemical staining of the cerebellum from a non-infected mouse, (b) from a mouse infected with AAV9-WT, (c) from a mouse infected with anti-TfR VHH(CMV-GFP-WPRE)-AAV9, (d) from a mouse infected with anti-TfR VHH(CAG-GFP-WPRE)-AAV9, (e) from a mouse infected with anti-TfR VHH(CBh-GFP-WPRE)-AAV9, and (f) from a mouse infected with anti-TfR VHH(PGK-GFP-WPRE)-AAV9. (a) shows the results of immunohistochemical staining of the whole brain of a mouse infected with an uninfected mouse, (b) with AAV9-WT, (c) with anti-TfR VHH(CAG-GFP-WPRE)-AAV9, or (d) with anti-TfR Fab-[(ALFANb ALFAtag)(CAG-GFP-WPRE)-AAV9]. This figure shows the results of a pharmacological test of rAAV particles using MPS-II model mice. The vertical axis shows the heparan sulfate concentration (μg / dry tissue weight) in the brain of an MPS-II model mouse administered with rAAV particles. This figure shows the results of a pharmacological test of rAAV particles using MPS-II model mice. The vertical axis shows the quantitative value of the rAAV genome (vg / gDNA) in the brain of an MPS-II model mouse administered with rAAV particles.

[0033] Figure 1 shows the results of a pharmacological test of rAAV particles using GM-1 gangliosidosis model mice. The vertical axis shows the concentration of Lyso-GM1 (μg / dry tissue weight) in the brains of GM-1 gangliosidosis model mice administered with rAAV particles.

[0034] Figure 1 shows the results of a pharmacological test of rAAV particles using GM-1 gangliosidosis model mice. The vertical axis shows the concentration of GLB1 (μg / wet tissue weight) in the brains of GM-1 gangliosidosis model mice administered with rAAV particles.Figure showing the results of immunohistochemical staining of brain myelin. ssAAV indicates anti-TfR VHH(CAG-ASPA-WPRE)-AAV9, and scAAV indicates anti-TfR VHH(CBh-ASPA)-scAAV9. H indicates hippocampus, TH indicates thalamus, Midbrain indicates midbrain, Cerebellum indicates cerebellum, and BS indicates brainstem. Arrowheads indicate cerebellar white matter. The scale bar indicates 500 μm. This figure shows the results of an evaluation of motor function in demyelinating disease model mice administered with rAAV particles. (a) shows the results of a mouse grip strength test. The vertical axis indicates grip strength measurements (kfg). (b) shows the results of a rotarod test. The vertical axis indicates the duration of movement (seconds). Figure showing the results of comparing the productivity of AAV9-WT and anti-TfR VHH(455-460)-AAV9. The vertical axis shows the quantitative value of the rAAV genome, and the white circles show the actual measured values ​​(N=2). Figure showing the results of examining the stability of rAAV particles. The vertical axis shows the ratio of cells infected with rAAV particles (AAV positive rate). The horizontal axis shows the amount of rAAV particles added to the medium as a concentration (vg / mL). Figure showing the ratio of the fusion protein between capsid protein and VHH in the capsid protein of rAAV particles. The white bar shows the ratio of the fusion protein between VP2 and VHH (VP2-VHH), and the black bar shows the ratio of the fusion protein between VP3 and VHH (VP3-VHH). Figure showing the yield of rAAV particles into which point mutations have been introduced into the Loop-8 region. The vertical axis indicates the relative yield when the yield of anti-TfR VHH(455-460)-AAV9[1] is set to 1.(x) is anti-TfR VHH(455-460)-AAV9[1], (a) is anti-TfR VHH(455-460)Q592I-AAV9[6], (b) is anti-TfR VHH (455-460) Q592A-AAV9 [7], (c) is anti-TfR VHH (455-460) Δ586S-AAV9 [9], (d) is anti-TfR VHH (455-460) Δ587A-AAV9

[10] , (e) is anti-TfR VHH(455-460)Δ588Q-AAV9

[11] , (f) is anti-TfR VHH(455-460)Δ589A-AAV9

[12] , (g) is anti-TfR VHH(455-460)Δ591A-AAV9

[13] , (h) is anti-TfR VHH (455-460) Δ592Q-AAV9

[14] , (i) is anti-TfR VHH (455-460) Δ593T-AAV9

[19] , (j) is anti-TfR VHH(455-460)Δ594G-AAV9

[19] , (k) is anti-TfR VHH(455-460)Δ595W-AAV9[9], (l) is anti-TfR (m) shows the relative yield of VHH(455-460)Δ596V-AAV9

[10] , (m) shows the relative yield of anti-TfR VHH(455-460)Δ597Q-AAV9

[11] , and (n) shows the relative yield of anti-TfR VHH(455-460)Δ598N-AAV9

[12] . This figure shows the expression level of GFP in the brains of mice infected with rAAV particles containing point mutations in the Loop-8 region. The vertical axis shows the relative yield, with the expression level of GFP following infection with anti-TfR VHH(455-460)-AAV9[1] set to 1.(x) is anti-TfR VHH(455-460)-AAV9 [1], (a) is anti-TfR VHH(455-460)Q592I-AAV9 [6], (b) is anti-TfR VHH(455-460)Q592A-AAV9 [7], (c) is anti-TfR VHH(455-460)Δ586S-AAV9 [9], (d) is anti-TfR VHH(455-460)Δ587A-AAV9

[10] , (e) is anti-TfR VHH(455-460)Δ588Q-AAV9

[11] , (f) is anti-TfR VHH(455-460)Δ589A-AAV9

[12] , (g) is anti-TfR VHH(455-460)Δ591A-AAV9

[13] , (h) is anti-TfR VHH(455-460)Δ592Q-AAV9

[14] , (i) is anti-TfR VHH(455-460)Δ593T-AAV9

[19] , (k) is anti-TfR VHH (455-460) Δ594G-AAV9 [9], (l) is anti-TfR VH H (455-460) Δ595W-AAV9

[10] , (m) is anti-TfR (n) shows the relative yield of VHH(455-460)Δ596V-AAV9

[11] , (n) shows the relative yield of anti-TfR VHH(455-460)Δ597Q-AAV9

[12] , and (o) shows the relative yield of anti-TfR VHH(455-460)Δ598N-AAV9

[13] . This figure shows the expression level of GFP in the liver of mice infected with rAAV particles containing point mutations in the Loop-8 region. The vertical axis shows the relative yield, with the expression level of GFP in the mouse infected with anti-TfR VHH(455-460)-AAV9[1] set to 1.(x) is anti-TfR VHH(455-460)-AAV9 [1], (a) is anti-TfR VHH(455-460)Q592I-AAV9 [6], (b) is anti-TfR VHH(455-460)Q592A-AAV9 [7], (c) is anti-TfR VHH(455-460)Δ586S-AAV9 [9], (d) is anti-TfR VHH(455-460)Δ587A-AAV9

[10] , (e) is anti-TfR VHH(455-460)Δ588Q-AAV9

[11] , (f) is anti-TfR VHH(455-460)Δ589A-AAV9

[12] , (g) is anti-TfR VHH(455-460)Δ591A-AAV9

[13] , (h) is anti-TfR VHH(455-460)Δ592Q-AAV9

[14] , (i) is anti-TfR VHH(455-460)Δ593T-AAV9

[19] , (k) is anti-TfR VHH(455-460)Δ594G-AAV9[9], (l) is anti-TfR VHH(455-460)Δ595W-AAV9

[10] , (m) is anti-TfR (n) shows the relative yield of anti-TfR VHH(455-460)Δ596V-AAV9

[11] , (n) shows the relative yield of anti-TfR VHH(455-460)Δ597Q-AAV9

[12] , and (o) shows the relative yield of anti-TfR VHH(455-460)Δ598N-AAV9

[13] . This figure shows the relationship between the ratio of the amount of a plasmid carrying a nucleic acid sequence encoding a ligand-modified (VP1 / VP2 / VP3) to the total amount of the plasmid and the plasmid carrying a nucleic acid sequence encoding VP1 / VP2 / VP3 (horizontal axis) and the ratio of the amount of mRNA encoding the ligand-modified VP3 to the total amount of the mRNA and the mRNA encoding VP1 / VP2 / VP3 (vertical axis) during transfection into host cells in the production process of a ligand-modified AAV vector. The square, circle, and triangle marker lines indicate the anti-TfR VHH, anti-TfR VHH (VP1 / 2 unmodified), and FMDV peptide modification, respectively. Figure 1 shows a schematic diagram of two types of plasmids used for transfection into host cells in the production process of ligand-modified AAV vectors.A is a plasmid encoding VP1 / 2 / 3 in which amino acid residues 456 to 462 from the N-terminus of VP1 have been substituted with a ligand, and the initiation codon of VP1 / 2 has been mutated to render it nonfunctional. B is a plasmid encoding VP1 / 2 / 3. This figure shows the relationship between the ratio (horizontal axis) of a plasmid carrying a nucleic acid sequence encoding ligand-modified VP3 to the total amount of said plasmid and a plasmid carrying a nucleic acid sequence encoding VP1 / VP2 / VP3, and the productivity of AAV vectors (vertical axis), during transfection into host cells in the production process of a ligand-modified AAV vector. The vertical axis shows the total amount of viral genome contained in the culture supernatant. This figure shows SDS-PAGE of culture supernatants obtained by changing the ratio (horizontal axis) of a plasmid carrying a nucleic acid sequence encoding ligand-modified VP3 to the total amount of said plasmid and a plasmid carrying a nucleic acid sequence encoding VP1 / VP2 / VP3, during transfection into host cells in the production process of a ligand-modified AAV vector. A to H show the results for samples obtained when the proportion of plasmid containing a nucleic acid sequence encoding ligand-modified VP3 was 0, 1, 3, 5, 10, 20, 30, and 50%, respectively. The arrows indicate the band of ligand-modified VP3. (Left) A diagram showing the infection efficiency of AAV vectors prepared with different plasmid ratios. The vertical axis shows the percentage of infected cells, and the horizontal axis shows the amount of AAV vector treated (amount of viral genome contained per mL of medium). (Right) A diagram showing the infection efficiency of AAV vectors (1E11 viral genomes / mL) prepared with different plasmid ratios. The vertical axis shows the percentage of infected cells. A diagram showing the expression level of GFP in the brain and liver of mice infected with rAAV particles containing point mutations introduced into the loop region. The vertical axis shows the relative level, with the expression level in the brain and liver following infection with anti-TfR VHH(455-460)-AAV9 set to 1. Same as Figure 33A. Same as Figure 33A. A diagram showing the amount of viral mRNA detected in the (left) liver and (right) brain of mice infected with rAAV particles in which a point mutation was introduced into the loop region. The vertical axis shows the relative amount, with the mRNA amount following infection with anti-TfR VHH(455-460)-AAV9 set to 1. Same as Figure 34.37. Same as FIG. 37.

[0047] FIG. 37 shows the amount of viral mRNA detected in the (left) liver and (right) brain of mice infected with rAAV particles having a point mutation introduced into the loop region. The vertical axis shows the relative amount when the amount of mRNA following infection with anti-TfR VHH(455-460)-AAV9 is set to 1.

[0048] FIG. 37 shows the amount of viral genome detected in the (left) liver and (right) brain of mice infected with rAAV particles having a point mutation introduced into the loop region. The vertical axis shows the relative amount when the amount following infection with anti-TfR VHH(455-460)-AAV9 is set to 1.

[0049] Same as FIG. 37. Same as FIG. 37.

[0050] The vertical axis shows the relative amount when the amount of each virus when infected with anti-TfR VHH(455-460)-AAV9 is set to 1. Same as Figure 40. Diagram showing the amount of viral genome detected in the plasma of mice infected with POD peptide-modified rAAV particles in which point mutations were introduced into the loop region. The vertical axis shows the amount of viral genome contained in mL of plasma. (Left) Diagram showing the infection efficiency of POD peptide-modified AAV vectors prepared with different plasmid ratios. The vertical axis shows the percentage of infected cells, and the horizontal axis shows the amount of AAV vector treated (amount of viral genome contained per mL of medium). (Right) Diagram showing the infection efficiency of AAV vectors prepared with different plasmid ratios (1E11 viral genome / mL). The vertical axis shows the percentage of infected cells. Same as Figure 42-2. 1 shows the viral genome amount detected in the (left) heart, (center) quadriceps, and (right) liver of mice infected with FMDV peptide-modified rAAV particles in which a point mutation has been introduced into the loop region. The vertical axis shows the relative amount when the viral genome amount in infection with anti-TfR VHH(455-460)-AAV9 is set to 1. 1 shows the viral genome amount detected in the plasma of mice infected with anti-TfR VHH-modified rAAV particles in which a point mutation has been introduced into the loop region. The vertical axis shows the viral genome amount contained in mL of plasma. 1 shows the test configuration for an infection efficiency test using monkeys. 1 shows the amount of GFP and mCherry mRNA in each tissue of monkeys administered with each AAV vector.The vertical axis shows the relative amount of mRNA when the mRNA amount in AN1 Cerebrum cortex is set to 1. The black and white bars show the mRNA amounts of GFP and mCherry, respectively. Same as Figure 46. Same as Figure 46. Same as Figure 46. A diagram showing the viral genome amount per cell in each tissue of monkeys administered each AAV vector. The vertical axis shows the amount of viral genome encoding GFP and mCherry detected per cell. The black and white bars show the mRNA amount per cell of viral genome encoding GFP and mCherry, respectively. Same as Figure 50. Same as Figure 50. Same as Figure 50. Same as Figure 50.

[0019] The present disclosure will now be described with reference to the best mode. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0020] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0021] (Definitions) As used herein, the term "about" refers to ±10% of the numerical value that follows, or refers to significant figures. For example, "about 20" includes "18 to 22." A range of numerical values ​​includes all numerical values ​​between and at the endpoints. When "about" refers to a range, it applies to both endpoints of the range. Thus, for example, "about 20 to 30" includes "18 to 33." It should be noted that, even when the term "about" is not used in this specification, all statements provided refer to significant figures.

[0022] As used herein, the term "surface" refers to being present on the surface when expressed in a cell.

[0023] As used herein, the term "ligand" refers to a molecule that exhibits specific binding affinity for a specific molecule or group of molecules. Ligands can be composed of molecular species with diverse chemical properties, such as proteins, peptides, antibodies and their fragments, nucleic acids, sugar chains, lipids, and small molecules. The ligands described herein are particularly presented on the surface of recombinant adeno-associated virus (rAAV) vectors and specifically bind to specific receptors or cell surface antigens expressed on target cells, thereby conferring targeting. The ligands used herein preferably have specific binding ability to target cells. Such specificity can be assessed by simple analytical methods such as ELISA, flow cytometry, and surface plasmon resonance (SPR) after mixing a candidate ligand molecule with target cells or non-target cells. For example, a molecule can be determined to be a ligand if it exhibits a clearly higher binding signal relative to control cells when mixed with target cells. Specific examples of ligands in the present disclosure include antibodies or antibody fragments (e.g., scFv, Fab, VHH antibodies), natural or synthetic peptides that bind to cell surface receptors (e.g., RGD peptides, peptides derived from cell adhesion factors), cytokines and their receptor-binding domains (e.g., interleukin-2, interferon-γ), growth factors (e.g., EGF, FGF, VEGF, etc.), sugar chains (e.g., galactose, mannose), or small molecule ligands that exhibit affinity for specific receptors on target cells (e.g., folic acid, biotin, retinoic acid), etc. By genetically fusing or chemically binding these ligands to the surface of the AAV capsid, the recombinant adeno-associated virus vectors of the present invention enable efficient and specific gene transfer into target cells.

[0024] As used herein, the terms "adeno-associated virus" and its abbreviation "AAV" are used interchangeably and refer to viruses belonging to the genus Dependovirus in the family Parvoviridae. The term "virus" is used in the same sense as commonly used in the art, and will be understood by those skilled in the art depending on the context. AAV is generally a non-pathogenic virus with a diameter of approximately 20-25 nm, containing a single-stranded DNA genome within a capsid. AAV cannot efficiently replicate alone, and can only replicate efficiently in the presence of a helper virus such as an adenovirus or a herpesvirus. As used herein, "adeno-associated virus" or "AAV" includes wild-type AAV and its derivatives and mutants, as well as recombinant adeno-associated viruses (rAAV). In particular, recombinant adeno-associated viruses (rAAVs) are AAVs produced by genetic recombination and are vectors used for gene transfer, etc., constructed by replacing part or all of the AAV genome sequence with a gene of interest (such as a therapeutic gene or a reporter gene). Specific AAV serotypes or variants include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, AAV-DJ, and AAV-LK03, which are selectively used based on properties such as affinity for target tissues, immunogenicity, and gene transfer efficiency. Because these AAV serotypes or variants share similarities in gene sequence and structure, they may be collectively and interchangeably referred to herein as "AAV." Therefore, when the term "adeno-associated virus" or "AAV" is used in this specification, it should be construed as including these viruses and their recombinant forms, unless otherwise specified by the context.

[0025] As used herein, the terms "recombinant adeno-associated virus," "recombinant AAV," or "rAAV" are used interchangeably and refer to a viral vector for gene transfer constructed by artificially modifying part or all of the genome sequence of an adeno-associated virus (AAV) to include a foreign gene (such as a therapeutic gene, a reporter gene, or a target sequence). Generally, recombinant AAV (rAAV) is constructed by removing both or either the Rep gene required for replication and the Cap gene required for capsid formation from the viral genome and inserting a foreign gene cassette to be introduced instead. This modification makes rAAV nonpathogenic while retaining its infectivity, allowing it to be used as a safe and efficient gene delivery tool.

[0026] As used herein, "virus-like particle" or "VLP (Virus-Like Particle)" refers to a structure that mimics the structural characteristics of a virus but does not contain a viral genome or lacks replication ability, and is defined as a safe nanoparticle that is non-infectious. VLPs are particles formed by self-assembly of capsid proteins of natural viruses (e.g., VP1, VP2, VP3, etc. in adeno-associated viruses). Externally, VLPs have nearly the same size, shape, and symmetry as viruses, but differ in that they lack pathogenicity and replication ability. Because VLPs mimic the three-dimensional structure of virus particles, they exhibit biological behavior similar to that of natural viruses with respect to the host's immune system and cellular receptors. However, because they do not undergo gene replication, they ensure high safety in applications such as gene transfer, vaccines, and drug delivery. As used herein, the term "virus-like particle" refers primarily to a capsid structure based on adeno-associated virus (AAV) and broadly encompasses particles having a capsid structure composed of VP proteins as an outer shell, regardless of whether a gene of interest is encapsulated therein. Specifically, the following particles qualify as "virus-like particles": rAAV particles that encapsulate a gene of interest but are replication-incompetent; "empty capsid" structures that do not contain genetic information; and VLPs derived from capsids derived from other viruses (e.g., HBV, HPV, etc.) as well as AAV-based VLPs. In one embodiment, VLPs are often prepared as rAAV particles carrying a therapeutic recombinant gene, and may have a configuration in which the surface loop structure of VP3 has been modified (e.g., ligand modification or mutation introduction). This allows VLPs to function as targeting drug delivery particles or vaccine carriers responsible for antigen presentation.

[0027] Furthermore, as used herein, "virus-like particles" or "VLPs" include structures that meet any of the following requirements: they contain viral capsid proteins and form a particle structure by self-assembly; they are morphologically similar to infectious viruses but lack proliferation or pathogenicity; and / or the outer shell structure retains the ability to bind to receptors on the surface of target cells. Therefore, "virus-like particles" or "VLPs" as used herein are bionanoparticles that structurally mimic the external shape of viruses but have excellent safety and therapeutic applicability, and are positioned as various functional carriers in the present invention, such as nucleic acid delivery vehicles, immune stimulators, and targeting devices. A representative example of "virus-like particles" or "VLPs" in the present disclosure is recombinant adeno-associated virus particles (rAAV particles), and unless specifically mentioned with respect to AAV, both refer to the same subject.

[0028] As used herein, "recombinant adeno-associated virus particle" or "rAAV particle" refers to an artificially constructed particle that has a capsid structure derived from an adeno-associated virus (AAV), but, unlike naturally occurring AAV viruses, lacks viral replication ability and encapsulates a gene of interest for therapeutic or experimental use. In other words, rAAV particles are non-infectious gene carriers used to introduce a gene of interest into host cells, and are virus-like nanoparticles that are highly safe and stable. In certain aspects of the present specification, rAAV particles may also be referred to as "recombinant adeno-associated virus vectors" or "rAAV vectors." Where applicable, descriptions of rAAV particles may also be applied to rAAV vectors, and those skilled in the art will appreciate their applicability depending on the context. However, rAAV particles can encapsulate nucleic acid molecules containing nucleic acid sequences encoding desired proteins (e.g., proteins for medical purposes such as therapeutic or preventive purposes), and in that sense, can be considered vectors for delivering nucleic acid molecules containing nucleic acid sequences encoding proteins for medical purposes such as therapeutic or preventive purposes.

[0029] As used herein, rAAV particles have an outer shell (capsid) formed from capsid-constituting proteins such as VP1, VP2, and VP3, and encapsulate a desired gene sequence (e.g., a therapeutic gene, a reporter gene, etc.) inside. These VP proteins are derived from the AAV cap gene, and are expressed and self-assembled in host cells such as HEK293 cells by co-transfection with a group of recombinant plasmids to form virus-like particles. The encapsulated gene sequence is sandwiched between AAV ITR (inverted terminal repeat) sequences, allowing for long-term and stable expression in host cells. The rAAV particles used herein have the following characteristics:

[0030] rAAV lacks replication ability and is intended only for transient or sustained expression of therapeutic genes; unlike wild-type AAV, it contains a vector genome that does not contain rep / cap genes; it is highly safe due to almost no random integration into the host genome; the introduction of tissue-specific promoters or regulatory sequences enables controlled gene expression in target cells; and / or modification of the capsid surface structure (e.g., loop region) confers selective infection (tropism) to specific cell types or tissues. Herein, for example, rAAV particles can be modified to have appropriate serotypes and structural modifications depending on the type of disease or treatment target, such as nerve cells, muscle cells, liver cells, ocular cells, or immune cells, enabling precise and efficient gene transfer. Also included are embodiments in which reduced immunogenicity and improved cell selectivity can be achieved through substitution or deletion of amino acid sequences in specific loop regions.

[0031] Furthermore, the term "rAAV particles" as used herein also includes so-called "empty capsid" particles that do not carry a gene of interest, as well as particles used in comparative experiments, evaluation of target recognition ability, verification of manufacturing processes, etc. Therefore, "recombinant adeno-associated virus particles" or "rAAV particles" are defined herein as safe and functional virus-like particles that have a structure derived from AAV and are artificially constructed for therapeutic, diagnostic, and experimental purposes, and have extremely high applicability as gene transfer vehicles.

[0032] As used herein, "serotype X adeno-associated virus (AAV)" refers to a specific type of AAV, where X is a specific number, letter, or combination thereof, and may be expressed as "AAVX" or the like. The "serotype" depends primarily on the amino acid sequence of the capsid proteins (VP1, VP2, VP3) on the surface of the virus particle, and exhibits differences in reactivity to different neutralizing antibodies, tissue tropism, cell entry pathway, etc. In other words, "serotype X AAV" refers to a specific type corresponding to X among AAVs classified based on the structure and antigenicity of the capsid protein. As used herein, the term "serotype X" corresponds to all known AAV serotypes, and includes, for example, the following serotypes: AAV1, AAV2, AAV3 (including AAV3a and AAV3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAV10, AAVrhlO, AAV11, AAVpo1, AAVpo2, AAVhu11, AAVhu37, AAVDJ, AAVLK03, AAVPHP.B, AAVPHP.eB, and AAVAnc80L65. These include natural types derived from humans (e.g., AAV2, AAV5, and AAV9), as well as mutant and chimeric types derived from non-human primates, pigs, or obtained by artificial synthesis or capsid shuffling. Serotype determination is primarily performed by sequence analysis of the capsid protein gene (Cap gene), cross-reactivity tests with neutralizing antibodies, comparison of infection tropism, and structural analysis of virus particles (electron microscopy, X-ray crystallography, etc.). Phylogenetic analysis of capsid amino acid sequences is also being used to classify closely related types. For example, AAV2 is a classic human-derived AAV with high neuronal infection efficiency. On the other hand, AAV9 can cross the blood-brain barrier and be delivered to the central nervous system. AAV8 and AAVrhlO have excellent liver tropism, while AAV5 has the property of efficiently infecting respiratory tract and brain neurons. Furthermore, AAVPHP. It has been reported that while AAVPHP.B and AAVPHP.eB are capable of widespread delivery to the mouse brain, this efficiency has not been replicated in humans.Therefore, as used herein, the term "adeno-associated virus of serotype X" refers comprehensively to adeno-associated viruses whose types are identified based on a specific capsid structure, including AAVs belonging to the serotypes listed above, or artificially modified AAVs derived from or having high sequence homology thereto.

[0033] As used herein, the term "vector" refers to a platform for transporting or delivering some factor (e.g., a nucleic acid sequence encoding a gene, a desired protein, a nucleic acid molecule containing a nucleic acid sequence encoding a desired protein, etc.). This term often refers to the transport of a nucleic acid sequence, such as a nucleic acid sequence encoding a gene, in which case the vector itself is often composed of nucleic acids. However, as used herein, other factors (e.g., proteins), such as rAAV vectors, can also be used as component factors. As used herein, the term may also refer to a nucleic acid molecule or complex thereof used to introduce and express a foreign gene sequence (e.g., a therapeutic gene, a reporter gene, a regulatory sequence, an RNA interference sequence, etc.) into cells using genetic engineering techniques, such as a viral vector. A vector serves to stably or transiently deliver a target gene to a target, such as a host cell, and to control gene expression in the cell. Specific examples of vectors used herein include virus-derived vectors and non-viral vectors. Examples of virus-derived vectors include adeno-associated virus vectors (AAV vectors), adenovirus vectors, retrovirus vectors, lentivirus vectors, and herpes virus vectors. Examples of non-viral vectors include plasmid DNA, minicircle DNA, artificial chromosome vectors, RNA vectors, and complexes using liposomes or polymers (lipid nanoparticles, polyplexes, lipoplexes, etc.). The vector particularly used herein is a recombinant adeno-associated virus (rAAV) vector. This rAAV vector is advantageous in that it provides specific target cell targeting, low immunogenicity, and stable gene expression.

[0034] As used herein, the terms "recombinant adeno-associated virus vector," "recombinant AAV vector," or "rAAV vector" are used interchangeably and refer to a virus-derived vector in which the genome of an adeno-associated virus (AAV) has been genetically modified and an exogenous gene sequence (such as a therapeutic gene, a reporter gene, or a regulatory sequence) has been incorporated. These vectors are constructed for the purpose of gene delivery to target cells. Specific examples of recombinant AAV vectors include, but are not limited to, those constructed by removing or replacing the Rep and Cap gene regions present in the wild-type AAV genome and instead introducing a desired exogenous gene cassette. This allows for safe gene transfer without the vector itself autonomously replicating. Strictly speaking, an rAAV vector is a vector that carries a desired exogenous protein, and may also be interpreted as referring to other parts (in the narrow sense); however, in this specification, it generally refers to the entire vector, including the exogenous protein. Therefore, when including the exogenous protein, the term is substantially synonymous with recombinant adeno-associated virus particle or rAAV particle in this specification.

[0035] This refers to the act of artificially introducing nucleic acid molecules, proteins, small molecular weight compounds, or carriers containing them (e.g., plasmids, vectors, liposomes, nanoparticles, etc.) into target cells (e.g., host cells, target cells, primary cells, cell lines, etc.) from the outside. In this specification, the term "introduction" is used as a broader concept encompassing any biomolecule or construct introduced into cells. "Introduction" encompasses various subconcepts depending on the purpose or type of target molecule, such as "gene introduction (introduction of foreign nucleic acids)," "protein introduction (introduction of exogenous proteins)," and "small molecule introduction (introduction of drugs, fluorescent dyes, etc.)." These are used for research, therapeutic, diagnostic, or cell engineering purposes. Various methods of introduction are applied depending on the physicochemical properties of the target molecule, the type of target cell, and the durability and efficiency of the introduction. Specifically, these include methods using viral vectors (e.g., adenovirus, adeno-associated virus (AAV), lentivirus, etc.) and non-viral methods (e.g., electroporation, lipofection, microinjection, nanoparticle carrier, peptide-based transfection, etc.). The "transduction" of particular interest herein refers to a technique for efficiently and selectively delivering a target foreign gene or regulatory molecule into cells, and a viral-based transduction method using recombinant adeno-associated virus (rAAV) is a particularly preferred embodiment. Due to its low immunogenicity, safety, long-term expression capacity, and tissue specificity, rAAV vectors have been widely applied in gene therapy, model animal production, or molecular transduction for disease modification. In this specification, the term "transduction" refers to a broad transduction procedure, including the viral and non-viral methods described above, unless otherwise specified. When the transduction target is a nucleic acid molecule, it is understood to be an aspect of "gene transduction."

[0036] As used herein, "gene transfer" refers to the artificial introduction of a foreign gene sequence of interest (such as a therapeutic gene, a reporter gene, a regulatory sequence, or an RNA interference sequence) into a host cell and the expression of the gene within the host cell. As a result of gene transfer, proteins or RNA molecules encoded by the gene of interest are transiently or continuously produced in the host cell. Gene transfer is primarily performed for the purposes of gene therapy, gene editing, basic research, or protein production. Specific gene transfer methods include, for example, viral methods using recombinant viral vectors (such as adeno-associated virus (AAV) vectors) and non-viral methods using plasmid DNA or RNA (such as electroporation, lipofection, and nanoparticle methods). A gene transfer method of particular interest herein uses a recombinant adeno-associated virus (rAAV) vector, which has the characteristic of enabling specific and efficient gene transfer into target cells. In this specification, the term "gene transfer" is used in a broad sense, including the viral and non-viral methods described above, unless otherwise specified.

[0037] As used herein, "VP" is an abbreviation for "virion protein" and refers to proteins that constitute the capsid of adeno-associated virus (AAV). The AAV capsid is mainly composed of three structural proteins: VP1, VP2, and VP3. These VP proteins are produced from a common capsid gene (Cap gene) and have different translation initiation sites, resulting in different molecular weights. VP1 has the largest molecular weight, followed by VP2 and the smallest by VP3, and these are known to form the capsid at a specific molar ratio (generally VP1:VP2:VP3 = approximately 1:1:10). In particular, among the VP proteins, VP3 is the main component of the capsid and plays an important role in functions such as vector particle formation, binding to target cells, intracellular entry, and immunogenicity. Furthermore, in the present invention, the targeting and infection specificity of AAV vectors can be improved by genetically modifying VP proteins and introducing or presenting specific ligands. Therefore, unless otherwise specified, the term "VP" used herein refers to the capsid-constituting proteins of adeno-associated viruses (VP1, VP2, VP3, etc.).

[0038] As used herein, the term "modification" in "ligand-modified VP3" refers to the direct or indirect introduction, binding, or fusion of a specific ligand molecule to VP3, a capsid structural protein of adeno-associated virus (AAV), using genetic engineering or chemical techniques. As used herein, "ligand-modified VP3" is also referred to as "ligand-containing VP3," and unless otherwise specified, the two terms are used interchangeably and have the same meaning. Therefore, "modification" includes any manner of substitution, insertion, addition, and the like. The modification enables the presentation of a ligand having binding affinity for a target cell receptor or a specific molecule on the surface of the AAV capsid containing the VP3 protein. Specific "modification" methods include the following: (1) a genetic engineering fusion method in which a gene sequence encoding a ligand is directly fused to the VP3 gene sequence; (2) a chemical modification method in which a specific chemically reactive group (such as an azide group, an alkyne group, biotin, or a tag sequence) is genetically engineered onto the VP3 protein, followed by chemical binding of a ligand to the group; and (3) a tag-mediated modification method in which a ligand is indirectly introduced into the VP3 protein via a specific affinity tag (such as a His tag, a FLAG tag, or a SpyTag). Unless otherwise specified, the term "modification" used herein is used in a broad sense to include manipulations to add a ligand molecule to VP3 using these methods, thereby improving the target cell tropism, specificity, and infection efficiency of the capsid.

[0039] As used herein, the term "nucleic acid molecule" includes both singular and plural, unless otherwise specified, and encompasses single-stranded or double-stranded DNA molecules, RNA molecules, DNA-RNA hybrid molecules, as well as mutants, derivatives, or modifications thereof. Specifically, the term "nucleic acid molecule" is used to encompass a wide range of nucleic acids, including genomic DNA, plasmid DNA, mitochondrial DNA, viral genomes, artificially synthesized nucleic acids, oligonucleotides, antisense nucleic acids, siRNA, mRNA, miRNA, and sgRNA. Unless otherwise specified, the term "nucleic acid molecule" as used herein includes artificial or naturally occurring nucleic acids obtained by genetic engineering techniques, chemical synthesis techniques, enzymatic techniques, or a combination thereof. The term also includes nucleic acid molecules with chemical or biochemical modifications, such as methylation, phosphate group modifications, sugar backbone modifications (phosphorothioates, phosphoramidates, 2'-O-methylation, etc.), and base modifications (methylated cytosine, uridine derivatives, etc.). Therefore, when the term "nucleic acid molecule" is used in this specification, it should be construed as a concept that broadly encompasses these nucleic acid molecules and includes both the singular and plural forms, unless otherwise expressly limited by the context.

[0040] As used herein, the term "host cell" refers to a cell capable of introducing, maintaining, replicating, and expressing a foreign nucleic acid molecule (gene). Host cells are used as a biological system for stably or transiently introducing and expressing a specific gene or nucleic acid molecule. The host cells used in the present disclosure contain the elements necessary for producing adeno-associated virus particles. Specific host cells include, but are not limited to, human-derived cells (e.g., HEK293 cells, HeLa cells, Jurkat cells, etc.), non-human mammalian-derived cells (e.g., CHO cells, COS cells, mouse fibroblasts, etc.), insect cells (e.g., Sf9 cells, Sf21 cells, etc.), yeast cells (e.g., Saccharomyces cerevisiae, Pichia pastoris, etc.), bacterial cells (e.g., Escherichia coli, lactic acid bacteria (Lactobacillus genus), etc.), and the like. In this specification, unless otherwise specified, the term "host cell" is to be construed as a concept that broadly encompasses packaging cells used in the production of recombinant adeno-associated virus (rAAV) vectors, cells that produce target gene products, cells for gene function analysis, and the like.

[0041] As used herein, "conditions for the expression of a protein encoded by a nucleic acid molecule" refers to a cell culture environment or in vivo environment in which a gene sequence (e.g., a therapeutic gene, a reporter gene, a regulatory RNA, etc.) encoded by an introduced nucleic acid molecule is transcribed or translated in a host cell to produce a functional gene product (protein or functional RNA (including mRNA)). Specific examples of conditions for the expression of a nucleic acid molecule include a temperature suitable for the host cell (e.g., approximately 37°C for mammalian cells), an appropriate culture medium and nutrients (amino acids, glucose, vitamins, serum, etc.), optimal gas conditions (e.g., 5% CO2, adjusted oxygen concentration), and an appropriate culture time (several hours to several weeks). Furthermore, expression conditions for a nucleic acid molecule may include factors such as promoters, enhancers, inducing factors (drugs, hormones, cytokines, specific compounds), and physical stimuli (light, heat, mechanical stimuli, etc.) that control gene expression. Unless otherwise specified herein, the term "conditions for the expression of a nucleic acid molecule" is used in a broad sense to encompass a set of conditions or factors necessary for the effective and sufficient production of a desired gene product from a nucleic acid molecule in a host cell or in vivo. Examples of conditions under which a nucleic acid molecule is expressed are exemplified in the Examples of this specification, but are not limited thereto.

[0042] As used herein, "conditions for producing recombinant adeno-associated virus particles" refer to conditions (including a cell culture environment or an in vivo environment) under which a nucleic acid molecule containing a nucleic acid sequence encoding a protein constituting a recombinant adeno-associated virus particle is expressed, and a nucleic acid molecule containing a nucleic acid sequence encoding the desired protein is encapsulated in the expressed recombinant adeno-associated virus particle. This can be achieved under conditions under which host cells normally grow, provided that the necessary elements are appropriately introduced into the host cell. Examples of conditions for producing recombinant adeno-associated virus particles include a temperature appropriate for the host cell (e.g., approximately 37°C for mammalian cells), an appropriate culture medium and nutrients (amino acids, glucose, vitamins, serum, etc.), optimal gas conditions (e.g., 5% CO2, adjusted oxygen concentration), and an appropriate culture time (several hours to several weeks). Furthermore, expression conditions for nucleic acid molecules may also include factors such as promoters, enhancers, and inducible factors (drugs, hormones, cytokines, specific compounds) that control gene expression, as well as physical stimuli (light, heat, mechanical stimulation, etc.).

[0043] As used herein, the terms "a (e.g., first) nucleic acid sequence to be expressed" and "a nucleic acid sequence to be rendered expressible" are used interchangeably and refer to a nucleic acid sequence that, when introduced into a host cell, is transcribed or translated under the control of expression regulators such as an appropriate promoter or regulatory sequence, or under the control of stimuli such as chemicals, to directly or indirectly produce a gene product such as a protein or a functional RNA molecule. The term "first" is used for illustrative purposes only, and one or more nucleic acid sequences may be used in the present disclosure. Specifically, such a nucleic acid sequence includes a translated region of the encoded protein or RNA molecule and, if necessary, an untranslated region (5'UTR, 3'UTR). For expression, the nucleic acid sequence must usually be linked to a functional promoter sequence, and may further include regulatory sequences such as enhancer sequences, polyadenylation sequences, and intron sequences to improve expression efficiency and stability. For example, in the context of the present disclosure, when a nucleic acid sequence encodes a therapeutic gene (e.g., an enzyme, receptor, or cytokine) or a reporter gene (e.g., GFP, luciferase, or β-galactosidase), it is expected that the gene will be appropriately expressed in the host cell into which it is introduced and will exert its desired function. Therefore, when used herein, the phrase "a (first) nucleic acid sequence that can be made expressible" is intended to be interpreted as a concept that broadly encompasses one or more nucleic acid sequences having a structure that allows expression in a host cell, unless otherwise specified.

[0044] As used herein, the term "polypeptide" refers to a molecule in which amino acids are linearly polymerized via peptide bonds, and is used to encompass proteins or fragments thereof having a specific function or structure. That is, "polypeptide" includes naturally occurring full-length proteins, recombinant proteins, artificially synthesized proteins, as well as subsequences, fragments, derivatives, or variants thereof. As used herein, polypeptides often have specific biological functions (e.g., enzymatic activity, receptor-binding activity, antibody activity, signal transduction ability, etc.), and are used for therapeutic, diagnostic, targeting, and other applications through these functions. Furthermore, polypeptides also include those with post-translational modifications (e.g., phosphorylation, glycosylation, methylation, etc.) or tag sequences (e.g., His tag, FLAG tag, etc.). Furthermore, there are no particular limitations on the peptide length, and the term encompasses a wide range of proteins, from short oligopeptides of about 10 residues to long polypeptides of 100 residues or more, and even multimeric proteins consisting of multiple subunits. Therefore, when the term "polypeptide" is used in this specification, unless otherwise specified, it is to be construed as a broad term that includes functional proteins, protein fragments, peptide sequences, and modifications thereof, whether naturally or artificially derived.

[0045] Furthermore, the identity between the amino acid sequence of an original protein and the amino acid sequence resulting from the mutation can be easily calculated using well-known homology calculation algorithms. Examples of such algorithms include BLAST (Altschul S. F. J. Mol. Biol. 215, 403-10 (1990)), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA. 85, 2444 (1988)), and the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2, 482-9 (1981)). Furthermore, throughout this specification, "identity" refers to the identity calculated using these algorithms. Note that, in this specification, the terms "amino acid sequence homology" and "amino acid sequence identity" are used interchangeably.

[0046] In the present disclosure, when a protein is prepared by substituting amino acids in the amino acid sequence of a wild-type protein with other amino acids, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of a wild-type protein are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Mutations combining these amino acid substitutions and deletions can also be added. When amino acids are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added within the amino acid sequence of the wild-type protein or to the N-terminus or C-terminus. Mutations combining these amino acid additions, substitutions, and deletions can also be added. The amino acid sequence of the mutated protein preferably exhibits 85% or more identity, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity to the amino acid sequence of the corresponding wild-type protein.

[0047] As used herein, examples of amino acid families related to "conservative amino acid substitutions" include the following: (1) acidic amino acids aspartic acid and glutamic acid, (2) basic amino acids histidine, lysine, and arginine, (3) aromatic amino acids phenylalanine, tyrosine, and tryptophan, (4) amino acids having a hydroxyl group (hydroxyamino acids) serine and threonine, (6) neutral hydrophilic amino acids cysteine, serine, threonine, asparagine, and glutamine, (7) amino acids that affect the orientation of the peptide chain glycine and proline, (8) amide amino acids (polar amino acids) asparagine and glutamine, (9) aliphatic amino acids alanine, leucine, isoleucine, and valine, (10) amino acids with small side chains alanine, glycine, serine, and threonine, (11) amino acids with particularly small side chains alanine and glycine, (12) The branched chain amino acids valine, leucine, and isoleucine.

[0048] As used herein, "specific affinity" refers to the ability of a molecule (e.g., a ligand, antibody, receptor, or enzyme) to selectively and preferentially bind to another molecule (e.g., a target antigen, cell surface receptor, substrate, etc.), and refers to the property of exhibiting significantly higher binding affinity compared to nonspecific or unrelated molecule binding. "Specific affinity" is distinct from simple physical adsorption or nonselective interactions and involves selective binding based on intermolecular conformational compatibility or chemical complementarity. Such affinity is generally achieved through highly selective biomolecular interactions such as antigen-antibody reactions, ligand-receptor binding, and substrate-enzyme interactions. As used herein, for example, a ligand having "specific affinity" for a particular cell surface marker means that the ligand preferentially binds to the marker of the target cell compared to other cell types, or that the binding affinity (e.g., Kd value) is significantly lower (i.e., stronger). Furthermore, "specific affinity" can be quantitatively or qualitatively evaluated using parameters such as affinity constants (Ka, Kd), binding ratio, selectivity, etc., by experimental techniques such as ELISA, flow cytometry, surface plasmon resonance (SPR), etc. Therefore, when described herein as "specific affinity," unless otherwise specified, it means the property of having preferential and selective binding ability to a target molecule, and is interpreted as including affinity binding that can be clearly distinguished from binding to non-targets.

[0049] As used herein, the terms "capsid protein" (also written as "kapusido" in Japanese, but both are written as "capsid" in English) and "CAP protein" are used interchangeably and refer to the constituent proteins that form the outer shell structure of adeno-associated virus (AAV) and are the main components that make up the capsid of the virus particle (virion). CAP proteins protect the viral genome and also play central functions in the entire infection process, such as interaction with cell surface receptors, intracellular entry, uncoating, and immune recognition. AAV capsid proteins are primarily composed of three types of virion proteins (VPs): VP1, VP2, and VP3. All of these VPs are translated from a common Cap gene and are produced as polypeptides of different sizes due to differences in initiation codons. VP3 is the most important component, accounting for 80-90% of the total, while VP1 and VP2 have auxiliary functions. Furthermore, the CAP protein has regions that are displayed on the capsid surface, and in the present invention, fusing ligands or tags to these regions makes it possible to regulate the targeting and in vivo kinetics of recombinant adeno-associated virus vectors. By modifying the amino acid sequence of specific sites in the CAP protein, functional improvements such as altering serotype specificity, reducing immunogenicity, and enhancing tissue tropism can also be achieved. Therefore, unless otherwise specified, the term "capsid protein (CAP protein)" used herein is to be interpreted in a broad sense as including a group of proteins including VP1, VP2, and VP3 that constitute the AAV capsid, as well as mutants, derivatives, fusion products, and the like thereof.

[0050] As used herein, "VP1" refers to the structural protein with the highest molecular weight among the capsid-forming proteins of adeno-associated viruses (AAVs). Compared to VP2 and VP3, it encompasses the full-length sequences of these proteins and further possesses a VP1-specific N-terminal extension region. The VP1-specific region contains a phospholipase A2 (PLA2)-like active domain, which is believed to be involved in viral escape from endosomes during the infection process. Like VP2 and VP3, VP1 is translated from the cap gene, but is synthesized using an alternative initiation codon (alternative splicing). VP1 translation initiation is controlled by an upstream non-conventional ATG (e.g., ACG), and while VP1 expression is lower than that of other VPs, its functional importance is generally considered to be extremely high. The 60-mer capsid structure of AAV particles is thought to contain approximately 5-10 VP1 molecules, which influences local and global particle stability and the intracellular internalization process. The PLA2 activity of VP1 is Ca 2+ VP1 is dependent on AAV vectors, and its activity expressed in the endosomal environment during infection changes the cell membrane structure and induces the genome release process. VP1 also contains a nuclear localization signal (NLS)-like sequence and is thought to be involved in transporting the viral genome into the nucleus. Therefore, VP1 is considered herein to be an important regulatory factor in designing the gene transfer efficiency, safety, and tissue specificity of AAV vectors.

[0051] As used herein, "VP2" refers to a medium-molecular-weight structural protein that constitutes the capsid of adeno-associated virus (AAV). At the sequence level, it refers to a protein that contains the entire amino acid sequence of VP3 plus a VP2-specific N-terminal region. Like VP1, VP2 is translated from the cap gene (alternative splicing) and typically initiates at a translation initiation codon (e.g., a non-canonical ATG) located in the middle of the gene. While the physiological role of VP2 has not been fully elucidated, it may cooperate with VP1 and VP3 to ensure capsid structural stability and support the functional expression of VP1. Approximately 5-10 VP2 molecules are believed to exist in native AAV particles, and, like VP1, they are often localized. While VP2 does not possess as pronounced PLA2 activity or NLS as VP1, its structure is identical to the major portion of VP3, and it forms a stable tertiary structure through its interaction with VP3 during particle assembly. Its expression level is lower than that of VP3, and it tends to be translated at a low rate, similar to VP1. Recent studies have shown that although viral particle formation is possible even when VP2 is deleted, this may result in changes in infection efficiency and stability. In this specification, VP2 is positioned as one of the design parameters useful for optimizing the physicochemical properties and intracellular kinetics of rAAV vectors by adjusting the ratio of VP1 to VP3.

[0052] As used herein, "VP3" refers to the smallest and most abundant structural protein constituting the capsid structure of adeno-associated virus (AAV), translated from the downstream ATG initiation codon of the cap gene (by alternative splicing). VP3 accounts for approximately 80-90% of the capsid protein, and approximately 45-50 molecules of the total 60-mer are composed of VP3. VP3 shares the C-terminal sequence with VP1 and VP2 and functions as the basic framework for capsid formation. VP3 alone can form capsid particles, potentially improving packaging efficiency using a simple expression system, particularly in rAAV vector design. While VP3 primarily plays a structural role, it is also believed to contribute to resistance to external environments (e.g., acidic pH and proteases) and to the formation of binding sites for cell surface receptors. The tertiary structure of VP3 is deeply involved in AAV serotype specificity, and specific amino acid substitutions can regulate tissue tropism and immune responsiveness. Furthermore, VP3 contains a region that functions as an epitope for neutralizing antibodies, making it an important target for immune-evasive vector design. Therefore, in this specification, VP3 is positioned as a core component for structural stability and an extremely important component for regulating target tissue tropism and immunogenicity.

[0053] As used herein, "VHH" refers to the variable domain of a heavy-chain antibody derived from a camelid (e.g., llama, alpaca, camel, etc.), and is a single-chain antibody fragment also commonly referred to as a "nanobody." VHHs are significantly smaller (approximately 12-15 kDa) than conventional antibodies and are composed of a single domain structure. Despite possessing antigen-binding ability, they are characterized by functioning solely via the variable domain of the heavy chain (VH), without requiring a light chain. Furthermore, due to their excellent thermal stability and solubility, and their high permeability into cells and deep tissues, they are attracting attention for a wide range of applications, including diagnosis, therapy, and targeting carriers. In the context of the present invention, VHHs can be used as ligands to fuse or modify the capsid surface of adeno-associated viruses (AAVs) to confer binding specificity to specific cell surface antigens or receptors, thereby improving the targeting and gene transfer efficiency of recombinant AAV vectors. Therefore, unless otherwise specified, the term "VHH" used herein should be interpreted in a broad sense as meaning a single-domain antibody fragment such as those described above, including naturally or artificially obtained mutants, fusion products, modified products, etc.

[0054] As used herein, "proteins present on the surface of vascular endothelial cells" refers to membrane proteins or membrane-bound glycoproteins that are localized on the cell membrane of endothelial cells lining the luminal surface of blood vessels and are expressed so as to be in direct contact with the extracellular environment. These proteins are involved in the diverse physiological functions of vascular endothelial cells, such as substance transport, cell-cell adhesion, signal transduction, and immune response regulation, and play a central role in maintaining vascular homeostasis and in the response mechanisms in pathological conditions. The identification and confirmation of surface localization of such proteins are generally performed by flow cytometry, immunofluorescence staining, biotinylation of cell surface proteins followed by Western blot analysis, or mass spectrometry. In particular, flow cytometry analysis using fluorescently labeled antibodies allows for quantitative and highly sensitive evaluation of the level or presence of expression on the cell membrane. For these analyses, cell lines or primary cultured cells derived from vascular endothelial cells, such as human umbilical vein-derived endothelial cells (HUVEC) or human arterial endothelial cells, are suitable. In the present disclosure, the term "proteins present on the surface of vascular endothelial cells" is limited to those experimentally confirmed to be localized on the cell membrane, and therefore excludes proteins confirmed only to be expressed by mRNA or localized in the cytoplasm. Furthermore, proteins localized on the membranes of intracellular organelles such as the endoplasmic reticulum, Golgi apparatus, and mitochondria are not included in this definition.Representative examples include transferrin receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transporter 1, organic anion transporters (e.g., OATP-F, and MCT-8 as a monocarboxylate transporter), monocarboxylate transporter, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 8, and membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor, PECAM-1 (Platelet Endothelial Cell Adhesion Molecule-1) involved in adhesion between vascular endothelial cells, vascular endothelial growth factor receptor (VEGFR2) involved in angiogenesis, and ICAM-1 (Intercellular Adhesion Molecule-1) and VCAM-1 (Vascular Cell Adhesion Molecule-1) mediating adhesion with leukocytes during inflammation. Adhesion Molecule-1) and the like. Various commercially available antibodies are available for these proteins, and their expression on the surface of vascular endothelial cells can be easily confirmed by the above-mentioned analytical methods.

[0055] As used herein, "a protein having affinity for a protein present on the surface of vascular endothelial cells" refers to a protein capable of selectively or specifically binding to a specific membrane protein or glycoprotein localized on the cell membrane of vascular endothelial cells. Such proteins generally bind to target endothelial surface proteins with affinity via ligand-receptor interactions, antigen-antibody recognition, or noncovalent interactions based on structural complementarity. Typical examples of proteins with such affinity include monoclonal antibodies, antibody fragments (e.g., scFv, Fab), natural or modified ligand peptides, or fusion proteins thereof. These proteins exhibit binding activity to specific antigens or receptors on vascular endothelial cells and may be used for targeted drug delivery or as diagnostic probes. The "affinity" of the protein can be evaluated in vitro by SPR (surface plasmon resonance), ELISA (enzyme-linked immunosorbent assay), flow cytometry, cell immunostaining, or co-immunoprecipitation. In particular, quantitative evaluations generally use the Kd (dissociation constant) as an indicator, and a protein is deemed to have "affinity" when it exhibits a binding affinity on the order of nanomolar to picomolar. Furthermore, a protein is deemed to have higher specificity when selective binding to vascular endothelial cells is confirmed in a cell-based assay, while low or no binding to non-endothelial cells is observed. In the present disclosure, "proteins with affinity" include not only proteins that exist in nature but also high-affinity proteins artificially designed or selected using antibody engineering techniques, peptide libraries, protein design techniques, etc. For example, for the transferrin receptor, transferrin or anti-transferrin receptor antibodies are used. Examples of such antibodies include single-domain antibodies, single-chain antibodies such as ScFv, and Fab, which have affinity for TfR. Such proteins bind with high selectivity based on structural complementarity and chemical affinity with the target endothelial surface protein. Examples include an anti-VEGFR2 antibody that exhibits high affinity for VEGFR2, which is selectively expressed at sites of angiogenesis, and an anti-ICAM-1 scFv that specifically binds to ICAM-1, which is induced during inflammation.These proteins can be used as vascular targeting therapeutic agents, molecular imaging agents, or components of DDS (drug delivery systems), and can achieve selective action on targets on the surface of vascular endothelial cells.

[0056] As used herein, "loop" refers to a variable region in the primary structure of a polypeptide or protein that does not correspond to the secondary structures of α-helix or β-sheet, and is a structural portion that is primarily flexible while connecting adjacent secondary structural elements. Loops are generally exposed to the outside and are involved in various biological functions, such as intermolecular interactions, antigen recognition, formation of enzyme active sites, and providing structural flexibility. Loop structures are continuous regions of the primary structure in an amino acid sequence, are highly flexible in molecular dynamics, and often form spatially protruding structures. In many cases, loops are identified as regions sandwiched between adjacent stable secondary structural elements (e.g., β-strands) in three-dimensional structural analyses (X-ray crystallography, NMR, or cryo-EM). As a result, loops form longer, more complex, and irregular structures while containing structural "turns." Loops can be identified by protein structure prediction algorithms, structural modeling based on the PDB database, or three-dimensional structural analysis methods such as Ramachandran plots. The functional role of loop regions is evaluated by mutagenesis experiments, antibody epitope mapping, ligand binding assays, and the like. In the present disclosure, loops are defined based on structural characteristics and are not simply non-conserved amino acid sequence regions, but also have significance as structural regions with functional plasticity. For example, the Complementary-Determining Region (CDR) in immunoglobulin variable regions (IgV domains) is a typical loop, playing a central role in antigen recognition. Therefore, the term "loop" as used herein includes not only variable structural sites present in natural proteins, but also artificial loop structures in designed and modified peptides, which are used to confer specific configurations or functionality.

[0057] As used herein, "Loop-4" of VP refers to the variable loop region corresponding to the fourth of the loop structures present in the three-dimensional structure of the capsid-constituting protein VP in adeno-associated virus (AAV). VP is a major component constituting the capsid surface of AAV and is an important structural protein involved in viral antigenicity, interaction with neutralizing antibodies, binding to cell surface receptors, and tissue tropism. Loop-4 is a flexible region that protrudes mainly between the β-strands constituting the β-barrel structure in the tertiary structure of VP, and is known to be exposed on the outer surface of the virus particle. Loop-4 is functionally important in interactions with antibodies and cellular receptors and has attracted widespread attention as a region particularly involved in the serotype specificity and immunogenicity of AAV. The specific sequence position of Loop-4 varies depending on the serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), but when aligned to AAV9, it corresponds to approximately positions 435 to 465 (e.g., 459) of SEQ ID NO: 3 in the amino acid sequence of VP, and amino acid substitutions or insertions in this region have been reported to affect the ability to escape from neutralizing antibodies and to confer new tissue tropism. Identification of Loop-4 was performed by three-dimensional structural analysis based on known AAV capsid three-dimensional structures (e.g., PDB ID: 1LP3, 3NG9, 7KNP, etc.), and positioning and structural visualization at the amino acid level were possible using various modeling software (PyMOL, SWISS-MODEL, etc.). Furthermore, functional analysis can be performed to verify its biological function and the region involved by site-directed mutation, antibody epitope mapping, cell infection assays, receptor binding assays, etc. As used herein, "VP Loop-4" includes not only the native sequence, but also mutant Loop-4 regions that have been artificially mutated, inserted, substituted, or modified, which are designed for the purposes of altering tissue tropism, improving immune evasion, or conferring novel targeting.Therefore, "Loop-4 of VP" refers to a structurally exposed flexible region on the surface of the AAV capsid, and as a functional region involved in antigenicity and cell tropism, it is an important site that can be targeted for structural design and functional modification.

[0058] As used herein, "Loop-5" of VP refers to a highly flexible region that protrudes from the surface of the VP, sandwiched between the β-barrel structures, and has different sequences, lengths, and configurations depending on the serotype. The length varies depending on the serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), but is consistent with the length, length, and configuration of AAV9 or other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03). When aligned with AAV9, Loop-5 corresponds to approximately amino acid residue position 500 (e.g., 489 to 510 of SEQ ID NO: 3) (e.g., 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506), and it has been reported that amino acid substitution, insertion, or deletion in this region significantly changes the virus's ability to escape from neutralizing antibodies, its ability to bind to target cells, and its infection efficiency.

[0059] Identification of Loop-5 can be performed by three-dimensional structural analysis based on known AAV capsid structures (e.g., AAV2 PDB ID: 1LP3, AAV9 PDB ID: 3UX1, etc.). Sequence comparison, structural modeling, and molecular dynamics analysis have identified it as a protruding loop between β-strands and distinguished it from other loops (Loop-1 to Loop-4). These analyses are performed using structural visualization and modeling tools such as PyMOL, UCSF Chimera, and SWISS-MODEL. The functional significance of the Loop-5 region will be elucidated through mutagenesis experiments to evaluate neutralizing antibody evasion, cell infection experiments, receptor binding assays, and other methods. In particular, Loop-5 is known to be involved in interactions with heparan sulfate proteoglycans (HSPGs) and other cell surface glycans in AAV2 and AAV9. Furthermore, in the present disclosure, "Loop-5 of VP" includes not only sequences derived from natural AAV serotypes, but also artificially designed mutants, such as Loop-5 sequences modified by amino acid substitution, insertion, deletion, or chimeric structures with different serotypes, which can confer new immune evasion properties, target cell selectivity, or tissue tropism.

[0060] Therefore, "VP Loop-5" is a structurally flexible and functionally important variable region present on the surface of the AAV capsid, and represents a potential site of modification for regulating the infectivity and immunological properties of the virus.

[0061] As used herein, "Loop-8" of VP refers to the eighth variable region among the loop structures present in the three-dimensional structure of the capsid-constituting protein VP of adeno-associated virus (AAV). VP is a major component forming the outer shell of AAV particles, and is based on a "β-barrel" structure in which multiple β-strands are folded into a barrel shape. The loop structures protruding outward from between these strands are involved in the antigenicity, receptor binding, and tissue tropism of the virus. Loop-8 is one such variable loop, and is a structural region present on the surface of the AAV capsid that is exposed to the external environment. The sequence and length vary depending on the serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), but may be different from, for example, AAV9 or other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11). When the AAVs (AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-DJ, AAV-LK03) are aligned with AAV9, Loop-8 is located approximately at amino acid residue 590 (e.g., near residues 570 to 610) (e.g., 589, 590, 591, 592, 594, 595, etc.) in SEQ ID NO: 3. This region can serve as a major epitope for neutralizing antibodies and is also involved in interactions with cell surface glycans and receptor proteins. The three-dimensional structure and functional contribution of Loop-8 have been elucidated by X-ray crystallography, cryo-electron microscopy (Cryo-EM), and comparative modeling, and Loop-8 is particularly positioned as a region overlapping with the loop structure common to the VP3 domain. Loop-8 is one of the promising target regions for structural modification aimed at controlling immunogenicity and modifying target tissue selectivity. The functional properties of this region are analyzed through site-directed mutagenesis, receptor binding assays, viral infection efficiency assessment tests, and antibody neutralization tests. For example, specific amino acid substitutions in Loop-8 may be able to evade neutralizing antibody binding or confer infection ability to new cell types.As used herein, "VP Loop-8" includes not only sequences contained in natural AAV serotypes, but also artificially modified sequences, such as regions constructed by chimerization with other types of Loop-8, or by substitution, insertion, or deletion of amino acid sequences. This makes it possible to optimize the tissue specificity and immune evasion ability of modified AAV vectors. Therefore, "VP Loop-8" is one of the functionally and structurally important variable regions in the AAV capsid structure, and in the present invention, it is positioned as a design target that enables control of infection characteristics, immune responses, drug delivery efficiency, and the like. Structural modifications targeting Loop-8 are useful for controlling the tissue tropism and immune evasion ability of AAV, and are particularly useful in developing AAV vectors aimed at improving nervous system delivery efficiency. For example, AAV-PHP.B, an AAV9-based mutant, and its improved variant AAV-PHP.B are examples of such modifications. By introducing specific amino acid substitutions into the Loop-8 region, eB can efficiently cross the mouse blood-brain barrier (BBB) ​​and enable highly efficient gene delivery to the central nervous system. In these mutants, modification of capsid surface residues located in Loop-8 has been shown to enhance neuronal tropism via the Ly6a receptor. A specific example of a mutation is the substitution deletion of amino acid residues located in Loop-8 in AAV9, which has the effect of improving selective delivery and immune tolerance without significantly impairing the capsid structure. Furthermore, AAV-PHP.S has been optimized for delivery to the peripheral nervous system and peripheral organs by similar modifications to residues around Loop-8, demonstrating that Loop-8 is a key region for controlling tissue selectivity. Furthermore, attempts have been made to confer new target cell selectivity to ligand-modified AAVs by inserting peptide motifs into the loop region containing Loop-8. For example, by inserting peptide sequences for integrins or EGFR into the Loop-8 region, it is possible to artificially design targeting of AAV vectors to tumor cells or specific tissues. These modified AAV vectors are currently being used in preclinical and clinical trials, and constitute an important technological platform for centrally targeted gene therapy, particularly for neurodegenerative diseases (e.g., ALS, SMA, Alzheimer's disease) and retinal diseases.Therefore, Loop-8 is not merely a structural region, but is extremely useful as a modification point for controlling the functionality of AAV vectors and achieving highly accurate delivery to therapeutic targets. In the present invention, too, by introducing specific mutations into the capsid region containing Loop-8, novel AAV vectors with desirable tissue tropism or immunological properties can be provided. Preferred mutations in various LOOPs are described elsewhere in this specification and demonstrated in the Examples.

[0062] As used herein, the term "linker" refers to a sequence or molecular structure used to physically or chemically link two or more components, such as peptides, protein domains, nucleic acid sequences, drugs, nanoparticles, etc. Linkers are designed to adjust the conformation, flexibility, functional independence, and interactivity between the linked components, and their length, structural units, flexibility, stability, etc., significantly affect the functionality of the linked components. Linkers in peptide or protein molecules are typically composed of specific amino acids, and sequences rich in glycine or serine, such as repeats of Gly-Gly-Gly-Gly-Ser, are preferred to provide flexibility and mobility. Specific examples are exemplified herein in SEQ ID NOS: 79-91, but are not limited to these. Alternatively, linkers may be designed for specific purposes, such as more rigid linkers, enzymatically cleavable linkers (linkers containing a protease cleavage site), or linkers containing disulfide bonds that are cleaved under reducing conditions. Linkers in nucleic acids function as spacers between oligonucleotides and can be any nucleotide sequence or non-nucleotide structure (e.g., PEG linkers, alkyl chains). In pharmaceuticals and biomaterials, chemical structures that enable reversible binding or environmentally responsive cleavage (e.g., acid-hydrolyzable linkers, photocleavable linkers) are used as linkers. Linker identification and characterization are performed by sequence analysis, mass spectrometry, structural analysis (NMR, X-ray crystallography), etc. Furthermore, comparative evaluation of the biological activity, stability, intracellular localization, etc. of the linked complex is useful for functional validation. Specific examples of linkers in the present disclosure include linkers between variable regions in bispecific antibodies, linkers between scaffolds that are components of CAR-T cells, and chemical linkers that connect drugs and antibodies in protein-drug conjugates (ADCs). In the present disclosure, a "linker" refers to a structural intermediate that effectively connects functional units and maintains or controls a desired biological function.Examples of linkers in the present disclosure are described and exemplified elsewhere in the specification, and those skilled in the art can refer to them as appropriate. It is understood that linkers known in the art that are not explicitly described in the specification can also be used as appropriate.

[0063] As used herein, the term "anchor" refers to a structure or sequence for physically or functionally fixing or localizing a specific molecule, component, or complex to a predetermined location or structure. As used herein, the term "anchor" refers to the other protein (A) used in a fusion protein, which must be capable of exerting the function of the other protein (A). Anchors are used for purposes such as membrane localization of biomolecules, targeting to intracellular organelles, immobilization to substrate surfaces, or positional control in higher-order complex formation. In biological contexts, anchors can have various origins, such as proteins, peptides, lipids, sugar chains, or artificial synthetic structures, and their structure and function are designed according to the desired localization environment. For example, membrane anchors with transmembrane domains are used as a means of immobilizing proteins on cell membranes. Meanwhile, glycosylphosphatidylinositol (GPI) anchors are added to the C-terminus of proteins to bind them to the lipid bilayer on the outer surface of the cell membrane. Known chemical anchors include high-affinity tags, such as biotin-streptavidin binding systems, and immobilization techniques based on silane coupling agents and click chemistry. These anchors are widely used for surface modification of biomaterials, diagnostic chips, and other devices. The presence and function of anchors can be confirmed by localization analysis (immunofluorescence staining, confocal microscopy), membrane fractionation analysis, immobilization efficiency evaluation, and biomolecular interaction analysis (SPR, ELISA, etc.). These methods enable quantitative and qualitative verification of whether a target molecule is stably positioned at a predetermined location via an anchor. Components used as "anchors" in the present invention include domains that control the membrane surface localization of proteins, high-affinity binding sites between ligands and receptors, tags for fixation to nanoparticle surfaces, or cross-linking sites with polysaccharide substrates. This enables control of the spatial arrangement, in vivo kinetics, and localized drug release of the complex. Therefore, an "anchor" is a structural relay element designed to spatially or functionally stabilize or localize a functional molecule, and its positioning plays an essential role in the functional expression of the present invention.Examples of anchors in this disclosure are described and exemplified elsewhere in the specification, and those skilled in the art can refer to them as appropriate, and it is understood that anchors known in the art that are not explicitly described in the specification can also be used as appropriate.

[0064] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a specific antigen, and is used in a broad sense to include not only naturally occurring full-length antibodies, but also their functional substructures, variants, fusions, and engineered molecules. In other words, "antibody" includes not only full-length antibodies belonging to immunoglobulin isotypes such as IgG, IgA, IgM, IgD, and IgE, but also all molecular forms that retain antigen-binding ability. Antibodies are typically composed of two heavy chains (H chains) and two light chains (L chains), each of which has a variable region and a constant region. The variable regions contain complementarity-determining regions (CDRs) involved in antigen binding, and the antigen specificity is determined by the amino acid sequence of these regions. In the present invention, antibody fragments, i.e., Fab (antigen-binding fragment), Fab', and F(ab') are also included. ２The term "antibody" also includes polyvalent antibody structures such as Fv (variable fragment), scFv (single-chain variable fragment) in which these are linked in a single chain, and diabodies, trimerbodies, tandem scFv (taFv) and other dimerized structures for stabilization. Also included are single-chain antibody domains derived from camelids, known as VHH (variable domain of heavy-chain antibodies) or nanobodies, and humanized VHH antibodies engineered based on these. Furthermore, antibodies artificially modified in species origin or sequence composition, such as chimeric antibodies (e.g., mouse variable region + human constant region), humanized antibodies (e.g., only CDR regions are mouse-derived, while framework and constant regions are human-derived), and fully human antibodies (e.g., derived from human antibody libraries or transgenic animals), are also included. Additionally, Fc-mutated antibodies in which effector functions (e.g., ADCC, CDC) are reduced or enhanced by introducing mutations into the Fc region, pH-sensitive antibodies, and antibodies with extended half-lives, as well as antibody fusions and antibody-drug conjugates (ADCs) in which other molecules (e.g., enzymes, drugs, peptides, nucleic acids, tags) are fused or conjugated to antibodies, are also included. The affinity and specificity of antibodies are measured by methods such as ELISA, flow cytometry, surface plasmon resonance (SPR), biolayer interferometry (BLI), immunoprecipitation, immunostaining, and antigen neutralization assays, and the results confirm specific and high-affinity binding to the target antigen. Specific examples include anti-EGFR scFv designed as scFv, anti-PD-L1 VHH, humanized anti-CD3ε antibody, fully human anti-IL-6R antibody, Fc-silenced anti-CD47 antibody, trastuzumab emtansine (T-DM1) which is a HER2-targeting ADC, and blinatumomab (CD3 x CD19) which is a bispecific antibody. For diagnostic purposes, antibody-reporter fusions fused with HRP (peroxidase) or fluorescent proteins are also included.Therefore, the term "antibody" as used herein broadly encompasses structures that have an immunoglobulin-like domain structurally or sequentially and can specifically bind to a desired antigen, and refers to functional molecules in general that have specific recognition ability, regardless of their origin, form, or whether or not they have been modified. As used herein, "anti-XXX antibodies," such as "anti-transferrin receptor antibodies," refer to any antibody identified with XXX as an antigen, and their descriptions are provided herein and can be appropriately recognized by those skilled in the art using publicly known information in the field. Furthermore, when the target is another entity YYY (e.g., VHH), it is referred to as "anti-XXXYYY," and a specific example is "anti-transferrin receptor VHH."

[0065] As used herein, the "ratio" of the "number of molecules" of a first nucleic acid sequence to a second nucleic acid sequence, etc., refers to the quantitative relationship between the abundance, specifically the number of molecules, molar amount, or concentration, of two or more types of molecules having different sequences, such as a first nucleic acid sequence and a second nucleic acid sequence, of each nucleic acid molecule. Such a ratio is typically expressed in a format where the number of molecules of the first nucleic acid sequence is the numerator and the number of molecules of the second nucleic acid sequence is the denominator, i.e., "first nucleic acid sequence:second nucleic acid sequence = X:Y" or "X / Y." This "ratio" is typically used to define the component ratio in cell introduction, in vitro reaction systems, or nucleic acid recombinant constructs, and is an important design parameter that affects the expression level, translation efficiency, or expression of a desired biological function of each nucleic acid. The ratio is expressed as a molecular copy ratio, molar ratio, or weight ratio, but unless otherwise specified herein, it is interpreted as a molecular copy ratio based on a molar ratio. Measurement and confirmation of this ratio can be performed using quantitative analysis by real-time PCR (qPCR), digital PCR, or next-generation sequencing (NGS), or by quantitative scanning after electrophoresis, spectrophotometer, or fluorescence measurement. Control may be based on the mixing ratio before introduction or when added to the reaction system, or the actual ratio may be evaluated based on the expression level or copy number after introduction. For example, if a first nucleic acid sequence encodes an expression regulator and a second nucleic acid sequence encodes a polypeptide of interest, and the former is co-introduced with the latter at a ratio of 1:5, this means that there is one molecule of the first sequence and five molecules of the second sequence. Conversely, a 1:1 ratio is intended to ensure equal expression balance between the two. In the present invention, a specific ratio may be an important parameter for achieving a desired biological activity (e.g., induction of cell function, optimization of protein expression, control of differentiation, etc.). Therefore, the "ratio" is not merely a description of the amount present, but has technical significance as a constituent element intended for functional optimization.

[0066] As used herein, the "molecular number ratio" of mRNA refers to the relative relationship between the number of each molecule among multiple types of messenger RNA (mRNA) molecules. The "molecular number" here refers to the quantitatively measured number of mRNA copies or its equivalent molar amount (mol), and indicates the amount of one mRNA and another mRNA present at a specific time point or state. The "mRNA molecular number ratio" can be expressed, for example, as the ratio of a first mRNA to a second mRNA in the form of "first mRNA:second mRNA = X:Y" or "X / Y." Herein, however, it is appropriately expressed as a percentage (wt % or % by number of molecules, although in the case of mRNAs with similar molecular weights, as in the present disclosure, both values ​​will be approximately the same). This is typically a design parameter for optimizing the quantitative control of translation products in cells, the balance of protein expression, or the activation level of signaling pathways. Unless otherwise specified, the "mRNA molecular number ratio" is interpreted as referring to a ratio based on the number of moles or copy numbers. This ratio can be determined as the mixture ratio at the time of mRNA introduction into cells, or evaluated by measuring the amount of mRNA actually expressed in cells. In the former case, this refers to the mixture ratio of in vitro transcription products or the composition ratio of mRNA pharmaceutical formulations, while in the latter case, it is measured using quantitative molecular biology techniques such as real-time RT-PCR (qRT-PCR), digital PCR, and RNA-Seq. For example, in an mRNA vaccine that induces an immune response, when mRNA encoding an antigen and mRNA encoding an immunostimulatory factor (e.g., CD80, CD86, GM-CSF, etc.) are simultaneously administered, the molecular ratio of the two mRNAs can be an important parameter for achieving optimal immune induction. It is known that blending antigen mRNA and adjuvant mRNA at a ratio of 5:1, 1:1, or 1:3, respectively, results in differences in the degree of immune cell activation and the durability of the response. Furthermore, in the present disclosure, for the purpose of simultaneously expressing multiple proteins, it is desirable to control the molecular ratio between mRNAs while taking into account the translation efficiency and stability of each mRNA.This makes it possible to precisely adjust the quantitative balance of each protein produced within the cell, thereby enabling the efficient formation of functional multiprotein complexes and the expression of downstream physiological activities.

[0067] As used herein, the term "length" of an amino acid refers to the number of amino acid residues constituting a polypeptide or protein, and is defined as the total number of consecutive amino acids contained in a specific amino acid sequence. That is, it refers to the number of positions in the amino acid sequence (the number of residues from the N-terminus to the C-terminus) in the primary structure generated by translation, and is used as an indicator for the design of specific structural domains, epitopes, functional motifs, linker sequences, etc. This "length" is the number of amino acid residues in the polypeptide sequence, and is usually expressed as an integer value (e.g., 15 amino acids, 35 amino acids, 120 amino acids, etc.). Length can be estimated by simply counting the number of residues based on sequence information; when the amino acid sequence is explicitly stated, the length of the sequence directly corresponds to the "length."

[0068] In this specification, "length" refers to the total number of residues comprising the translated contiguous sequence from the initiating methionine residue at the N-terminus to the final residue at the C-terminus. For example, if a protein is composed of a total of 300 residues, its length is considered to be 300 amino acids. Furthermore, when a peptide fragment is described as having a length of 15 residues, this refers to a peptide composed of 15 amino acids. "Length" is an extremely important design parameter that takes into account the impact on functionality and three-dimensional structure in antigen peptide design, linker sequence design, structural domain excision, tag sequence addition, and the like. For example, it is known that epitope peptide lengths that induce immune responses are suitable for MHC class I and MHC class II, respectively. Furthermore, for artificially designed linker sequences, lengths of approximately 10 to 30 amino acids may be preferred to ensure flexibility. On the other hand, shorter (3 to 5 residues) or longer (50 or more residues) linkers may be used for purposes such as controlling the three-dimensional configuration of binding sites or functional separation. Therefore, the term "amino acid length" is a concept that quantitatively indicates a structural or functional unit defined by the number of amino acid residues contained in a sequence, and in the present invention, it is a basic design element for functional construction and optimization of a target polypeptide.

[0069] As used herein, the term "average number of ligands in an AAV particle population" refers to the number average, unless otherwise specified, and is a value measured by the following method. However, when the ligands are the same or similar, the weight average may also be used since the molecular weights are approximately similar. That is, the number of ligands attached to each particle among all AAV particles present in a particle population is tallied and the total value is divided by the number of particles. This number average value is useful as an index for quantitatively evaluating the attachment state of the entire population, even when ligands are not attached uniformly to individual particles. The average number of ligands can be measured by the following methods: 1) A method in which AAV particles whose mobility has changed due to ligand attachment are separated using electrophoresis (e.g., SDS-PAGE or isoelectric focusing), and the degree of ligand attachment is estimated based on the area ratio of each obtained peak. 2) A method in which the amount of ligand in a sample is calculated based on comparison with a dilution series of a standard specimen using an immunoassay such as ELISA using a specific antibody. This makes it possible to derive the average amount of ligand attached per particle. 3) A method in which the proportion of particles with ligands attached (the percentage of attached particles) is calculated separately using bead pull-down or immunoprecipitation in addition to quantification by electrophoresis, and the average number of attached particles is corrected based on this. 4) A method in which the presence or absence of ligands on individual particles is visually confirmed by observation using a transmission electron microscope (TEM) or cryo-electron microscope (cryo-EM), and the average number of attached particles is estimated by statistically processing the frequency of such observations. 5) A method in which the average number of attached ligands is determined by fractionating particles according to the number of attached ligands using affinity column chromatography or size exclusion chromatography, and then quantifying the composition ratio of each fraction. These measurement methods can be used alone, or combining multiple methods can enable more accurate estimation of the average number of attached ligands. The method selected is appropriate depending on the sample properties, analytical purpose, required accuracy, and experimental system constraints. The "number average" in this definition is a statistical quantity distinct from weight average and area average, and is an important indicator in analyses that take into account variations in physicochemical properties.

[0070] (Description of Preferred Embodiments) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. It is also understood that the following embodiments can be used alone or in combination.

[0071] (Summary of the Present Disclosure) In gene therapy using AAV, a recombinant AAV genome (rAAV genome) in which a part of the wild-type AAV genome has been replaced with a foreign gene is administered to a patient in the form of a recombinant AAV particle (rAAV particle) encapsulated in a capsid protein. The rAAV genome is, for example, a wild-type AAV genome in which a region including the Rep gene and the Cap gene has been replaced with a gene encoding a foreign protein. The term "recombinant AAV genome" is synonymous with and interchangeable with the rAAV genome. The term "recombinant AAV particle" is synonymous with and interchangeable with the terms rAAV particle, recombinant AAV virion, and rAAV virion. Depending on the context, "recombinant AAV particle" may also be simply referred to as "AAV particle" or "AAV virion." The term "capsid protein" refers to the protein that constitutes the capsid of the rAAV particle. The term "CAP protein" includes VP1, VP2, and VP3, as well as fusion proteins of these proteins with another protein (A). The term "CAP protein" refers to all of the proteins that function as components of the capsid of rAAV particles, or any one or more of these proteins. However, in this specification, the term "CAP protein" also includes fusion proteins of another protein (A) depending on the context.

[0072] [Production Method] In one aspect, the present disclosure provides a method for producing a recombinant adeno-associated virus particle or recombinant adeno-associated virus vector having a ligand on its surface. More specifically, the present disclosure provides a method for producing a recombinant adeno-associated virus particle or recombinant adeno-associated virus vector having a ligand on its surface, comprising the steps of: (A) introducing into a host cell one or more nucleic acid molecules containing a VP nucleic acid sequence that, upon transfection, enables expression of VP1, VP2, VP3, and VP3 modified with the ligand, and, if necessary, a nucleic acid molecule containing a nucleic acid sequence encoding a desired protein; and (B) subjecting the host cell to conditions under which the recombinant adeno-associated virus particle is produced. Alternatively, in another aspect, the present disclosure provides a method for producing recombinant adeno-associated virus particles having a ligand on their surface, the method comprising the steps of: (A) introducing into a host cell (1) a first nucleic acid sequence that, upon introduction, enables expression of VP1, VP2, and VP3; (2) a second nucleic acid sequence that, upon introduction, enables expression of ligand-modified VP3; and (3) a nucleic acid sequence encoding a desired protein; and (B) subjecting the host cell to conditions under which the recombinant adeno-associated virus particles are produced. The host cell used in the present disclosure contains the elements necessary for producing recombinant adeno-associated virus-like particles (referred to as rAAV particles, which correspond to a type of VLP in the present disclosure). In a preferred embodiment, the first nucleic acid sequence and the second nucleic acid sequence may be introduced as separate nucleic acid molecules. The elements necessary for producing adeno-associated virus particles are described in detail below. A nucleic acid sequence encoding a desired protein is incorporated into the recombinant adeno-associated virus particle under conditions that result in the particle being produced, and then expressibly incorporated into the particle.

[0073] In certain embodiments, the host cell used in the methods of the present disclosure contains the elements necessary for producing adeno-associated virus particles. In certain embodiments, the necessary elements include a nucleic acid sequence encoding a Rep protein. More particularly, the nucleic acid sequence encoding the Rep protein and the VP nucleic acid sequence may be located between at least two inverted terminal repeats (ITRs), either individually or together. Alternatively / in addition, the necessary elements used in the present disclosure further include a nucleic acid sequence encoding a protein responsible for helper function. Specifically, the protein responsible for helper function includes at least one, two, three, four, or all five proteins selected from the group consisting of E1A, E1B, E2A, VA1, and E4. In specific embodiments, the protein responsible for helper function, when two or more proteins are involved, is located between at least two inverted terminal repeats (ITRs), either individually or together. In another embodiment, the nucleic acid sequence encoding a desired protein is located between at least two inverted terminal repeats (ITRs). In particular embodiments, desired proteins include therapeutic proteins, proteins for genome editing, experimental proteins, and the like.

[0074] A nucleic acid sequence encoding a desired protein used in the present disclosure is incorporated into the recombinant adeno-associated virus particle under conditions that result in the particle being produced, and then incorporated into the particle in an expressible state.

[0075] Three types of plasmids are typically used to produce rAAV particles: (1) a plasmid (plasmid 1) having a structure containing a nucleotide sequence including a first inverted terminal repeat (ITR) and a nucleotide sequence including a second inverted terminal repeat (ITR) derived from a virus such as AAV, and a gene encoding a desired protein located between these two ITRs; (2) a plasmid (plasmid 2) containing an AAV Rep gene (Rep region) that has the functions necessary for integrating the nucleotide sequence of the region sandwiched between the ITR sequences (including the ITR sequence) into the genome of a host cell, and a gene encoding an AAV capsid protein (Cap region); and (3) a plasmid (plasmid 3 or helper plasmid) containing the E2A region, E4 region, and VA1 RNA region of adenovirus. Accordingly, the host cells of the present disclosure may contain or be introduced with these plasmids. Furthermore, the Cap (capsid) gene refers to a single gene region encoding capsid-constituting proteins (virion proteins, VP) VP1, VP2, and VP3, and three types of VP (VP1, VP2, VP3) are generated from the Cap gene due to differences in splicing and translation initiation sites, and therefore, it can be said that the features of the present disclosure are features of plasmid 2. Furthermore, when modifying the function or recombining VP, the basic principle is to manipulate the Cap gene.

[0076] Generally, to produce recombinant adeno-associated virus (rAAV) virions, these three types of plasmids are first introduced into host cells, such as HEK293 cells, whose genomes contain adenovirus E1a and E1b genes. Then, a region containing a base sequence including a first inverted terminal repeat (ITR), a base sequence including a second inverted terminal repeat (ITR), and a gene encoding a desired protein located between these two ITRs is integrated into the genome of the host cell. Single-stranded DNA is replicated from this region and packaged into AAV capsid proteins to form recombinant adeno-associated virus (rAAV) virions. These recombinant adeno-associated virus (rAAV) virions are infectious and can be used to introduce foreign genes into cells, tissues, or living organisms. In the present disclosure, the foreign gene or the foreign protein encoded thereby can be used as the desired protein.

[0077] In one embodiment of the present invention, rAAV particles are produced by introducing three types of plasmids, plasmids 1 to 3, into host cells. In another embodiment, a plasmid in which two of the above plasmids 1 to 3 are ligated is used. The ligated plasmids may be any combination of plasmids 1 and 2, plasmids 2 and 3, or plasmids 1 and 3. rAAV particles can be produced by introducing this ligated plasmid and the remaining two types of plasmids into host cells. In yet another embodiment, a plasmid in which three of the above plasmids 1 to 3 are ligated is used. In this case, rAAV particles can be produced by introducing only this ligated plasmid into host cells.

[0078] In either case, a nucleic acid molecule comprising: (a) a nucleotide sequence encoding an adeno-associated virus Rep protein or a functional equivalent thereof; (b) a nucleotide sequence encoding an adeno-associated virus CAP protein or a functional equivalent thereof; (c) a nucleotide sequence comprising a first inverted terminal repeat (ITR); (d) a nucleotide sequence comprising a second inverted terminal repeat (ITR); (e) a nucleotide sequence encoding a foreign protein located between the first and second ITRs; (f) a nucleotide sequence encoding an adenoviral E2A protein or a functional equivalent thereof; (g) a nucleotide sequence encoding an adenoviral E4 protein or a functional equivalent thereof; and (h) a nucleotide sequence encoding an adenoviral VA1 RNA or a functional equivalent thereof is introduced into a host cell.

[0079] Furthermore, in one embodiment of the present invention, a base sequence including a first gene expression control site that controls the expression of the Rep protein, a base sequence including a second gene expression control site that controls the expression of the CAP protein, and a base sequence including a third gene expression control site that controls the expression of the foreign protein are introduced into a host cell.

[0080] The Rep protein of adeno-associated virus (AAV) is encoded by the Rep gene of AAV. The Rep protein has a function necessary for integrating, for example, the AAV genome into the genome of a host cell via the ITRs present in the genome. There are multiple subtypes of Rep proteins, but two types, Rep68 and Rep78, are required for the integration of the AAV genome into the genome of a host cell. Rep68 and Rep78 are translation products of two types of mRNA transcribed by alternative splicing from the same gene. In the present invention, the term "AAV Rep protein" includes at least the two types of proteins, Rep68 and Rep78.

[0081] In one embodiment of the present invention, the nucleotide sequence encoding the Rep proteins of adeno-associated virus refers to a nucleotide sequence encoding at least Rep68 and Rep78, or a nucleotide sequence containing a mutation therein. The Rep proteins are preferably those of AAV serotype 2, but are not limited thereto, and may be those of any of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0082] Furthermore, as long as Rep68 exerts its function, it may be modified by substitution, deletion, addition, or the like in the amino acid sequence of wild-type Rep68 of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In the present invention, Rep68 with these mutations is also included in Rep68.

[0083] When amino acids in the amino acid sequence of wild-type Rep68 are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of wild-type Rep68 are deleted, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Rep68 obtained by adding mutations that combine these amino acid substitutions and deletions is also included in Rep68. When amino acids are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus of wild-type Rep68. Rep68 obtained by adding mutations that combine these amino acid additions, substitutions, and deletions is also included in Rep68. The amino acid sequence of the mutated Rep68 preferably exhibits 85% or more identity to the amino acid sequence of wild-type Rep68, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.

[0084] Furthermore, as long as Rep78 exerts its function, it may be modified by substitution, deletion, addition, or the like in the amino acid sequence of wild-type Rep78 of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In the present invention, Rep78 with these mutations is also included in Rep78.

[0085] When amino acids in the amino acid sequence of wild-type Rep78 are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of wild-type Rep78 are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Rep78 obtained by adding mutations that combine these amino acid substitutions and deletions is also included in Rep78. When amino acids are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus of wild-type Rep78. Rep78 obtained by adding mutations that combine these amino acid additions, substitutions, and deletions is also included in Rep78. The amino acid sequence of the mutated Rep78 preferably exhibits 85% or more identity to the amino acid sequence of wild-type Rep78, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.

[0086] Substitution of an amino acid in the amino acid sequence of Rep68 with another amino acid occurs within a family of amino acids that are related, for example, by their side chains and chemical properties, and is predicted to not significantly alter the function of anti-hRep68 (i.e., be a conservative amino acid substitution).

[0087] A functional equivalent of an AAV REP protein refers to a substance that can be used functionally in place of Rep68, in the case of Rep68, and refers to a substance that can be used functionally in place of Rep78, in the case of Rep78.

[0088] In one embodiment of the present invention, a nucleotide sequence encoding an adeno-associated virus CAP protein refers to a nucleotide sequence encoding at least VP1, a protein constituting the AAV capsid, or a nucleotide sequence containing a mutated nucleotide sequence thereof. VP1 is preferably that of AAV serotype 9, but is not limited thereto and may be any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 10, or 11. For example, VP1 of wild-type serotype 6 AAV has the amino acid sequence set forth in SEQ ID NO: 1. The nucleotide sequence encoding VP1 of wild-type serotype 8 AAV has the amino acid sequence set forth in SEQ ID NO: 2. VP1 of wild-type serotype 9 AAV has the amino acid sequence set forth in SEQ ID NO: 3. When referring to the amino acid positions of VPs in the present disclosure, unless otherwise specified, the positions are those aligned with the serotype 9 sequence as a reference. Therefore, it will be understood that even for other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV10, AAVrhlO, AAV11, AAV11, AAV-DJ, AAV-LK03, etc.), the amino acid positions should be understood as being replaced with the amino acid positions when aligned with AAV9.

[0089] In one embodiment of the present invention, the term "adeno-associated virus inverted terminal repeat (ITR)" refers to a base sequence essential for integration of an adeno-associated virus gene into the genomic sequence of a host cell by non-homologous recombination. In one embodiment of the present invention, two adeno-associated virus inverted terminal repeats (ITRs) are present in a nucleic acid molecule, and are referred to as a first adeno-associated virus inverted terminal repeat (ITR) and a second adeno-associated virus inverted terminal repeat (ITR), respectively. Here, when a gene encoding a foreign protein is placed between the two ITRs, the ITR located on the 5' side is referred to as the first adeno-associated virus inverted terminal repeat (ITR), and the ITR located on the 3' side is referred to as the second adeno-associated virus inverted terminal repeat (ITR).

[0090] The inverted terminal repeats (ITRs) are preferably those of AAV serotype 2, but may be any of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10 or 11, without limitation.

[0091] Furthermore, as long as the inverted terminal repeat (ITR) can exhibit its function, it may be one in which the nucleotide sequence of a wild-type ITR of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 has been modified by substitution, deletion, addition, or the like. ITRs with these mutations are also included in the ITR.

[0092] When bases in the base sequence of a wild-type ITR are substituted with other bases, the number of substituted bases is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When bases in the base sequence of an ITR are deleted, the number of deleted bases is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. An ITR with a mutation that combines these base substitutions and deletions is also considered an ITR. When bases are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 bases are added to the base sequence or the 5' end or 3' end of the wild-type ITR. An ITR with a mutation that combines these base additions, substitutions, and deletions is also included in the ITR. The base sequence of the mutated ITR preferably exhibits 85% or more identity with the base sequence of the wild-type ITR, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.

[0093] A functional equivalent of an AAV ITR refers to a substance that can be used functionally in place of an AAV ITR. In addition, an ITR artificially constructed based on an AAV ITR is also considered a functional equivalent of an AAV ITR as long as it can replace an AAV ITR.

[0094] In one embodiment of the present invention, a base sequence for inserting a base sequence encoding a foreign protein and / or a base sequence encoding a foreign protein is present between the inverted terminal repeat (ITR) of the first adeno-associated virus and the inverted terminal repeat (ITR) of the second adeno-associated virus.

[0095] In one embodiment of the present invention, the "nucleotide sequence for inserting a nucleotide sequence encoding a foreign protein" refers to a nucleotide sequence containing a nucleotide sequence that can be specifically cleaved with a restriction enzyme. This also includes so-called multicloning sites. This nucleotide sequence can be cleaved with a restriction enzyme, and a nucleic acid molecule encoding a desired foreign protein can be inserted into the cleavage site.

[0096] In one embodiment of the present invention, the foreign protein that can be encoded in a nucleic acid molecule is not particularly limited. When the foreign protein is derived from a specific biological species, the biological species is not particularly limited, and the protein may be a protein encoded in the genome of a prokaryotic or eukaryotic cell. Examples of eukaryotic cells include fungi, yeast, insects, protozoa, amphibians, reptiles, birds, mammals, and plants. Furthermore, when the biological species is mammalian, examples include humans, non-human primates, livestock such as cows, horses, pigs, and sheep, and pets such as cats and dogs. Furthermore, the foreign protein may be a protein obtained by adding mutations such as substitutions, deletions, or additions to the amino acid sequence of a wild-type protein derived from a specific biological species. Furthermore, the foreign protein may be an artificial protein containing an amino acid sequence that does not exist in nature.

[0097] When a foreign protein substitutes amino acids in the amino acid sequence of a wild-type protein with other amino acids, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of a wild-type protein are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added in the amino acid sequence or to the N-terminus or C-terminus of the wild-type protein. Mutations that combine these amino acid additions, substitutions, and deletions can also be added. The amino acid sequence of the mutated protein preferably exhibits 85% or more identity, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity to the amino acid sequence of the corresponding wild-type protein.

[0098] In one embodiment of the present invention, the foreign protein is not particularly limited, and examples thereof include proteins that are partially or completely functionally deficient in genetic diseases. Examples of such genetic diseases include lysosomal diseases, cystic fibrosis, and hemophilia. Other foreign proteins include growth hormone, somatomedin, insulin, glucagon, lysosomal enzymes, cytokines, lymphokines, blood coagulation factors, antibodies, fusion proteins of antibodies with other proteins, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), macrophage-colony-stimulating factor (M-CSF), erythropoietin, darbepoetin, tissue plasminogen activator (t-PA), thrombomodulin, follicle-stimulating hormone (FSH), gonadotropin-releasing hormone (GnRH), gonadotropin, DNase I, thyroid-stimulating hormone (TSH), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), and glial Examples include cell line neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor α receptor (TNF-α receptor), enzymes having the activity of degrading beta-amyloid, etanercept, pegvisomant, metreleptin, abatacept, asfotase, and GLP-1 receptor agonists, aspartacylase (ASPA).

[0099] Examples of foreign proteins include mouse antibodies, humanized antibodies, human-mouse chimeric antibodies, and human antibodies. Further examples of foreign proteins include anti-IL-6 antibodies, anti-beta amyloid antibodies, anti-BACE antibodies, anti-EGFR antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-HER2 antibodies, anti-PCSK9 antibodies, and anti-TNF-α antibodies.

[0100] When the foreign protein is a lysosomal enzyme, the gene for the foreign protein may be any of α-L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N Examples include N-acetylgalactosaminidase, acid sphingomyelinase, α-galactosidase A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2.

[0101] The foreign protein may be a fusion protein of an antibody and another protein. In such a fusion protein, the antibody is any one of a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, and a human antibody, and the other protein is a growth hormone, a lysosomal enzyme, a cytokine, a lymphokine, a blood coagulation factor, an antibody, a fusion protein of an antibody and another protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), macrophage-colony-stimulating factor (M-CSF), erythropoietin, darbepoietin, tissue plasminogen activator (t-PA), thrombomodulin, follicle-stimulating hormone, DNase I, thyroid-stimulating hormone (TSH), neurotrophin, steroid hormone (NSH), steroid hormone (SHR ... Examples of the fibroblast growth factor include growth factor (NGF), ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor α receptor (TNF-α receptor), and enzymes having the activity of degrading beta-amyloid.

[0102] The foreign protein may be a fusion protein of an antibody and a lysosomal enzyme. In such a fusion protein, the antibody is any one of a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, and a human antibody, and the lysosomal enzyme is any one of α-L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosidase, and the like. Examples of the enzyme include sphingomyelinase, α-galactosaminidase, acid sphingomyelinase, α-galactosidase A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2.

[0103] When the foreign protein is a fusion protein of an antibody and another protein or an antibody and a lysosomal enzyme, the antibody has specific affinity for a protein present on the surface of vascular endothelial cells. Examples of such proteins include transferrin receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transporter 1, organic anion transporter, monocarboxylate transporter, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 8, and the membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor. Further examples of organic anion transporters include OATP-F, and examples of monocarboxylate transporters include MCT-8.

[0104] A gene encoding a foreign protein is placed under the control of a promoter between the inverted terminal repeats (ITR) of the first adeno-associated virus and the inverted terminal repeats (ITR) of the second adeno-associated virus. The promoter is not particularly limited as long as it can express the foreign protein in host cells, but the CAG promoter and the CBh promoter are preferably used. These promoters are particularly preferred when expressing a foreign protein in brain tissue, with the CAG promoter being particularly preferred.

[0105] Adenovirus provides the functions necessary for the replication and packaging of the AAV genome into capsids to form viral virions in host cells, which are performed by the E1, E2A, E4, and VA1 RNA regions of the adenoviral genome.

[0106] In one embodiment of the present invention, the term "recombinant AAV particles (rAAV particles)" refers to AAV capsid proteins (including functional equivalents thereof) packaged with a nucleic acid molecule, such as an rAAV genome, in which a wild-type AAV genome has been modified. Here, the term "rAAV genome (recombinant AAV genome)" refers to a nucleic acid molecule in which a wild-type AAV genome has been modified. The rAAV genome packaged in rAAV particles is single-stranded DNA. Examples of such nucleic acid molecules include single-stranded DNAs that, from the 5' end, contain a base sequence containing an inverted terminal repeat (ITR) of a first adeno-associated virus or a functional equivalent thereof, a region containing a base sequence encoding a foreign protein, and an inverted terminal repeat (ITR) of a second adeno-associated virus. However, the nucleic acid molecule not packaged in rAAV particles can also be referred to as an rAAV genome. Therefore, the rAAV genome may be single-stranded or double-stranded DNA.

[0107] The function of the E1 region of adenovirus is required for the formation of rAAV particles in host cells. Generally, host cells containing all or part of the E1 region are used to produce rAAV particles. HEK293 cells are known as such cells. The genome of HEK293 cells contains at least the coding regions of E1A and E1B.

[0108] When a host cell having all or a part of the E1 region is used, the regions of the adenovirus genome necessary for the AAV genome to be replicated and packaged into capsids to form viral virions are the E2A region, the E4 region, and the VA1 RNA region. These regions encode proteins and RNAs required for AAV replication. Regarding the functions provided by the E4 region, the E4 34 kDa protein encoded by open reading frame 6 (ORF6) of the E4 region is required for AAV replication.

[0109] In one embodiment of the present invention, the E2A region may be that of any adenovirus of serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24 to 30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3, as long as it exhibits the original functions required for AAV replication. The E2A region of serotype 2 adenovirus is one of the preferred regions in the present invention.

[0110] Furthermore, the E2A region may be a wild-type adenovirus E2A region modified by substitution, deletion, addition, or the like, as long as the region exhibits its inherent function. In one embodiment of the present invention, the E2A region includes such mutated E2A regions.

[0111] When bases in the base sequence of the wild-type E2A region are substituted with other bases, the number of substituted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence of the E2A region are deleted, the number of deleted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. E2A regions that have been mutated by combining these base substitutions and deletions are also included in the E2A region. When bases are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 bases are added to the base sequence or to the 5' end or 3' end of the wild-type E2A region. E2A regions that have been mutated by combining these base additions, substitutions, and deletions are also included in the E2A region. The base sequence of the mutated E2A region preferably exhibits 85% or more identity to the base sequence of wild-type E2A, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.

[0112] In one embodiment of the present invention, the E4 region may be that of any adenovirus of serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24 to 30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3, as long as it exhibits the original functions required for AAV replication. The E4 region of serotype 2 adenovirus is one of the preferred regions in the present invention.

[0113] Furthermore, the E4 region may be a wild-type adenovirus E4 region modified by substitution, deletion, addition, or the like, as long as the E4 region exhibits its inherent function. In one embodiment of the present invention, the E4 region includes such mutated E4 regions.

[0114] When bases in the base sequence of the wild-type E4 region are substituted with other bases, the number of substituted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence of the E4 region are deleted, the number of deleted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Furthermore, an E4 region mutated by a combination of these base substitutions and deletions is also included in the E4 region. When bases are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 bases are added to the wild-type E4 base sequence or to the 5'-end or 3'-end. E4 mutated by a combination of these base additions, substitutions, and deletions is also included in the E4. The mutated E4 base sequence preferably exhibits 85% or more identity, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity to the wild-type E4 base sequence.

[0115] In one embodiment of the present invention, VA1 The RNA region may be that of any adenovirus of serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3, as long as it exerts the original functions required for AAV replication. The VA1 RNA region of serotype 2 adenovirus is one of the preferred regions in the present invention.

[0116] Furthermore, the VA1 RNA region may be a wild-type adenovirus VA1 RNA region modified by substitution, deletion, addition, or the like, as long as the region exhibits its inherent function. In one embodiment of the present invention, the VA1 RNA region also includes such mutated VA1 RNA regions.

[0117] When bases in the base sequence of the wild-type VA1 RNA region are substituted with other bases, the number of substituted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence of the VA1 RNA region are deleted, the number of deleted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. VA1 RNA regions that have been mutated by combining these base substitutions and deletions are also VA1 RNA regions. When bases are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 bases are added to the base sequence or to the 5' end or 3' end of the wild-type VA1 RNA region. VA1 RNA regions that have been mutated by combining these base additions, substitutions, and deletions are also included in the VA1 RNA region. The base sequence of the mutated VA1 RNA region preferably exhibits 85% or more identity, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity to the base sequence of the wild-type VA1 RNA region.

[0118] In a preferred embodiment of the present invention, the E2A region, E4 region, and VA1 RNA region may be located in any order. A base sequence including the E2A region, E4 region, and VA1 RNA region is called a helper region, and a plasmid including such a helper region is called a helper plasmid.

[0119] A preferred embodiment of the helper plasmid includes a nucleotide sequence in which the E4 region is located downstream of the E2A region and the VA1 RNA region is located further downstream. A helper plasmid in which such a nucleotide sequence has been modified by substitution, deletion, addition, or the like can also be used as long as the E2A region, E4 region, and VA1 RNA region each exhibit their original functions.

[0120] In one embodiment of the present invention, the base sequence that can be used as the base sequence containing the first gene expression regulatory site that controls the expression of the REP protein is not particularly limited, as long as it is controlled by the protein and RNA encoded in a region containing the E2A region, E4 region, and VA1 RNA region, but is preferably the AAV p5 promoter. When the first gene expression regulatory site is an AAV p5 promoter, it is preferably the p5 promoter of AAV serotype 2, but is not limited thereto, and may be any of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, and 11.

[0121] Furthermore, the AAV p5 promoter may be one in which the nucleotide sequence of the wild-type p5 promoter of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 has been modified by substitution, deletion, addition, or the like, so long as it still exhibits its function. In one embodiment of the present invention, p5 promoters with these mutations are also included in the p5 promoter. A region comprising the p5 promoter or a functional equivalent thereof and a nucleotide sequence encoding the adeno-associated virus REP protein or a functional equivalent thereof is referred to as the Rep region.

[0122] In one embodiment of the present invention, the base sequence that can be used as the base sequence containing the second gene expression regulatory site that controls the expression of the CAP protein is not particularly limited, as long as it is controlled by the protein and RNA encoded by the region containing the E2A region, E4 region, and VA1 RNA region, but is preferably the AAV p40 promoter. In the case of an AAV p40 promoter, it is preferably the p40 promoter of AAV of serotype 2, but is not limited thereto, and may be any of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, and 11.

[0123] Furthermore, the AAV p40 promoter may be one obtained by modifying the nucleotide sequence of the wild-type p40 promoter of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, such as by substitution, deletion, or addition, so long as it still exhibits its function. In one embodiment of the present invention, p40 promoters with these mutations are also included in the p40 promoter. When bases in the nucleotide sequence of the wild-type p40 promoter are substituted with other bases, the number of substituted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the nucleotide sequence of the wild-type p40 promoter are deleted, the number of deleted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Furthermore, p40 promoters with mutations that combine these base substitutions and deletions are also p40 promoters. When bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence or the 5'-end or 3'-end of the wild-type p40 promoter. Mutations of p40 promoters that combine these base additions, substitutions, and deletions are also included in the p40 promoter. The base sequence of the mutated p40 promoter preferably exhibits 85% or more identity, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity to the base sequence of the wild-type p40 promoter.

[0124] The p40 promoter or a functional equivalent thereof is usually located upstream of a base sequence encoding the adeno-associated virus CAP protein or a functional equivalent thereof. The region containing the p40 promoter or a functional equivalent thereof and the base sequence encoding the adeno-associated virus CAP protein or a functional equivalent thereof is called the Cap region.

[0125] In one embodiment of the present invention, the serotype of AAV from which the Cap region is derived is not particularly limited, and the serotype may be any of 1, 3, 4, 5, 6, 7, 8, 9, 10, or 11, but the Cap regions of serotypes 8 and 9 are preferably used.

[0126] Modifications such as substitution, deletion, and addition to the base sequence of the Cap region can also be used as the Cap region, as long as they still exhibit the original function of the Cap region. When bases in the base sequence of the Cap region are substituted with other bases, the number of substituted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence are deleted, the number of deleted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Furthermore, mutations that combine these base substitutions and deletions can also be used as the Cap region. When bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence or to the 5' end or 3' end. Mutations that combine these base additions, substitutions, and deletions can also be used as the Cap region. The base sequence of the mutated Cap region preferably exhibits 85% or more identity to the original base sequence, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.

[0127] In a preferred embodiment of the present invention, the Rep region may be located upstream or downstream of the Cap region. A region containing the Rep region and the Cap region is called a Rep-Cap region. In one embodiment, plasmid 2 contains this Rep-Cap region.

[0128] As described above, three types of plasmids are typically used to produce recombinant adeno-associated virus (rAAV) virions used to introduce foreign genes into cells, tissues, or living organisms: (1) a plasmid (plasmid 1) having a structure containing a nucleotide sequence including a first inverted terminal repeat (ITR) and a nucleotide sequence including a second inverted terminal repeat (ITR) derived from a virus such as AAV, and a gene encoding a desired protein located between these two ITRs; (2) a plasmid (plasmid 2) containing an AAV Rep gene having the functions necessary for integrating the nucleotide sequence of the region flanked by the ITR sequences (including the ITR sequences) into the genome of a host cell, and a gene encoding an AAV capsid protein; and (3) a plasmid (plasmid 3 or helper plasmid) containing the adenovirus E2A region, E4 region, and VA1 RNA region. Two of these plasmids 1 to 3 can be ligated together to be used as a single plasmid, or all three can be ligated together to be used as a single plasmid. Furthermore, without being limited to these, four or more types of plasmids containing these genes can be used as long as all of the genes necessary for rAAV particles contained in these three plasmids are introduced into host cells. For example, plasmid 2 is usually a plasmid containing the AAV Rep gene and a gene encoding an AAV capsid protein, but a plasmid containing the AAV Rep gene and a plasmid containing a gene encoding an AAV capsid protein can also be prepared and used for rAAV particles. Note that the plasmid containing the gene encoding the AAV capsid protein is referred to as plasmid 2 in this specification.

[0129] The rAAV produced in one embodiment of the present invention has a protein with a desired function on its surface. There are two types of rAAVs with a protein with a desired function on their surface: (1) a capsid containing a fusion protein of at least one of the capsid-constituting proteins (capsid proteins) VP1, VP2, and VP3 with another protein (A), wherein the other protein (A) contains a protein with a desired function; or (2) a capsid containing a fusion protein of at least one of the capsid proteins VP1, VP2, and VP3 with another protein (A), wherein a separately prepared protein with a desired function is bound to the capsid via the other protein (A). In either case, it is preferred that the rAAV contains a fusion protein of all of the capsid proteins VP1, VP2, and VP3 with the other protein (A). However, the rAAV may be one whose capsid contains only a fusion protein of VP1 and another protein (A), one whose capsid contains only a fusion protein of VP2 and another protein (A), or one whose capsid contains only a fusion protein of VP1 and VP2 and another protein (A). Alternatively, the rAAV may be one whose capsid contains only a fusion protein of VP3 and another protein (A), one whose capsid contains only a fusion protein of VP2 and VP3 and another protein (A), or one whose capsid contains only a fusion protein of VP1 and VP3 and another protein (A).

[0130] To produce rAAV particles containing a fusion protein of at least one of the capsid proteins VP1, VP2, and VP3 with another protein (A), a plasmid 2 is used, in which a nucleotide sequence encoding the other protein (A) is added to the nucleotide sequence of the Cap region so that the nucleotide sequence is in-frame with the nucleotide sequence encoding the capsid protein. Such a plasmid 2 can express the fusion protein of the capsid protein and the other protein (A) in a host cell.

[0131] The site to which the other protein (A) should be added in the fusion protein is described in detail below, taking as an example a case where the Cap region is derived from AAV8. When the other protein (A) consists of 100 or more amino acid residues, for example, when the other protein (A) is a VHH, the addition site is preferably the C-terminal side of any amino acid residue contained in the variable region IV of VP1 consisting of the amino acid sequence of SEQ ID NO: 157, more preferably the C-terminal side of amino acid residues 445 to 477, 450 to 465, or 456 to 462 from the N-terminus of VP1, for example, the C-terminal side of amino acid residue 455, 457, or 462. Furthermore, for example, the C-terminal side of any amino acid residue contained in variable region VIII of VP1 consisting of the amino acid sequence of SEQ ID NO: 158 is preferred, and the C-terminal side of amino acid residues 584 to 602, 586 to 600, or 588 to 600 from the N-terminus of VP1 is more preferred, for example, the C-terminal side of amino acid residues 588 or 599. Another example is the C-terminal side of amino acid residue 501 from the C-terminus of VP1. Another preferred form of addition is one in which at least one amino acid residue, for example, 1 to 7, 1 to 6, 2 to 7, or 2 to 6 amino acid residues, in the amino acid sequence of amino acids 455 to 460 or 456 to 462 from the N-terminus of VP1 is substituted with the amino acid sequence of the other protein (A).

[0132] When the other protein (A) consists of less than 100 amino acid residues, for example, when the other protein (A) consists of 3 to 100, 10 to 100, 20 to 100, 10 to 80, 20 to 80, 10 to 50, or 10 to 30 amino acid residues, in addition to the positions as in the case where the other protein (A) consists of 100 or more amino acid residues, it can also be added to the C-terminal side of any amino acid residue contained in the variable region IX of VP1 consisting of the amino acid sequence of SEQ ID NO: 159, for example, the C-terminal side of the 707th to 717th amino acid residues, the 707th to 712th amino acid residues, or the 707th amino acid residue from the N-terminus of VP1.

[0133] The amino acid sequence of VP1 of AAV8 is shown in SEQ ID NO: 2. rAAV particles obtained by introducing a nucleic acid molecule encoding such a fusion protein into a host cell contain a fusion protein of another protein (A) and VP1 as a capsid protein. VP1, VP2, and V3 are expressed by transcription of mRNA generated by alternative splicing of a single gene. Therefore, from a nucleic acid molecule to which a nucleotide sequence encoding another protein (A) has been added so as to express the above-mentioned fusion protein of VP1 and another protein (A), a fusion protein of VP2 and another protein (A) and a fusion protein of V3 and another protein (A) are expressed, but normal VP2 and VP3 are not expressed.

[0134] The above detailed description of the site in the fusion protein to which the other protein (A) should be added is given taking the example of a case in which the Cap region is derived from AAV8, but the same applies to other serotypes of AAV, such as AAV9. Among the amino acid sequences of VP1 of AAV of other serotypes, those corresponding to the variable regions IV, VIII, and IX of the amino acid sequence of VP1 of AAV8 can be easily determined by comparing these amino acid sequences.

[0135] In one embodiment of the present invention, rAAV particles containing a fusion protein of a capsid protein and another protein (A) as the capsid protein are those in which a portion of the capsid protein is the fusion protein. Such rAAV particles can be obtained by introducing into a host cell a nucleic acid molecule encoding a normal capsid protein in addition to a nucleic acid molecule encoding the fusion protein of the capsid protein and another protein (A). Here, as plasmid 2, a plasmid 2 containing a nucleotide sequence encoding the fusion protein of the capsid protein and another protein (A) and a plasmid 2 encoding the normal capsid protein may be separately prepared and then introduced into the same host cell. Alternatively, a plasmid 2 containing a nucleotide sequence encoding the fusion protein of the capsid protein and another protein (A) and a plasmid 2 encoding the normal capsid protein may be prepared and then introduced into a host cell.

[0136] In one embodiment, a nucleic acid molecule encoding a normal capsid protein is referred to as a first nucleic acid molecule, and a nucleic acid molecule encoding a fusion protein of the capsid protein and another protein (A) is referred to as a second nucleic acid molecule. In a host cell into which the first and second nucleic acid molecules have been introduced, both the fusion protein of the capsid protein and another protein (A) and the normal capsid protein are expressed, and by using such a host cell, rAAV particles in which the capsid protein is composed of the fusion protein and the normal capsid protein can be obtained.

[0137] Here, the ratio of the fusion protein to the capsid protein constituting the rAAV particle is important because an increase in the ratio of the fusion protein tends to decrease the yield of rAAV particles. If the yield of rAAV particles decreases, it becomes difficult to secure the required amount of rAAV particles and the cost required for their production also increases.

[0138] In one embodiment of the present invention, the ratio of the total number of molecules of normal VP1, VP2, and VP3 in the capsid proteins of rAAV particles to the total number of molecules of fusion proteins of each of these (i.e., VP1, VP2, and VP3) with another protein (A) is preferably 9.95:0.05 to 8.0:2.0, more preferably 9.9:0.1 to 9.0:1.0, for example, 9.9:0.1 to 9.2:0.8, or 9.85:0.15 to 9.5:0.5, when the other protein (A) consists of 100 or more amino acid residues, for example, when the other protein (A) is a VHH. To achieve a preferable ratio between the total number of molecules of normal VP1, VP2, and VP3 in the capsid proteins of rAAV particles and the total number of molecules of fusion proteins of each of these (i.e., VP1, VP2, and VP3) with another protein (A), the first nucleic acid molecule and the second nucleic acid molecule are introduced into host cells so that the ratio of their numbers of molecules is 9.9:0.1 to 8.0:2.0, for example, 9.8:0.2 to 8.7:1.3, 9.7:0.3 to 8.5:1.5, 9.5:0.5, 9.0:1.0, etc. The average total number of molecules of fusion proteins of VP1, VP2, and VP3 with another protein (A) contained in one AAV particle is preferably 0.5 to 12, for example, 0.5 to 9, 0.5 to 6, 0.75 to 9, 0.75 to 6, 1 to 9, 1 to 6, 2 to 6, 3 to 6, etc. The number of proteins constituting the capsid of an AAV particle is, for example, 60. These ratios can also be expressed as percentages. In such cases, those skilled in the art can easily perform the conversion based on the description herein.For example, when (1) a first nucleic acid sequence is introduced as a first nucleic acid molecule, such that when it is introduced, VP1, VP2, and VP3 are expressible, and (2) a second nucleic acid sequence is introduced as a second nucleic acid molecule, such that when it is introduced, VP3 modified with a ligand is expressible, and when it is expressed in the present disclosure as "transfecting" a host cell so that the ratio of the number of molecules of the second nucleic acid sequence to the sum of the number of molecules of the first nucleic acid sequence to be introduced and the number of molecules of the second nucleic acid sequence to be introduced is, for example, 1 to 50%, the former typically corresponds to the sum of the number of molecules of the fusion protein of VP3 and another protein (A) (which may serve as a ligand) in the absence of a fusion protein of VP1 and another protein (A), in the absence of a fusion protein of VP2 and another protein (A), and the sum of the number of molecules of these fusion proteins and the sum of the number of molecules of VP1, VP2, and VP3 corresponds to the latter.

[0139] In one embodiment of the present invention, the ratio of the total number of molecules of normal VP2 and VP3 in the capsid proteins of rAAV particles to the total number of molecules of the fusion proteins of each of these (i.e., VP2 and VP3) with another protein (A) is preferably 9.95:0.05 to 8.0:2.0, more preferably 9.95:0.1 to 9.0:1.0, for example, 9.9:0.1 to 9.2:0.8, or 9.85:0.15 to 9.5:0.5, when the other protein (A) consists of 100 or more amino acid residues, for example, when the other protein (A) is a VHH. To achieve a preferable ratio between the total number of normal VP2 and VP3 molecules in the capsid proteins of rAAV particles and the total number of molecules of the fusion proteins of each of these (i.e., VP2 and VP3) with another protein (A), the first nucleic acid molecule and the second nucleic acid molecule are introduced into host cells so that the ratio of their numbers of molecules is 9.9:0.1 to 8.0:2.0, for example, 9.8:0.2 to 8.7:1.3, 9.7:0.3 to 8.5:1.5, 9.5:0.5, 9.0:1.0, etc. The average total number of molecules of the fusion protein of VP2 and VP3 with another protein (A) contained in one AAV particle is preferably 0.5 to 12, for example, 0.5 to 9, 0.5 to 6, 0.75 to 9, 0.75 to 6, 1 to 9, 1 to 6, 2 to 6, 3 to 6, etc. The number of proteins constituting the capsid of an AAV particle is, for example, 60.

[0140] In one embodiment of the present invention, the ratio of the number of molecules of normal VP2 in the capsid protein of an rAAV particle to the number of molecules of the fusion protein of VP2 and another protein (A) is preferably 9.95:0.05 to 8.0:2.0, more preferably 9.9:0.1 to 9.0:1.0, for example, 9.9:0.1 to 9.2:0.8, or 9.85:0.15 to 9.5:0.5, when the other protein (A) consists of 100 or more amino acid residues, for example, when the other protein (A) is a VHH. To achieve a preferred ratio of the number of molecules of normal VP2 in the capsid protein of an rAAV particle to the total number of molecules of the fusion protein of VP2 and another protein (A), the first nucleic acid molecule and the second nucleic acid molecule are introduced into a host cell so that the ratio of their numbers of molecules is 9.9:0.1 to 8.0:2.0. For example, the ratio is 9.8:0.2 to 8.7:1.3, 9.7:0.3 to 8.5:1.5, 9.5:0.5, 9.0:1.0, etc. The average total number of molecules of the fusion protein of VP2 and another protein (A) contained in one AAV particle is preferably 0.25 to 6, for example, 0.25 to 4.5, 0.25 to 3, 0.4 to 4.5, 0.4 to 3, 0.5 to 4.5, 0.5 to 3, 1 to 3, 1.5 to 3, etc. The number of proteins constituting the capsid of an AAV particle is, for example, 60.

[0141] In one embodiment of the present invention, the ratio of the number of molecules of normal VP3 in the capsid protein of an rAAV particle to the number of molecules of the fusion protein of VP3 and another protein (A) is preferably 9.95:0.05 to 8.0:2.0, more preferably 9.9:0.1 to 9.0:1.0, for example, 9.9:0.1 to 9.2:0.8, or 9.85:0.15 to 9.5:0.5, when the other protein (A) consists of 100 or more amino acid residues, for example, when the other protein (A) is a VHH. To achieve a preferred ratio of the number of molecules of normal VP3 in the capsid protein of an rAAV particle to the total number of molecules of the fusion protein of VP3 and another protein (A), the first nucleic acid molecule and the second nucleic acid molecule are introduced into a host cell so that the ratio of their numbers of molecules is 9.9:0.1 to 8.0:2.0. For example, the ratio is 9.8:0.2 to 8.7:1.3, 9.7:0.3 to 8.5:1.5, 9.5:0.5, 9.0:1.0, etc. The average total number of molecules of the fusion protein of VP3 and another protein (A) contained in one AAV particle is preferably 0.25 to 6, for example, 0.25 to 4.5, 0.25 to 3, 0.4 to 4.5, 0.4 to 3, 0.5 to 4.5, 0.5 to 3, 1 to 3, 1.5 to 3, etc. The number of proteins constituting the capsid of an AAV particle is, for example, 60.

[0142] In one embodiment of the present invention, the ratio of the total number of molecules of normal VP1, VP2, and VP3 in the capsid protein of an rAAV particle to the total number of molecules of each of these (i.e., VP1, VP2, and VP3) and another protein (A) is, when the other protein (A) consists of less than 100 amino acid residues, for example, 3 to 100, 10 to 100, 20 to 100, or 30 to 100. When the polysaccharide consists of 1, 10 to 80, 20 to 80, 10 to 50, or 10 to 30 amino acid residues, the ratio is preferably 9.95:0.05 to 7.0:3.0, more preferably 9.95:0.1 to 8.0:2.0, 9.95:0.1 to 9.0:1.0, for example, 9.9:0.1 to 9.3:0.7, 9.9:0.1 to 9.2:0.8, or 9.85:0.15 to 9.5:0.5. To achieve a preferable ratio between the total number of molecules of normal VP1, VP2, and VP3 in the capsid proteins of rAAV particles and the total number of molecules of fusion proteins of each of these proteins (i.e., VP1, VP2, and VP3) with another protein (A), the first nucleic acid molecule and the second nucleic acid molecule are introduced into host cells so that the ratio of their numbers of molecules is 9.9:0.1 to 7.0:3.0, for example, 9.9:0.1 to 8.0:2.0, 9.8:0.2 to 8.7:1.3, 9.7:0.3 to 8.5:1.5, 9.5:0.5, 9.0:1.0, etc. The average total number of molecules of the fusion protein of VP1, VP2, and VP3 with another protein (A) contained in one capsid is preferably 1 to 18, for example, 1 to 12, 1.5 to 18, 1.5 to 12, 2 to 18, 2 to 12, 6 to 12, etc. The number of proteins constituting the capsid of an AAV particle is, for example, 60.

[0143] In one embodiment of the present invention, the ratio of the total number of normal VP2 and VP3 molecules in the capsid protein of an rAAV particle to the total number of molecules of the fusion protein of each of these (i.e., VP2 and VP3) with another protein (A) is, when the other protein (A) consists of less than 100 amino acid residues, for example, when the other protein (A) consists of 3 to 100, 10 to 100, 20 to 100, 10 When the first nucleic acid molecule is composed of up to 80, 20 to 80, 10 to 50, or 10 to 30 amino acid residues, the ratio is preferably 9.95:0.05 to 7.0:3.0, more preferably 9.95:0.1 to 8.0:2.0, 9.95:0.1 to 9.0:1.0, for example, 9.9:0.1 to 9.3:0.7, 9.9:0.1 to 9.2:0.8, or 9.85:0.15 to 9.5:0.5. In order to achieve a preferred ratio between the total number of normal VP2 and VP3 molecules in the capsid protein of an rAAV particle and the total number of molecules of fusion proteins of each of these (i.e., VP2 and VP3) with another protein (A), the first nucleic acid molecule and the second nucleic acid molecule are introduced into a host cell so that the ratio of their numbers of molecules is 9.9:0.1 to 7.0:3.0. For example, the ratio is 9.9:0.1 to 8.0:2.0, 9.8:0.2 to 8.7:1.3, 9.7:0.3 to 8.5:1.5, 9.5:0.5, 9.0:1.0, etc. The average total number of molecules of the fusion protein of VP2 and VP3 with another protein (A) contained in one capsid is preferably 1 to 18, for example, 1 to 12, 1.5 to 18, 1.5 to 12, 2 to 18, 2 to 12, 6 to 12, etc. The number of proteins constituting the capsid of an AAV particle is, for example, 60.

[0144] In one embodiment of the present invention, the ratio of the number of molecules of normal VP2 in the capsid protein of an rAAV particle to the number of molecules of the fusion protein of VP2 and another protein (A) is preferably 9.95:0.05-7.0:3.0, more preferably 9.95:0.1-8.0:2.0, or 9.95:0.1-9.0:1.0, for example, 9.9:0.1-9.3:0.7, 9.9:0.1-9.2:0.8, or 9.85:0.15-9.5:0.5, when the other protein (A) consists of less than 100 amino acid residues, for example, 3-100, 10-100, 20-100, 10-80, 20-80, 10-50, or 10-30 amino acid residues. To achieve a preferred ratio of the number of normal VP2 molecules to the total number of molecules of the fusion protein of VP2 and another protein (A) in the capsid protein of an rAAV particle, the first nucleic acid molecule and the second nucleic acid molecule are introduced into a host cell so that the ratio of these molecules is 9.9:0.1 to 7.0:3.0. For example, this ratio may be 9.9:0.1 to 8.0:2.0, 9.8:0.2 to 8.7:1.3, 9.7:0.3 to 8.5:1.5, 9.5:0.5, 9.0:1.0, etc. The average number of molecules of the fusion protein of VP2 and another protein (A) contained in one capsid is preferably 0.5 to 9, for example, 0.5 to 6, 0.75 to 9, 0.75 to 6, 1 to 9, 1 to 6, 3 to 6, etc. The number of proteins that constitute the capsid of an AAV particle is, for example, 60.

[0145] In one embodiment of the present invention, the ratio of the number of molecules of normal VP3 in the capsid protein of an rAAV particle to the number of molecules of the fusion protein of VP3 and another protein (A) is preferably 9.95:0.05-7.0:3.0, more preferably 9.95:0.1-8.0:2.0, or 9.95:0.1-9.0:1.0, for example, 9.9:0.1-9.3:0.7, 9.9:0.1-9.2:0.8, or 9.85:0.15-9.5:0.5, when the other protein (A) consists of less than 100 amino acid residues, for example, 3-100, 10-100, 20-100, 10-80, 20-80, 10-50, or 10-30 amino acid residues. To achieve a preferred ratio of the number of normal VP3 molecules to the total number of molecules of the fusion protein of VP3 and another protein (A) in the capsid protein of an rAAV particle, the first nucleic acid molecule and the second nucleic acid molecule are introduced into a host cell so that the ratio of these molecules is 9.9:0.1 to 7.0:3.0. For example, this ratio may be 9.9:0.1 to 8.0:2.0, 9.8:0.2 to 8.7:1.3, 9.7:0.3 to 8.5:1.5, 9.5:0.5, 9.0:1.0, etc. The average number of molecules of the fusion protein of VP3 and another protein (A) contained in one capsid is preferably 0.5 to 9, for example, 0.5 to 6, 0.75 to 9, 0.75 to 6, 1 to 9, 1 to 6, 3 to 6, etc. The number of proteins that constitute the capsid of an AAV particle is, for example, 60.

[0146] Hereinafter, a rAAV in which the capsid contains a fusion protein of at least one of the capsid proteins VP1, VP2, and VP3 with another protein (A), wherein the other protein (A) contains a protein having a desired function, will be described in detail.

[0147] To produce such rAAV, a nucleic acid molecule encoding a fusion protein of a capsid protein and another protein (A), where the other protein (A) has a desired function, is used as the second nucleic acid molecule. A nucleic acid molecule encoding a conventional capsid protein is used as the first nucleic acid molecule. Thus, the resulting rAAV contains conventional VP1, VP2, and VP3, as well as the fusion protein of the capsid protein and the other protein (A), in the capsid. The AAV serotype of the Cap region used here is not particularly limited, but is preferably derived from AAV8 or AAV9.

[0148] The fusion protein must be able to exhibit the function of the other protein (A). Therefore, it is preferable that the other protein (A) contains a first linker, a functional protein, and a second linker in this order from the N-terminus, or a functional protein and a second linker in this order from the N-terminus, but a linker is not essential. The first linker and the second linker, either in combination or alone, serve to enable the fusion protein to exhibit the function derived from the other protein (A). Note that a "protein having a function" can also be referred to as a "functional region," a "functional region," a "functional protein," etc. A functional protein itself has a specific physiological activity.

[0149] The size of the other protein (A) is not particularly limited, but it is preferably composed of 5 to 500 amino acids, for example, 5 to 300, 5 to 200, 5 to 150, 5 to 100, 10 to 300, 10 to 200, 10 to 150, 10 to 100, 20 to 300, 20 to 200, 20 to 150, or 20 to 100 amino acids.

[0150] The amino acid sequence of the peptide constituting the linker is not particularly limited, but the first linker is preferably composed of 0 to 50 amino acids, for example, 2 to 30 or 5 to 20. The second linker is preferably composed of 12 to 100 amino acids, for example, 12 to 50, 12 to 40, 12 to 30, 13 to 50, 13 to 40, 13 to 30, 15 to 50, 15 to 40, 15 to 30, 15, 20, etc. Preferred amino acid sequences for the first linker and the second linker are shown in Tables 1 and 2, respectively. However, the amino acid sequence of the first linker is not limited thereto, and may be an amino acid sequence consisting of 2 to 10 amino acid residues consisting of one glycine, one serine, GS, glycine, and serine, or an amino acid sequence in which any two or more of these or the amino acid sequences shown in Table 1 are linked together. The amino acid sequence of the second linker may be an amino acid sequence consisting of 2 to 10 amino acid residues consisting of glycine and serine, or an amino acid sequence in which any two or more of the amino acid sequences shown in Table 2 are linked together. Preferred amino acid sequences for the first linker and the second linker are shown in Tables 1 and 2, respectively. However, the linker is not limited to these, and may also be an amino acid sequence consisting of 2 to 10 amino acid residues consisting of GS, glycine, and serine, or an amino acid sequence consisting of 12 to 50 amino acid residues in which a two or more of these amino acid sequences and the amino acid sequences shown in Table 1 are linked together.

[0151]

[0152]

[0153] For example, preferred linker combinations include a combination of linkers in which the first linker is GGGGS×1 and the second linker is GGGGS×3, a combination of linkers in which the first linker is GGGGS×1 and the second linker is cIgG2a hinge, a combination of linkers in which the first linker is cIgG2a hinge and the second linker is GGGGS×3, a combination of linkers in which the first linker is cIgG2a hinge and the second linker is cIgG2a hinge, a combination of linkers in which the first linker is 2xEAAAK and the second linker is cIgG2a hinge, and a combination of linkers in which the first linker is GGGGS×3 and the second linker is GGGGS×3.

[0154] The functional protein constituting the other protein (A) itself has the desired physiological activity, and its type is not particularly limited. However, preferably, the functional protein has specific affinity for a protein present on the surface of vascular endothelial cells. The vascular endothelial cells are preferably human vascular endothelial cells. Here, the protein present on the surface of vascular endothelial cells is not particularly limited, but is preferably a transferrin receptor, an insulin receptor, a leptin receptor, an insulin-like growth factor I receptor, an insulin-like growth factor II receptor, a lipoprotein receptor, a glucose transporter 1, an organic anion transporter, a monocarboxylate transporter, a low-density lipoprotein receptor-related protein 1, a low-density lipoprotein receptor-related protein 8, or a membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor, particularly a transferrin receptor and an insulin receptor.

[0155] When the functional protein has specific affinity for the transferrin receptor (TfR), the functional protein is transferrin or a fragment of transferrin containing the TfR-binding region, or an antibody having affinity for TfR.

[0156] When the functional protein is an antibody having affinity for TfR, the form of the antibody is not particularly limited, and examples include single-domain antibodies and single-chain antibodies in which the light chain variable region and the heavy chain variable region are linked via a linker. Single-domain antibodies are antibodies that have the property of specifically binding to an antigen via a single variable region. Single-domain antibodies include antibodies whose variable region consists only of the heavy chain variable region (heavy-chain single-domain antibodies) and antibodies whose variable region consists only of the light chain variable region (light-chain single-domain antibodies). VHH, VNAR, and nanobody (trademark of Ablynx N.V.) are types of single-domain antibodies. As a single-chain antibody, scFv is preferred.

[0157] VNARs are described in detail below. Shark antibodies consist of two heavy chains linked by disulfide bonds. Antibodies consisting of these two heavy chains are called heavy chain antibodies. VNARs are antibodies consisting of a single heavy chain that includes the variable region of the heavy chain that constitutes a heavy chain antibody, or antibodies consisting of a single heavy chain that lacks the constant region (CH) that constitutes a heavy chain antibody. Antibodies in one embodiment of the present invention include those in which mutations have been added to the amino acid sequence of shark antibodies. Humanized shark antibodies are also one of the antibodies in one embodiment of the present invention.

[0158] VHHs are described in detail below. Some camelid antibodies consist of two heavy chains linked by disulfide bonds. Antibodies consisting of these two heavy chains are called heavy-chain antibodies. VHHs are antibodies consisting of a single heavy chain that includes the variable region of the heavy chain that constitutes the heavy-chain antibody, or antibodies consisting of a single heavy chain that lacks the constant region (CH) that constitutes the heavy-chain antibody. Antibodies in one embodiment of the present invention also include antibodies in which mutations have been added to the amino acid sequence of camelid antibodies (including VHHs) in order to reduce antigenicity when the camelid-derived antibody (including VHH) is administered to humans.

[0159] When the functional protein is a VHH with affinity for human TfR (hTfR), examples of suitable VHH amino acid sequences are shown in Table 3. The term "human transferrin receptor" or "hTfR" refers to a membrane protein having the amino acid sequence shown in SEQ ID NO: 4. In one embodiment, the anti-hTfR antibody of the present invention specifically binds to the portion of the amino acid sequence shown in SEQ ID NO: 1 from the N-terminus of the 89th cysteine ​​residue to the C-terminus of phenylalanine (the extracellular domain of hTfR). Furthermore, a VHH with affinity for human TfR (hTfR) preferably has a dissociation constant (KD) with hTfR of preferably 5 x 10 as measured by the method described in Example 44. －８ M or less, and more preferably 2×10 －８ M or less, for example, 1 × 10 －８ M or less, 5 × 10 －９ M or less, and 1 × 10 －９ For example, a suitable one is one having a dissociation constant of 5×10 －１１ M~1 x 10 －８ M is 2 x 10 －１１ M~1 x 10 －８ M, 1 x 10 －１０ M~1 x 10 －８ M, 1.0 x 10 －９ ~1.0 x 10 －８ It's M.

[0160]

[0161] The amino acid sequences of CDR1 to CDR3 of the VHHs shown in Table 3 are shown in Table 4. Regions other than the CDRs are called frame regions (FR). From the N-terminus, VHHs have the sequences of FR1, CDR1, FR2, CD2, FR3, CDR3, and FR4.

[0162]

[0163] The amino acid sequences of VHHs shown in Table 3 may be mutated, as long as the VHHs can specifically bind to hTfR. When such mutations are added, the amino acid sequence of the VHHs after mutation preferably has 80% or more identity, more preferably 85% or more identity, even more preferably 90% or more identity, and even more preferably 95% or more identity, for example, 98% or more identity, to the original amino acid sequence.

[0164] When mutations are introduced into the amino acid sequence of a VHH, it is also possible to introduce mutations only into the FR without introducing mutations into the amino acid sequences of CDR1, CDR2, and CDR3. When introducing such mutations, the amino acid sequence of the variable region after the mutations preferably has an identity of 85% or more, more preferably 90% or more, even more preferably 95% or more, for example, 98% or more, to the original amino acid sequence. Furthermore, when mutations are introduced into the amino acid sequence of the variable region, it is also possible to introduce mutations only into the CDR region without introducing mutations into the amino acid sequence of the FR. When introducing such mutations, the amino acid sequence of the variable region after the mutations has an identity of preferably 90% or more, more preferably 95% or more, for example, 98% or more, to the original amino acid sequence.

[0165] When amino acids in the amino acid sequence of VHH are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 20, more preferably 1 to 15, even more preferably 1 to 10, and even more preferably 1 to 5, for example, 1, 2, or 3. When amino acids in the amino acid sequence of the variable region are deleted, the number of amino acids to be deleted is preferably 1 to 20, more preferably 1 to 15, even more preferably 1 to 10, and even more preferably 1 to 5, for example, 1, 2, or 3. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to the variable region, preferably 1 to 20, more preferably 1 to 15, even more preferably 1 to 10, and even more preferably 1 to 5 amino acids, for example, 1, 2, or 3 amino acids, are added to the amino acid sequence of the variable region or to the N-terminus or C-terminus. Mutations that combine these amino acid additions, substitutions, and deletions can also be added.

[0166] When mutations such as substitutions, deletions, and additions are made to the amino acid sequence of a VHH, mutations can be made only in the FR region without making mutations in the amino acid sequences of the CDR regions (CDR1, CDR2, and CDR3). When making substitutions only in the FR region, the number of amino acids to be substituted is preferably 1 to 12, more preferably 1 to 10, even more preferably 1 to 8, and even more preferably 1 to 4, for example, 1, 2, or 3. When amino acids are deleted only in the amino acid sequence of the FR region, the number of amino acids to be deleted is preferably 1 to 8, more preferably 1 to 4, even more preferably 1 to 3, and even more preferably 1 to 2, for example, 1 or 2. Mutations that combine these amino acid substitutions and deletions can also be made. Furthermore, when amino acids are added only to the amino acid sequence of the FR region, preferably 1 to 8, more preferably 1 to 4, even more preferably 1 to 3, and even more preferably 1 to 2 amino acids, for example, 1 or 2 amino acids, are added to the amino acid sequence of the variable region or to the N-terminus or C-terminus. Mutations that combine these amino acid additions, substitutions, and deletions can also be added.

[0167] When mutations are introduced only in the FR region of a VHH, a method is known in which amino acid residues in the FR region are replaced with corresponding amino acid residues in the FR region of a variable region of an IgG-type human antibody. Herein, this method is referred to as VHH humanization. Such a method is disclosed, for example, in Vincle C., et al., J. Biol. Chem. 284, 3273-84 (2009). If the original antibody is an alpaca antibody, it may be recognized as an antigen when administered to humans. Humanized antibodies are expected to have lower antigenicity than the original antibody. When mutations are introduced only in the FR region, humanization is a preferred embodiment. Herein, VHH also includes humanized VHH.

[0168] When mutations such as substitution, deletion, and addition are made to the amino acid sequence of VHH, mutations can be made only in the CDR regions without making mutations in the amino acid sequence of the FR region. When making substitutions only in the CDR regions, the number of amino acids to be substituted is preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 2, for example, 1 or 2. When amino acids are deleted only in the amino acid sequence of the CDR regions, the number of amino acids to be deleted is preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 2, for example, 1 or 2. Mutations that combine these amino acid substitutions and deletions can also be made. When amino acids are added only in the amino acid sequence of the FR region, preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 2 amino acids, for example, 1 or 2 amino acids, are added to the amino acid sequence of the variable region or to the N-terminus or C-terminus. Mutations that combine these amino acid additions, substitutions, and deletions can also be made.

[0169] Next, we will describe in detail rAAV, which contains a fusion protein of at least one of the capsid proteins VP1, VP2, and VP3 with another protein (A), and in which a separately prepared molecule with a desired function is bound to the capsid via this other protein (A).

[0170] To produce such rAAV, a nucleic acid molecule encoding a fusion protein of a capsid protein and another protein (A), which is used as the second nucleic acid molecule, is used. A nucleic acid molecule encoding a conventional capsid protein is used as the first nucleic acid molecule. Thus, the resulting rAAV contains conventional VP1, VP2, and VP3, as well as the fusion protein of the capsid protein and the other protein (A), in the capsid. The AAV serotype of the Cap region used here is not particularly limited, but is preferably derived from AAV8 or AAV9.

[0171] The fusion protein must be capable of exerting the function of the other protein (A). Here, the function of the other protein (A) is to allow a separately prepared molecule with a desired function to bind to the capsid via the other protein (A). Since the other protein (A) binds a separately prepared protein with a desired function, it can also be called an "anchor." There are no particular limitations on the amino acid sequence that can be used as this anchor, as long as it can exert the function. There are also no particular limitations on the number of amino acids constituting the anchor, but the number of amino acids is preferably 5 to 200, for example, 5 to 100, 5 to 50, 10 to 200, 10 to 100, 10 to 50, or 20 to 40.

[0172] An example of an anchor is ALFA Tag having the amino acid sequence of SEQ ID NO: 52. When the anchor sequence is bound to the capsid protein, a linker is not necessarily required. However, the above-mentioned linker may be bound to the N-terminus and / or C-terminus.

[0173] The separately prepared molecule having a desired function that is bound to the rAAV particle via the anchor is not limited to a protein, and may be a protein, a conjugate of a protein and a substance other than a protein, a substance other than a protein, or a conjugate of two substances other than proteins, as long as it can specifically bind to the anchor.

[0174] When the molecule having the desired function is a protein, the protein may be, for example, a fusion protein of a protein having the desired function and a protein capable of binding to an anchor sequence. Alternatively, the protein having the desired function and the protein capable of binding to an anchor sequence may be separately prepared and then chemically conjugated together. Furthermore, the protein may be a bifunctional antibody.

[0175] When the molecule having the desired function is a fusion protein of a protein having the desired function and a protein capable of binding to an anchor sequence, the two may be linked directly or via the above-mentioned linker sequence.

[0176] An example of a protein capable of binding to an anchor is an antibody against the anchor. There are no particular limitations on the form of such an antibody, but single-chain antibodies such as Fab and scFv, and single-domain antibodies such as VHH, VNAR, and nanobodies can be suitably used as such antibodies. When the anchor is an ALFA Tag, a suitable antibody against the anchor is a nanobody against the ALFA Tag having the amino acid sequence of SEQ ID NO: 53. As long as it has affinity for the ALFA Tag, antibodies with mutations (substitution, deletion, or addition) of one to three (e.g., one or two) amino acids in the amino acid sequence of SEQ ID NO: 53 can also be used as antibodies against the anchor.

[0177] An example of a case where the molecule having a desired function is a fusion protein of a protein having the desired function and a protein having the ability to bind to an anchor sequence is a fusion protein of a protein having specific affinity for a protein present on the surface of vascular endothelial cells and a protein having the ability to bind to an anchor sequence.

[0178] Here, the protein present on the surface of vascular endothelial cells is not particularly limited, but examples include transferrin receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transporter 1, organic anion transporter, monocarboxylate transporter, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 8, or a membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor.

[0179] Proteins having specific affinity for proteins present on the surface of these vascular endothelial cells include ligands or antibodies for these receptors. For example, for the transferrin receptor, transferrin or anti-transferrin receptor antibodies are used. Suitable antibodies that have affinity for TfR include, but are not limited to, single-domain antibodies, single-chain antibodies such as ScFv, and Fabs. Suitable single-domain antibodies include those shown in Tables 3 and 4. Examples of Fab include those having the amino acid sequence of the heavy chain variable region represented by SEQ ID NO: 54 and the amino acid sequence of the light chain variable region represented by SEQ ID NO: 55. The amino acid sequences of the CDRs of the heavy and light chain variable regions of this antibody are shown in Table 5. The amino acid sequence of the Fab may be mutated, as long as the Fab can specifically bind to hTfR. The above-mentioned method of mutating VHHs can also be applied to mutating Fabs.

[0180]

[0181] It includes a fusion protein of at least one of the capsid proteins VP1, VP2, and VP3 with another protein (A), and an AAV in which a separately prepared molecule having a desired function is bound to the capsid via this other protein (A) can be obtained by obtaining an AAV, mixing it with the molecule having the desired function to bind the two, and then purifying the bound product by means of chromatography or the like.

[0182] A method for producing rAAV particles according to one embodiment of the present invention is described below. First, a predetermined amount of host cells is cultured. Three types of plasmids are introduced into the host cells: (1) a plasmid (plasmid 1) having a structure containing a nucleotide sequence including a first inverted terminal repeat (ITR) and a nucleotide sequence including a second inverted terminal repeat (ITR) derived from a virus such as AAV, and a gene encoding a desired protein located between these two ITRs; (2) a plasmid (plasmid 2) containing an AAV Rep gene (Rep region) that has the functions necessary for integrating the nucleotide sequence of the region sandwiched between the ITR sequences (including the ITR sequence) into the genome of the host cell, and a gene encoding an AAV capsid protein (Cap region); and (3) a plasmid (plasmid 3 or helper plasmid) containing the E2A region, E4 region, and VA1 RNA region of adenovirus. The host cells used in this case are not particularly limited as long as they are capable of forming rAAV particles when the three types of plasmids are introduced into the cells. However, cells having the E1A and E1B genes are preferred, and cells having the E1A and E1B genes and expressing the large T antigen gene of the SV40 virus, such as HEK293T cells, which are a cell line derived from human embryonic kidney cells, are more preferred.

[0183] Two types of plasmid 2 are used: one having a normal Rep region and Cap region, and the other having a gene encoding another protein (A) incorporated into the Cap region so that a fusion protein with the capsid protein is formed. These two types of plasmid 2 are introduced into host cells so that the ratio of the former to the latter is, for example, 9.9:0.1 to 8.0:2.0. In the absence of a fusion protein of VP1 and VP2, this corresponds to the ratio of the number of molecules of the second nucleic acid sequence to the sum of the number of molecules of the first nucleic acid sequence to be introduced and the number of molecules of the second nucleic acid sequence to be introduced in the present disclosure. In this case, the relevant calculation is possible, with 9.9:0.1 roughly corresponding to 1%, and 8.0:2.0 corresponding to 20%.

[0184] A solution containing plasmids 1 to 3 and polyethyleneimine is added to a culture vessel containing host cells and a medium containing valproic acid. The weight ratio of plasmids 1 to 3 to polyethyleneimine at this time is, for example, plasmid 1:plasmid 2:plasmid 3:polyethyleneimine=4.5-5.5:5.5-6.5:9-11:40-44, e.g., 5:6:10:42.

[0185] After introducing the plasmids 1 to 3, the host cells are cultured for 2 to 7 days, for example, 3 to 4 days. After the culture is completed, a solution containing an endonuclease and a surfactant is added to the culture solution. The endonuclease is used to decompose DNA and RNA contained in the culture solution. Benzonase is one such endonuclease. ＴＭ can be suitably used. The resulting cell lysate is centrifuged to obtain a supernatant. rAAV particles are purified from the supernatant using affinity column chromatography using a ligand with affinity for rAAV particles. These purified rAAV particles include empty AAV particles that do not contain an AAV genome. Empty AAV particles are removed from these purified rAAV particles by density gradient centrifugation to obtain a purified rAAV particle product.

[0186] In this aspect of the present disclosure, the process of genetically introducing into host cells one or more nucleic acid molecules containing VP nucleic acid sequences that, upon genetic introduction, enable the expression of VP1, VP2, VP3, and VP3 modified with one or more ligands (which may be the same or different), as well as nucleic acid molecules containing nucleic acid sequences encoding desired proteins, if necessary, can be achieved in various forms. The nucleic acid molecules of interest here are those that enable the expression of at least VP1, VP2, VP3, and VP3 modified with a specific ligand (also referred to herein as modified VP3). These can be introduced in a single molecule, or in two molecules, each expressing, for example, a wild-type and a modified VP, or in a manner in which each molecule is expressed individually, or in combinations thereof. (1) In a single-molecule, multiple sequences are present in the VP1, VP2, VP3, and modified VP3 sequences linked together on a single nucleic acid molecule. VP1, VP2, and VP3 can be expressed by alternative splicing. It is also conceivable to incorporate a modified VP3 into this. Alternatively, each sequence may be separated by an internal ribosome entry site (IRES) or a self-cleaving peptide sequence (2A sequence), and designed as a multigene expression cassette. This allows multiple proteins to be simultaneously expressed with a single introduction procedure, which has the advantage of simplifying cell manipulation.

[0187] A configuration of multiple molecules, for example, two molecules, is also possible. In this embodiment, two nucleic acid molecules are introduced separately, each with a different role. One is a molecule encoding wild-type VP1, VP2, or VP3, and alternative splicing is usually utilized in this case. The other is a molecule encoding modified VP3 in which a ligand modification site has been added to VP3. For modified forms, in configurations containing the VP1 or VP2 sequence, the start codon may be disabled by point mutation or the like to prevent alternative splicing, thereby intentionally suppressing translation. This enables selective control to functionally express only modified VP3 under specific conditions. In addition, expression regulation using exon selection based on alternative splicing can be combined with individual expression forms. In individual molecule introduction (single expression type) or a combination thereof, independent nucleic acid molecules encoding VP1, VP2, VP3, and modified VP3 are introduced individually, or two or three of them are introduced in combination. This allows for individual adjustment of expression levels, control of introduction timing, and the selection and use of different promoters, enabling the design of complex expression patterns. For example, the expression level can be adjusted by constitutively expressing VP1 under a constitutive promoter and regulatedly expressing modified VP3 under an inducible promoter. As an example, the first nucleic acid sequence and the second nucleic acid sequence of the present disclosure may be introduced as separate nucleic acid molecules.

[0188] In this aspect of the present disclosure, the "step of subjecting host cells to conditions for producing recombinant adeno-associated virus particles" refers to conditions (including a cell culture environment or an in vivo environment) under which a nucleic acid molecule containing a nucleic acid sequence encoding a protein constituting the recombinant adeno-associated virus particle is expressed, and a nucleic acid molecule containing a nucleic acid sequence encoding the desired protein is encapsulated in the expressed recombinant adeno-associated virus particle. This can be achieved under conditions under which host cells normally grow, provided that the necessary elements have been appropriately introduced into the host cell. Examples of conditions for producing recombinant adeno-associated virus particles include a temperature appropriate for the host cell (e.g., approximately 37°C for mammalian cells), an appropriate culture medium and nutrients (amino acids, glucose, vitamins, serum, etc.), optimal gas conditions (e.g., 5% CO2, adjusted oxygen concentration), and an appropriate culture time (several hours to several weeks). Furthermore, expression conditions for nucleic acid molecules may include factors such as promoters, enhancers, inducers (drugs, hormones, cytokines, specific compounds), and physical stimuli (light, heat, mechanical stimulation, etc.) that control gene expression. More specifically, it refers to a series of processing steps that place host cells in an appropriate environment so that rAAV particles can be produced using the elements introduced into the host cells. In other words, this process goes beyond simple nucleic acid introduction and includes the establishment and manipulation of conditions and procedures necessary to induce and promote substantial "expression" (production of transcription or translation products) of the introduced nucleic acid. The "expression conditions" in this process include the physical, chemical, cultural, and induction conditions exemplified below: (Regarding temperature conditions, incubation at 37°C is generally desirable for mammalian cells. A temporary temperature shift (e.g., to 32°C) may be performed immediately after introduction.) The medium composition can be appropriately determined by taking into consideration factors such as changing to a medium containing a nutrient source, buffer system, serum (e.g., FBS), and additives (e.g., in the absence of antibiotics) suitable for nucleic acid expression. The time required for incubation after introduction is, for example, several hours to several days, until expression stabilizes. When an inducible promoter is used, an inducer such as doxycycline, IPTG, or tamoxifen can be added at an appropriate concentration.The gas environment is appropriately adjusted by adjusting the O2 concentration (usually 5%) and O2 partial pressure. While cell density is optional, the post-transfection cell density can be appropriately controlled to optimize expression efficiency. Passaging procedures may be performed. For example, this can be done to re-seed cells at high density to sustain expression. Avoidance of inhibitory factors may be performed as desired. For example, cytokines, stress factors, pH fluctuations, and other factors that affect expression can be suppressed. For example, in gene transfer using rAAV vectors, stable gene expression is often confirmed by culturing in a complete medium containing FBS for approximately 72 hours after transfection. Furthermore, in expression-controlled transfection using the Tet-on system, the temporal expression of the target gene can be controlled by adjusting the timing and concentration (e.g., 1 μg / mL) of doxycycline addition. Therefore, this step is essential for host cells transfected with nucleic acid molecules to actually produce the target molecule and is considered one of the core technical means for effectively implementing the present invention.

[0189] In another aspect, a method for producing a recombinant adeno-associated virus vector having a ligand on its surface is provided, comprising: (A) the steps of: (1) introducing into a host cell (i.e., a first nucleic acid sequence that, when introduced, enables expression of VP1, VP2, and VP3); (2) the step of introducing into a host cell (i.e., a second nucleic acid sequence that, when introduced, enables expression of ligand-modified VP3); and (3) the step of introducing into a host cell a nucleic acid sequence encoding a desired protein; and (B) the step of subjecting the host cell to conditions under which the recombinant adeno-associated virus particles are produced.

[0190] In one embodiment, (A) the step of genetically introducing into a host cell (1) a first nucleic acid sequence that, upon genetic introduction, enables expression of VP1, VP2, and VP3, (2) a second nucleic acid sequence that, upon genetic introduction, enables expression of ligand-modified VP3, and (3) a nucleic acid sequence encoding a desired protein can be carried out in various forms, similar to the expression of one or more types of nucleic acid molecules described above. That is, in this step, nucleic acid sequences that enable expression of multiple virion proteins (VPs) involved in virus particle formation are genetically introduced into the host cell, where (1) a first nucleic acid sequence designed to express VP1, VP2, and VP3, and (2) a second nucleic acid sequence designed to express ligand-modified VP3 ("modified VP3") are introduced into the host cell. The first nucleic acid sequence may contain VP1, VP2, and VP3 as independent expression units, or may be designed so that they are expressed simultaneously or sequentially within a single expression cassette using mechanisms such as alternative splicing. The first nucleic acid sequence may be configured as a single nucleic acid molecule containing the coding sequences for these VPs, or may be divided into multiple nucleic acid molecules that are introduced together. The second nucleic acid sequence has a structure in which a sequence encoding a specific ligand molecule is fused to the VP3 gene sequence. This ligand may be a molecule that exhibits specific affinity for a desired target molecule or a cell surface receptor, and may be any molecule such as a partial molecule of an antibody, a ligand-binding domain, a peptide, or an aptamer. In the present disclosure, the first nucleic acid sequence and the second nucleic acid sequence may be introduced into host cells simultaneously at the same time, or sequentially at different times. In the case of sequential introduction, expression of VP1 to VP3 by the first nucleic acid sequence may be initiated in the host cell, followed by the additional introduction of the second nucleic acid sequence, or vice versa. The first nucleic acid sequence and the second nucleic acid sequence may be designed in the same plasmid or vector and introduced together, or may be introduced separately using separate vectors.Furthermore, any known method can be used for the introduction, including transfection in the form of plasmid DNA, introduction using viral vectors such as lentiviral vectors and adeno-associated viral (AAV) vectors, electroporation, lipid particle-mediated methods, etc. Thus, the process of the present invention allows standard VP3 and modified VP3 to be expressed simultaneously or sequentially in host cells, thereby efficiently and reliably producing functional viral particles that display a ligand on their surface.

[0191] In an embodiment of the production method of the present disclosure, it is preferable to introduce genes into the host cells so that the ratio of the number of molecules of the second nucleic acid sequence to the sum of the number of molecules of the first nucleic acid sequence to be introduced (sometimes referred to as the TF ratio) falls within a predetermined range. The predetermined ratio range is usually 1-50%, and typically 3-30% or 1% to 30%. This ratio has been found to provide good AAV productivity and infection efficiency, but is not limited thereto. A lower ratio is preferable when productivity is prioritized, and a higher ratio may be used when infection efficiency is prioritized. In a more preferred embodiment, this ratio (TF ratio) is 5% to 20%, with an upper limit of 50%, 45%, 40%, 35%, 30%, etc., and a lower limit of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.0%, 5%, etc., but is not limited thereto.

[0192] In another embodiment, under conditions in which the nucleic acid sequence is expressed in a host cell of the present disclosure, the proportion of ligand-modified VP3 mRNA in the sum of the number of VP3 mRNA molecules and the number of ligand-modified VP3 mRNA molecules in the host cell (also referred to as the modified VP3 mRNA ratio) is preferably within a predetermined range. The modified VP3 mRNA ratio is typically 0.2% to 30%, more typically 0.5% to 20%. Without wishing to be bound by theory, a ratio below the lower limit may result in reduced productivity, while a ratio above the upper limit may result in reduced infection efficiency. Preferably, the ratio is 1% to 15%, more preferably 2% to 10%.

[0193] In one embodiment, the ligand used in the present disclosure is preferably a polypeptide having a length of 41 amino acids or more. It was not anticipated that such long ligands could be used, and it is notable that the present disclosure is the first to demonstrate that long ligands are feasible. Such lengths may be 50 amino acids or more, 60 amino acids or more, 70 amino acids or more, 80 amino acids or more, 90 amino acids or more, 100 amino acids or more, etc., with an upper limit of 500 amino acids or less, 1000 amino acids or less, etc. The size of the ligand may also be expressed in terms of size, and the ligand used in the present disclosure may be one having a molecular weight greater than a predetermined value. The predetermined molecular weight may be, for example, a polypeptide or other substance having a size of 4.5 kDa or more, and may be 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, 10 kDa or more, etc. On the other hand, the upper limit may be, but is not limited to, 100 kDa or less, 50 kDa or less, 20 kDa or less, etc.

[0194] In another embodiment, the ligands that may be used may be, but are not limited to, VHH, VNAR (shark heavy chain antibody variable region), and the like.

[0195] Conservative amino acid substitutions referred to herein also apply to the substitution of an amino acid in the amino acid sequence of another protein such as VHH with another amino acid.

[0196] In one embodiment, the ligand used has a specific affinity for a protein present on the surface of vascular endothelial cells.

[0197] In another embodiment, the host cell used in the present disclosure does not express ligand-modified VP1 and / or ligand-modified VP2, where not expressing includes not expressing them at all, as well as expressing them at a lower level than normally expressed in nature.

[0198] In another embodiment, any one, two, three, or all of VP1, VP2, VP3, and ligand-modified VP3 used in the present disclosure are mutated VPs in which one or more amino acid residues in Loop-4 and / or Loop-5 and / or Loop-8 of the VP are absent and / or substituted with one or more other amino acid residues, respectively. The mutated VPs may have any of the forms detailed elsewhere in this specification. The ligand has a first linker on the N-terminus, a second linker on the C-terminus, or a first linker on the N-terminus and a second linker on the C-terminus. In the above, the amino acid residues in VP1 refer to serotype 9 adeno-associated virus amino acid residues, or in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), the corresponding amino acid residues of other serotypes of adeno-associated virus when aligned with serotype 9 adeno-associated virus amino acids.

[0199] In another embodiment, the ligand has a specific affinity for another molecule.

[0200] In another embodiment, more than one type of ligand is present on the surface.

[0201] In another embodiment, the ligand comprises an anti-transferrin receptor VHH.

[0202] In another embodiment, the present invention comprises a first linker having an amino acid sequence set forth in any one or more of SEQ ID NOs: 79-91, an amino acid sequence set forth in any one of SEQ ID NOs: 5-13, or a combination of the following: (1) a CDR1 having an amino acid sequence set forth in SEQ ID NO: 14, 15, 20, 21, 26, 27, 32, 33, 40, 41, 46, or 47; (2) a CDR2 having an amino acid sequence set forth in SEQ ID NO: 16, 17, 22, 23, 28, 29, 34, 35, 38, 39, 42, 43, 48, or 49; and (3) a CDR3 having an amino acid sequence set forth in SEQ ID NO: 18, 19, 24, 25, 30, 31, 36, 37, 44, 45, 50, or 51, or, preferably, (A1) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 14 or 15, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 16 or 17, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18 or 19; (A2) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 20 or 21, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 22 or 23, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 24 or 25; (A3) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26 or 27, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 28 or 29, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 30 or 31; (A4) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 32 or 33, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 34 or 35, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 36 or 37; (A5) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26 or 27, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 28 or 29, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 30 or 31; (A6) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26 or 27, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 38 or 39, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 30 or 31;(A7) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 40 or 41, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 42 or 43, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 44 or 45; (A8) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 40 or 41, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 42 or 43, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 44 or 45; or (A9) comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 46 or 47, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 48 or 49, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 50 or 51, and a second linker having the amino acid sequence shown in any one or more of SEQ ID NOs: 79 to 91.

[0203] [Host Cells] In another aspect, the present disclosure provides host cells that produce recombinant adeno-associated virus particles or recombinant adeno-associated virus vectors having VP3 modified with a ligand on their surface. The host cells used in the present disclosure contain the elements necessary for producing adeno-associated virus particles. The host cells are designed to improve targeting ability and maximize gene transfer efficiency into specific tissues or cells. The host cells are mammalian cell lines, particularly cells selected from HEK293 cells, CHO cells, or Sf9 insect cells. These cells are highly adaptable to genetic manipulation and have the intracellular machinery necessary for mass production of recombinant viruses. The host cells of the present disclosure are stably transformed with a first expression vector containing a DNA sequence encoding a fusion protein of a VP3 protein and a ligand. Various ligands are attached to the N-terminus, C-terminus, or specific amino acid residue positions (e.g., positions 587, 588, or 453) of the VP3 protein by genetic engineering techniques. The ligand may be selected from peptide ligands, non-peptide ligands, or synthetic ligands, and may specifically recognize cell surface receptors. Examples include peptides containing an RGD motif, integrin-binding domains, growth factors, cytokines, antibody fragments, aptamers, or small molecule compounds. The host cell is co-transformed with a second expression vector containing an expression cassette for the Rep and Cap genes required for AAV replication. These genes are essential for AAV replication and particle formation and are expressed under the control of a strong promoter. The host cell of the present invention is equipped with a third expression vector containing an AAV genome carrying a gene of interest. The gene of interest encodes a therapeutic protein, functional RNA, or gene editing system, etc., and is flanked by long terminal repeats (ITRs). A fourth expression vector providing helper functions derived from adenovirus or herpesvirus is also introduced into the host cell to support efficient AAV replication and particle formation.The host cells of the present invention have an expression system for ligand-modified VP3 controlled by a temperature-sensitive or inducible promoter, allowing the expression level to be optimized by adjusting the culture conditions. This enables stable production of high-titer AAV vectors. The host cells of the present invention are genetically modified to overexpress glycosyltransferases, sulfotransferases, or other post-translational modification enzymes, providing advanced post-translational modification capabilities to optimize ligand function. In conclusion, the host cells of the present invention enable efficient and large-scale production of adeno-associated virus vectors with improved target specificity, providing an innovative platform for gene therapy and biopharmaceutical development.

[0204] In another aspect, the present disclosure provides a host cell comprising (1) a first nucleic acid sequence that, upon expression, enables expression of VP1, VP2, and VP3, (2) a second nucleic acid sequence that, upon expression, enables expression of the ligand-modified VP3, and (3) a nucleic acid sequence encoding a desired protein, which, when configured, can produce a recombinant adeno-associated virus vector having a ligand-modified VP3 on its surface.

[0205] In another aspect, the present disclosure provides a host cell comprising one or more nucleic acid molecules encoding nucleic acid sequences that, upon expression, enable expression of VP1, VP2, VP3, and ligand-modified VP3, which host cell can then produce a recombinant adeno-associated virus vector bearing ligand-modified VP3 on its surface.

[0206] In one embodiment, in a host cell of the present disclosure, the first nucleic acid sequence and the second nucleic acid sequence are exogenous, and the ratio of the second nucleic acid molecules to the sum of the number of molecules of the first nucleic acid sequence and the number of molecules of the second nucleic acid sequence (sometimes referred to as the TF ratio) is usually 1-50%, and typically 3-30% or 1% to 30%. This ratio has been found to result in good AAV productivity and infection efficiency, but is not limited to this. A lower ratio is preferable when productivity is prioritized, and a higher ratio may be used when infection efficiency is prioritized. In a more preferred embodiment, this ratio (TF ratio) is 5% to 20%, with the upper limit being 50%, 45%, 40%, 35%, 30%, etc., and the lower limit being 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.0%, 5%, etc.

[0207] In one embodiment, the ligand possessed by the AAV particle or AAV vector produced by the host cell of the present disclosure is preferably a polypeptide having a length of 41 amino acids or more. It was not anticipated that such a long ligand could be used, and it is notable that the present disclosure is the first to demonstrate that long ligands are feasible. Such lengths may be 50 amino acids or more, 60 amino acids or more, 70 amino acids or more, 80 amino acids or more, 90 amino acids or more, 100 amino acids or more, etc., with an upper limit of 500 amino acids or less, 1000 amino acids or less, etc. The size of the ligand may also be expressed in terms of size, and the ligand used in the present disclosure may be one having a molecular weight greater than a predetermined value. The predetermined molecular weight may be, for example, a polypeptide or other substance having a size of 4.5 kDa or more, and may be 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, 10 kDa or more, etc. On the other hand, the upper limit may include, but is not limited to, 100 kDa or less, 50 kDa or less, 30 kDa, etc.

[0208] In another embodiment, the ligand carried by the host cell of the present disclosure can be a VHH, VNAR (shark heavy chain antibody variable region), and the like.

[0209] In one embodiment, the ligand possessed by the host cells of the present disclosure has a specific affinity for a protein present on the surface of vascular endothelial cells.

[0210] In another embodiment, the host cell of the present disclosure does not express ligand-modified VP1 and / or ligand-modified VP2, where not expressing includes not expressing them at all, as well as expressing them less than normally expressed in nature.

[0211] In a specific embodiment, the number of molecules of the ligand per recombinant adeno-associated virus-like particle (VLP) in a host cell of the present disclosure (sometimes referred to as the "actual modification rate (number / particle)") is a predetermined value, typically 1 to 50. This value (the "actual modification rate (number / particle)") is preferably 1 to 30, more preferably 1 to 22, 1 to 20, 1 to 16, 2 to 16, 2 to 13, or the like, molecules of the ligand per recombinant adeno-associated virus vector particle. Without wishing to be bound by theory, these values ​​provide good values ​​for both productivity and infection efficiency.

[0212] In another embodiment, the host cell of the present disclosure is a mutated VP in which any one, two, three, or all of VP1, VP2, VP3, and ligand-modified VP3 are mutated VPs in which one or more amino acid residues in Loop-4 and / or Loop-5 and / or Loop-8 of the VP of an adeno-associated virus of serotype 9, or amino acid residues corresponding thereto, are absent and / or substituted with one or more other amino acid residues. Any of the forms of mutated VPs described in detail elsewhere in this specification can be used.

[0213] In another embodiment, the ligand has a specific affinity for another molecule.

[0214] In another embodiment, more than one type of ligand is present on the surface.

[0215] [Recombinant Adeno-Associated Virus Vectors, Recombinant Adeno-Associated Virus Particles (Virus-Like Particles), and Compositions Thereof] In another aspect, the present disclosure provides recombinant adeno-associated (rAAV) virus particles or recombinant adeno-associated virus (rAAV) vectors having VP3 modified with a ligand on their surface. The rAAV particles or rAAV vectors of the present disclosure are used to produce VLPs (rAAV particles or rAAV vectors) containing a VP3 protein constituting the capsid modified with a ligand to enhance targeting to specific cells or tissues. This ligand preferably has specific affinity for a specific receptor on any cell membrane or a specific cell surface antigen. Examples of ligands include, but are not limited to, antibody fragments (e.g., scFv, Fab fragments, VHHs), peptide ligands, carbohydrate ligands, nucleic acid aptamers, cytokines, chemokines, growth factors, etc. The rAAV vectors of the present disclosure are extremely useful in the field of gene therapy, such as for selective delivery to specific diseased tissues and reducing side effects. Ligand modification can be performed at the N-terminus, C-terminus, or internal region of the VP3 protein, but modification of a region that is highly exposed to the capsid surface is particularly preferred, as this allows the ligand to interact with host cell receptors with high efficiency and achieves specific infection efficiency.

[0216] In another aspect, the present disclosure provides a composition comprising an rAAV particle or rAAV vector having VP3 modified with a ligand on its surface. An important aspect of this composition is that it can be used as a pharmaceutical by including a therapeutic or prophylactic protein as the desired protein. In the composition of the present disclosure, VP3, a major protein forming the capsid, is modified with a ligand capable of binding to specific cells or tissues. This improves selectivity and specific infection ability for the intended target cells, and suppresses infection of non-target cells. Such improved targeting is particularly advantageous for pharmaceutical applications, as it maximizes therapeutic efficacy and reduces the risk of side effects.

[0217] In another aspect, the present disclosure provides a virus-like particle (VLP) having VP3 modified with a ligand on its surface. An important aspect of this VLP is that it can be used as a pharmaceutical containing a therapeutic or prophylactic protein as the desired protein. In the VLP of the present disclosure, VP3, the main protein forming the capsid, is modified with a ligand capable of binding to specific cells or tissues. This improves selectivity and specific infectivity for the intended target cells, and suppresses infection of non-target cells. Such improved targeting is particularly advantageous for pharmaceutical applications, as it maximizes therapeutic efficacy and reduces the risk of side effects.

[0218] In another aspect, the present disclosure provides a composition comprising a virus-like particle having VP3 modified with a ligand on its surface. An important aspect of this composition is that it can be used as a pharmaceutical containing a protein for treatment or prevention as the desired protein. In the composition of the present disclosure, VP3, the main protein forming the capsid, is modified with a ligand capable of binding to specific cells or tissues. This improves the selectivity and specific infectivity of the composition toward the intended target cells, and can suppress infection of non-target cells. Such improved targeting is particularly advantageous for pharmaceutical applications, as it maximizes therapeutic efficacy and reduces the risk of side effects.

[0219] The recombinant adeno-associated virus vectors of the present disclosure can be described as blueprints that are introduced into host cells to provide recombinant AAV particles.

[0220] The VLPs produced by the recombinant adeno-associated virus vectors of the present disclosure are characterized in that they do not contain ligand-modified VP1 and ligand-modified VP2, or contain them at a lower rate than when expressed naturally.

[0221] The rAAV particles or rAAV vectors of the present disclosure are extremely useful in the field of gene therapy, such as for selective delivery to specific diseased tissues and reducing side effects. Ligand modification can be performed at the N-terminus, C-terminus, or internal region of the VP3 protein, but modification is particularly preferred in regions that are highly exposed to the capsid surface. This allows the ligand to interact with host cell receptors with high efficiency, achieving specific infection efficiency. Furthermore, rAAV vectors containing the ligand-modified VP3 of the present disclosure can maintain stable particle formation and efficient cell infection without impairing the function of the native AAV capsid protein. Furthermore, the method for producing the vector can be achieved through simultaneous or separate expression of nucleic acid sequences in host cells, resulting in excellent flexibility and efficiency of the production process. In certain embodiments, target cells can be selected arbitrarily depending on the therapeutic or research purpose, such as neurons, hepatocytes, muscle cells, cancer cells, immune cells, and endothelial cells. For example, cancer cell-specific infection can be achieved by using a tumor-specific antibody fragment as the ligand. Furthermore, by using a neuron-specific peptide ligand, it is possible to specifically transfer genes to the central nervous system. The rAAV vector of the present disclosure can achieve stable and long-term expression of target genes, and therefore has the potential to bring about groundbreaking therapeutic effects in the treatment of intractable diseases, rare diseases, and genetic diseases.

[0222] In one embodiment, the ligand contained in the rAAV particle or rAAV vector of the present disclosure is preferably a polypeptide having a length of 41 amino acids or more. It was not anticipated that such a long ligand could be used, and it is notable that the present disclosure is the first to demonstrate that long ligands are feasible. Such lengths may be 50 amino acids or more, 60 amino acids or more, 70 amino acids or more, 80 amino acids or more, 90 amino acids or more, 100 amino acids or more, etc., with an upper limit of 500 amino acids or less, 1000 amino acids or less, etc., but are not limited thereto. The size of the ligand may also be expressed in terms of size, and the ligand used in the present disclosure may be one having a molecular weight greater than a predetermined value. The predetermined molecular weight may be, for example, a polypeptide or other substance having a size of 4.5 kDa or more, and may be 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, 10 kDa or more, etc. On the other hand, the upper limit may be 100 kDa or less, 50 kDa or less, 30 kDa or less, etc., but are not limited thereto.

[0223] In another embodiment, the ligand used in the present disclosure can be, but is not limited to, a VHH, a VNAR (shark heavy chain antibody variable region), and the like.

[0224] In one embodiment, the ligand used in the present disclosure has a specific affinity for a protein present on the surface of vascular endothelial cells.

[0225] In another embodiment, the viral vector of the present disclosure does not have ligand-modified VP1 and / or ligand-modified VP2 on its surface. Here, the absence of these includes not only having less than that normally expressed in nature, but also not having these on the surface or not having them at all.

[0226] In certain embodiments, the number of molecules of the ligand per particle of the rAAV particle or rAAV vector of the present disclosure (sometimes referred to as the "actual modification rate (number / particle)") is a predetermined value, typically 1 to 50. This value (the "actual modification rate (number / particle)") may preferably be 1 to 30, more preferably 1 to 20, 1 to 16, or the like, molecules of the ligand per recombinant adeno-associated virus vector particle. Without wishing to be bound by theory, these values ​​provide good values ​​for both productivity and infection efficiency.

[0227] In another embodiment, in an rAAV particle or rAAV vector of the present disclosure, any one, two, three, or all of VP1, VP2, VP3, and ligand-modified VP3 are mutated VPs in which one or more amino acid residues in Loop-4 and / or Loop-5 and / or Loop-8 of the VP are absent and / or substituted with one or more other amino acid residues, respectively. The mutated VPs may take any of the forms described in detail elsewhere in this specification. Here, the amino acid residues in VP1 refer to the amino acid residues of adeno-associated virus of serotype 9, or, in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), the amino acid residues of adeno-associated virus of other serotypes that correspond to these when aligned with the amino acid residues of adeno-associated virus of serotype 9.

[0228] In one embodiment, in a composition comprising an rAAV particle or rAAV vector of the present disclosure having VP3 modified with a ligand on its surface, the actual modification rate of the number of molecules of the ligand in the VLP1 particles produced is a predetermined percentage. The actual modification rate is typically 1 to 50%. Here, the actual modification rate (%) is the ratio of VLPs having the modified VP to a vector comprising VLPs having VP3 modified with a ligand on their surface and VLPs having unmodified VP3.

[0229] In a preferred embodiment, the actual modification rate of the ligand of the present disclosure may be 3 to 30%, more preferably 4 to 25%, or 4 to 10%. It has been found that these actual modification rates result in good clearance rates and infection efficiencies.

[0230] In a specific embodiment, for a ligand contained in a composition of the present disclosure, the number of molecules of the ligand per VLP particle (the "actual modification rate (number / particle)") is a predetermined value, typically 1 to 50. This value (the "actual modification rate (number / particle)") is preferably 1 to 30, more preferably 1 to 22, 1 to 20, 1 to 16, 2 to 16, 2 to 13, or the like, molecules of the ligand per recombinant adeno-associated virus vector particle. Without wishing to be bound by theory, this is because these values ​​provide good values ​​for both productivity and infection efficiency.

[0231] In another embodiment, the ligand has a specific affinity for another molecule.

[0232] In another embodiment, more than one type of ligand is present on the surface.

[0233] [Reduced Hepatotoxicity VP] In another aspect, there is provided a mutated VP in which one or more amino acid residues in Loop-4 and / or Loop-5 and / or Loop-8 of VP are absent and / or substituted with one or more other amino acid residues, wherein the amino acid residues in VP1 represent the amino acid residues of serotype 9 adeno-associated virus, or in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), the amino acid residues of other serotypes of adeno-associated virus that correspond to these when aligned with the amino acid residues of serotype 9 adeno-associated virus. This mutated VP is characterized by the deletion or substitution of one or more amino acid residues in the Loop-4, Loop-5, and Loop-8 regions. In a specific embodiment, the VP includes a mutant in which one or more specific amino acid residues are deleted in the Loop-4 region of the VP. In another embodiment, the present disclosure also encompasses mutants in which amino acid residues in the Loop-5 region are substituted with other amino acid residues, such as alanine, serine, glycine, or threonine. In yet another embodiment, the present disclosure also encompasses mutant VPs in which amino acid residues in the Loop-8 region are deleted or substituted. These mutations can confer new features to the structural or functional properties of VP1. For example, mutations in these Loop regions are expected to improve properties such as host cell infection efficiency, tissue specificity, immunogenicity, or in vivo stability. In particular, the Loop-4, Loop-5, and Loop-8 regions are highly exposed on the surface of the capsid and are directly involved in interaction with host cell surface receptors and immune recognition, so introducing mutations into these regions may alter the infection specificity of target cells and recognition by the immune system. Furthermore, AAV vector particles produced using the mutated VPs of the present disclosure may have the potential to improve the transduction efficiency of target genes and are extremely useful in fields such as gene therapy, vaccine development, and gene expression regulation.Furthermore, vector particles containing the present mutated VP may have reduced immunogenicity in vivo compared to vector particles derived from conventional wild-type AAV9, enabling repeated administration. One specific example is a mutant vector particle with improved selective infection of neurons or muscle cells obtained by substituting specific amino acids in Loop-4 or Loop-5. Another specific example includes an embodiment in which hepatocyte-specific tropism is exhibited by amino acid deletion or substitution in the Loop-8 region. The present mutated VP can be produced, for example, by mutagenesis methods that identify the position of specific amino acids (site-directed mutagenesis). Specifically, it can be prepared by various genetic engineering techniques, such as site-directed mutagenesis using PCR, gene editing using CRISPR-Cas technology, and construction methods using synthetic gene fragments. The mutated VPs provided in these various embodiments significantly expand the range of therapeutic and research applications of rAAV vectors, contributing to improved therapeutic efficacy and safety for specific diseases, particularly in the field of gene therapy.

[0234] In one embodiment, the present invention relates to rAAV particles that suppress liver infection when administered intravenously or otherwise. Such rAAV particles have a mutation in the Loop-8 region of the capsid protein VP. Here, in the case of serotype 9 AAV, the Loop-8 region corresponds to amino acids 582 to 604 from the N-terminus of VP1, a region that nearly overlaps with the variable region.

[0235] Preferred mutations in the Loop-8 region include deletion of amino acids constituting the Loop-8 region. As long as infection of the liver is suppressed, the number of amino acids to be deleted is not particularly limited, but is preferably 1 to 8, for example, 1 to 6, 1 to 4, 1 to 3, or 1 to 2. A specific example of a preferred mutation is one in which the alanine residue at position 591, the glutamine residue at position 592, the threonine residue at position 593, and the glycine residue at position 594 are deleted from the N-terminus of VP1. Among these, one in which the alanine residue at position 591, the glutamine residue at position 592, and the threonine residue at position 593 are deleted from the N-terminus of VP1 is more preferred, and one in which the threonine residue at position 593 is deleted is particularly preferred. Alternatively, two, three, or four amino acid residues may be deleted by combining deletion of the alanine residue at position 591, the glutamine residue at position 592, the threonine residue at position 593, and the glycine residue at position 594 from the N-terminus of VP1. For AAV of other serotypes, mutations similar to those described above can be introduced into the region corresponding to the Loop-8 region of AAV of serotype 9. The amino acid sequences of the regions corresponding to the Loop-8 region of AAV of various serotypes are shown in the Sequence Listing as SEQ ID NOs: 160 to 173.

[0236] A preferred mutation in the Loop-8 region is the addition of an amino acid to the Loop-8 region. As long as infection of the liver is suppressed, the number of amino acids to be added is not particularly limited, but is preferably 1 to 8, for example, 1 to 6, 1 to 4, 2 to 6, or 2 to 4. A specific example of a suitable mutation is one in which the alanine residue at position 591, the glutamine residue at position 592, the threonine residue at position 593, and the glycine residue at position 594 are deleted from the N-terminus of VP1. Among these, one in which the alanine residue at position 591, the glutamine residue at position 592, and the threonine residue at position 593 are deleted from the N-terminus of VP1 is more preferred, and one in which the threonine residue at position 593 is particularly preferred. For AAV of other serotypes, mutations similar to those described above can be introduced into the region corresponding to the Loop-8 region of serotype 9 AAV.

[0237] When rAAV particles with such mutations are administered by intravenous injection or the like, infection of the liver is suppressed, and therefore the amount taken up by other organs can be increased.

[0238] The above-mentioned mutation in the capsid protein that suppresses liver infection of rAAV particles is one embodiment that can be used in combination with other embodiments. For example, the following two forms can be used to apply the mutation to rAAV particles whose CAP protein contains a fusion protein of the CAP protein and another protein: (1) the mutation is introduced into all of the CAP proteins, including the fusion protein of the CAP protein and another protein; and (2) the mutation is introduced only into the fusion protein of the CAP protein and another protein, but not into the other CAP proteins.

[0239] In another aspect, the present disclosure provides a method for producing a recombinant adeno-associated virus vector, comprising incorporating into an adeno-associated virus vector a mutated VP in which any one, two, or all of VP1, VP2, and VP3 lacks and / or substitutes with one or more other amino acid residues one or more of the amino acid residues in Loop-4 and / or Loop-5 and / or Loop-8 of the VP, respectively. The amino acid residues in VP1 represent the amino acid residues of serotype 9 adeno-associated virus, or, in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), the amino acid residues of the adeno-associated virus of the other serotype that correspond to these when aligned with the amino acid residues of serotype 9 adeno-associated virus.

[0240] The method includes the step of producing an rAAV vector containing, as a capsid component, a VP (hereinafter referred to as a "mutated VP") in which mutations have been introduced into any one, two, or all of VP1, VP2, and VP3 at amino acid residues exemplified herein in Loop-4, Loop-5, and Loop-8 derived from VP1, or amino acid residues corresponding to those residues in other serotypes. The amino acid residues in VP1 represent serotype 9 adeno-associated virus amino acid residues, or, in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, and AAV-LK03), represent adeno-associated virus amino acid residues of other serotypes that correspond to those residues when aligned with serotype 9 adeno-associated virus amino acids.

[0241] In the production method of the present disclosure, first, a nucleic acid containing a cap gene encoding the desired mutated VP is designed, and the gene sequence is incorporated into a plasmid vector. Mutation is achieved by site-directed mutagenesis or insertion of a synthetic gene fragment, resulting in the deletion or substitution of one or more amino acid residues in the loop region with other amino acids. Next, a plasmid containing the cap gene, an auxiliary plasmid containing a rep gene, and an rAAV vector genome plasmid containing the target gene are simultaneously or sequentially co-transfected into appropriate host cells (e.g., HEK293 cells). After transfection, expression of the VP protein and assembly and packaging of rAAV particles are induced in the cells. After culturing, rAAV particles are recovered from the resulting cells or culture supernatant, and purified (e.g., by density gradient centrifugation, affinity chromatography, etc.), allowing the production of highly purified rAAV vectors containing the mutated VP. The rAAV vector obtained by this method has a capsid structure modified by the mutated VP, and is endowed with therapeutically useful properties such as improved target tissue specificity, increased infection efficiency, or improved immune evasion ability.

[0242] In another aspect, the present invention provides a host cell that produces a recombinant adeno-associated virus vector in which any one, two, or all of VP1, VP2, and VP3 are mutated VPs in which one or more amino acid residues of Loop-4 and / or Loop-5 and / or Loop-8 of VP are absent and / or substituted with one or more other amino acid residues, respectively, and that has a gene cluster necessary for producing the recombinant adeno-associated virus vector. The amino acid residues in VP1 represent the adeno-associated virus amino acid residues of serotype 9, or, in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), the corresponding adeno-associated virus amino acid residues of other serotypes when aligned with the adeno-associated virus amino acids of serotype 9.

[0243] In another aspect, the present disclosure provides a recombinant adeno-associated virus vector in which any one, two, or all of VP1, VP2, and VP3 are mutated VPs that lack and / or substitute one or more amino acid residues of Loop-4 and / or Loop-5 and / or Loop-8 of the VP, respectively. The amino acid residues in VP1 represent the amino acid residues of serotype 9 adeno-associated virus, or, in the case of other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), the amino acid residues of the adeno-associated virus of the other serotype that correspond to these when aligned with the amino acid residues of serotype 9 adeno-associated virus.

[0244] In one preferred embodiment, the mutated VP, recombinant adeno-associated virus vector, method for producing the same, and host cell of the present disclosure do not have one or more amino acid residues at positions 496, 497, 498, 499, 502, 504, 591, 592, 593, 594, and 595 of VP1 or at positions corresponding thereto, when aligned with VP1, in the case of an adeno-associated virus of serotype 9 or other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03). In a preferred embodiment, in the case of serotype 9 or other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AVrhlO, AAV-DJ, AAV-LK03), it is preferable that the site for introducing the mutation does not have any of the amino acid residues at positions 589 and 590, 590 and 591, 591 and 592, and 594 and 595 of VP1, or any combination of amino acid residues at positions corresponding thereto, when aligned with adeno-associated virus VP1 of serotype 9.

[0245] In one preferred embodiment, the mutated VP, recombinant adeno-associated virus vector, production method thereof, and host cell of the present disclosure can be employed as an individual embodiment in any of the embodiments described in the present specification, such as [Production method], [Host cell], [Recombinant adeno-associated virus vector, recombinant adeno-associated virus particle (virus-like particle), and composition thereof], or in any combination thereof.

[0246] Although the present disclosure has been described above, the present disclosure is not limited to the above and various modifications are possible within the scope of the gist of the present disclosure. Hereinafter, the present disclosure will be described in more detail using examples, but the present disclosure is not limited to the following examples and can of course be implemented by making appropriate modifications within the scope that can conform to the above and below gist, and all of these modifications are included in the technical scope of the present disclosure.

[0247] The present invention will be described in more detail below with reference to examples, but it is not intended that the present invention be limited to these examples.

[0248] Example 1: Construction of pR2C8 Vector The pAAV-CMV-GFP vector having the nucleotide sequence of SEQ ID NO: 133 was cleaved with ClaI and BglII, and the R2C8 DNA fragment (SEQ ID NO: 67), synthesized so as to be sandwiched between ITRs, was inserted using the In-Fusion HD cloning kit (Clontech). Furthermore, the nucleotide sequence of the ITRs at both ends (SEQ ID NO: 68) was removed by inverse PCR to create a vector named pR2C8. pR2C8 contains, from upstream to downstream, the AAV2 Rep region (SEQ ID NO: 69), the AAV8 Cap region (SEQ ID NO: 70), and the p5 promoter (SEQ ID NO: 71), which functions as an enhancer. Furthermore, pR2C8 contains an ampicillin resistance gene and an origin of replication (ColE1 ori).

[0249] Example 2: Construction of pR2C9 Using pR2C8 prepared in Example 1 as a template, PCR was performed using Primer 1 (SEQ ID NO: 72) and Primer 2 (SEQ ID NO: 73) to synthesize a DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 74, which includes the AAV9 Cap region. This synthesized DNA fragment was then inserted into the PCR-amplified DNA fragment using an In-Fusion HD cloning kit (Clontech). The resulting vector was named pR2C9. pR2C9 contains, from upstream to downstream, the AAV2 Rep region, the AAV9 Cap region (SEQ ID NO: 75), and the p5 promoter (SEQ ID NO: 71), which functions as an enhancer. pR2C9 also contains an ampicillin resistance gene and an origin of replication (ColE1 ori).

[0250] Example 3: Preparation of pR2C8 Vector Incorporating a VHH with Affinity for Human TfR A DNA fragment having the nucleotide sequence of SEQ ID NO: 76 was synthesized, including a nucleotide sequence encoding an N-terminal linker, a nucleotide sequence encoding a VHH with affinity for human TfR, and a nucleotide sequence encoding a C-terminal linker. The amino acid sequence of the N-terminal linker was (GGGGGS x 1), the amino acid sequence of the C-terminal linker was (GGGGGS x 3), and the amino acid sequence of the VHH was SEQ ID NO: 9. This DNA fragment was inserted into the Cap region of pR2C8 using an In-Fusion HD cloning kit (Clontech) so as to be in frame with the gene encoding the capsid in the Cap region. After insertion of this DNA fragment, the vector encodes a fusion protein in which the amino acid sequence of a VHH with a linker attached (linker-attached VHH) is present within the amino acid sequence of VP1 (SEQ ID NO: 2) encoded in the Cap region. The position of the linker-attached VHH in the fusion protein is 269, 353, 359, 377, 387, 395, 434, 455, 457, 462, 468, 501, 552, 576, 588, 599, 656, 666, 709, or 719. Here, each number indicates the amino acid number counted from the N-terminus of the amino acid sequence of VP1, and the linker-attached VHH is located on the C-terminus side. The name of each vector is, for example, pR2C8(VHH269), indicating the position of the linker-attached VHH at the end.

[0251] Furthermore, pR2C8 (VHH456-462) was prepared by the following method. pR2C8 (VHH456-462): Using pR2C8 prepared in Example 1 as a template, PCR was performed with primer 3 (SEQ ID NO: 77) and primer 4 (SEQ ID NO: 78) to obtain a DNA fragment. This DNA fragment and the DNA fragment encoding the linker-attached VHH were ligated using an In-Fusion HD cloning kit (Clontech). The resulting vector lacks amino acid sequences 456 to 462 of the VP1 amino acid sequence (SEQ ID NO: 2) encoded in the Cap region, and instead encodes a fusion protein having the amino acid sequence of a linker-attached VHH (linker-attached VHH). This vector was designated pR2C8 (VHH456-462).

[0252] [Example 4] Preparation of pR2C9 vector incorporating VHH with affinity to human TfR pR2C9 incorporating VHH with affinity to human TfR, pR2C9 (VHH455-460), pR2C9 (VHH455), and pR2C9 (VHH2-455), were each prepared by the following method.

[0253] pR2C9 (VHH455-460): PCR was performed using pR2C9 prepared in Example 2, primer 11 (SEQ ID NO: 127), and primer 12 (SEQ ID NO: 128) to obtain a DNA fragment. This DNA fragment was ligated to the DNA fragment encoding the linker-attached VHH using an In-Fusion HD cloning kit (Clontech). The resulting vector encodes a fusion protein lacking amino acid sequences 455 to 460 of the VP1 sequence encoded in the Cap region, and instead containing the amino acid sequence of a linker-attached VHH (linker-attached VHH). This vector was designated pR2C9 (VHH455-460). Here, the amino acid sequence of the N-terminal linker is the cIgG2a Hinge linker of SEQ ID NO: 81, the amino acid sequence of the C-terminal linker is also the cIgG2a Hinge linker, and the amino acid sequence of the VHH is shown in SEQ ID NO: 9. Similarly, pR2C9 (VHH455) was also prepared in which a gene encoding a linker-added VHH was inserted at a position corresponding to the C-terminal side of the 455th amino acid residue from the N-terminus of VP1 encoded in the Cap region.

[0254] pR2C9 (VHH2-455): A DNA fragment was amplified by PCR using primers 11 and 13 (SEQ ID NOs: 127 and 129), which were designed to remove the nucleotide sequences encoding the VHH and C-terminal linker of pR2C9 (VHH455-460). A DNA fragment (SEQ ID NO: 130) encoding a VHH (VHH2) with affinity for anti-TfR and having the amino acid sequence shown in SEQ ID NO: 11 was synthesized, and this DNA fragment and the PCR amplification product were ligated using an In-Fusion HD cloning kit (Clontech). Using the resulting vector as a template, PCR was performed with primers 11 and 14 (SEQ ID NOs: 127 and 131), which were designed for the C-terminus of VHH2, to amplify a DNA fragment having a nucleotide sequence encoding a VHH2 with a linker sequence (SEQ ID NO: 132) added to the C-terminus, and this amplification product was allowed to self-anneal. The resulting vector was designated pR2C9 (VHH2-455), which encodes a fusion protein in which...

Claims

1. A method for producing recombinant adeno-associated virus particles having a ligand on their surface, comprising: (A) introducing into a host cell a nucleic acid molecule containing a VP nucleic acid sequence that, upon introduction, enables expression of VP1, VP2, VP3, and VP3 modified with the ligand, as well as a nucleic acid molecule containing a nucleic acid sequence encoding a desired protein; and (B) subjecting the host cell to conditions that produce the recombinant adeno-associated virus particles.

2. A method for producing recombinant adeno-associated virus particles having a ligand on their surface, comprising: (A) the steps of: (1) introducing into a host cell: (i) a first nucleic acid sequence that, when introduced, enables the expression of VP1, VP2, and VP3; (2) a second nucleic acid sequence that, when introduced, enables the expression of VP3 modified with a ligand; and (3) a nucleic acid sequence encoding a desired protein; and (B) subjecting the host cell to conditions that produce the recombinant adeno-associated virus particles.

3. The method of claim 1 or 2, wherein the host cell contains the elements necessary to produce adeno-associated virus particles.

4. The method according to claim 2 or 3, wherein the first nucleic acid sequence and the second nucleic acid sequence are introduced as separate nucleic acid molecules, and are introduced into the host cell so that the ratio of the number of molecules of the second nucleic acid sequence to the sum of the number of molecules of the first nucleic acid sequence to be introduced and the number of molecules of the second nucleic acid sequence to be introduced is 1 to 50%.

5. The method according to any one of claims 2 to 4, wherein the first nucleic acid sequence and the second nucleic acid sequence are introduced as separate nucleic acid molecules and are introduced into the host cell so that the ratio of the number of molecules of the second nucleic acid sequence to the sum of the number of molecules of the first nucleic acid sequence to be introduced and the number of molecules of the second nucleic acid sequence to be introduced is 5% to 20%.

6. The method according to any one of claims 1 to 5, wherein, under conditions for producing the recombinant adeno-associated virus particles, the proportion of ligand-modified VP3 mRNA in the sum of the number of VP3 mRNA molecules and the number of ligand-modified VP3 mRNA molecules in the host cells is 0.5% to 20%.

7. The method of any one of claims 1 to 6, wherein the ligand is a polypeptide having a length of 41 amino acids or more.

8. The method of any one of claims 1 to 7, wherein the ligand is a polypeptide having a size of 4.5 kDa or greater.

9. The method according to any one of claims 1 to 8, wherein the ligand is a VHH.

10. The method according to any one of claims 1 to 9, wherein the ligand has a specific affinity for a protein present on the surface of vascular endothelial cells.

11. The method of any one of claims 1 to 10, wherein the host cell does not express ligand-modified VP1 and / or ligand-modified VP2.

12. The method according to any one, two, three or all of the VP1, the VP2, the VP3 and the ligand-modified VP3 are mutated VPs in which one or more amino acid residues of Loop-4 and / or Loop-5 and / or Loop-8 of VP are absent and / or substituted with one or more other amino acid residues.

13. A host cell that produces recombinant adeno-associated virus particles having VP3 modified with a ligand on their surface.

14. The host cell of claim 13, comprising: (1) a first nucleic acid sequence that, when expressed, enables expression of VP1, VP2, and VP3; (2) a second nucleic acid sequence that, when expressed, enables expression of VP3 modified with the ligand; and (3) a nucleic acid sequence encoding a desired protein.

15. Recombinant adeno-associated virus (rAAV) particles having VP3 modified with a ligand on their surface.

16. The rAAV particle of claim 15, wherein the adeno-associated virus particle comprises elements necessary for constructing the particle.

17. The rAAV particle of claim 15 or 16, wherein the ligand is a polypeptide having a length of 41 amino acids or more.

18. The rAAV particle of any one of claims 15 to 17, wherein the ligand is a polypeptide having a size of 4.5 kDa or greater.

19. The rAAV particle of any one of claims 15 to 18, wherein the ligand is a VHH.

20. The rAAV particle of any one of claims 15 to 19, which is substantially free of ligand-modified VP1 and ligand-modified VP2.

21. The rAAV particles according to any one of claims 15 to 20, which are configured so that the number of molecules of the ligand per rAAV particle is 1 to 50.

22. The rAAV particle of any one of claims 15 to 21, wherein the number of molecules of the ligand per recombinant adeno-associated virus particle is 1 to 16.

23. The rAAV particle of any one of claims 15 to 22, wherein any one, two, three, or all of VP1, VP2, VP3, and ligand-modified VP3 are mutated VPs in which one or more amino acid residues in Loop-4 and / or Loop-5 and / or Loop-8 of VP are absent and / or substituted with one or more other amino acid residues.

24. The rAAV particle described in any one of claims 15 to 23, wherein the ligand has a first linker on the N-terminus side, or a second linker on the C-terminus side, or a first linker on the N-terminus side and a second linker on the C-terminus side.

25. The rAAV particle of any one of claims 156-24, wherein the ligand has a specific affinity for another molecule.

26. The rAAV particle of any one of claims 15 to 25, wherein the ligand comprises an anti-transferrin receptor VHH.

27. A pharmaceutical composition comprising the rAAV particles of any one of claims 15 to 26.

28. A recombinant adeno-associated virus (rAAV) particle comprising a mutated VP in which any one, two, or all of VP1, VP2, and VP3 are absent and / or have one or more amino acid residues in Loop-4 and / or Loop-5 and / or Loop-8 of the VP substituted with one or more other amino acid residues, respectively.

29. A mutagenized VP in which one or more amino acid residues in Loop-4 and / or Loop-5 and / or Loop-8 of the VP are absent and / or substituted with one or more other amino acid residues.

30. A virus-like particle (VLP) comprising a mutated VP in which one or more amino acid residues in Loop-4 and / or Loop-5 and / or Loop-8 of the VP are absent and / or substituted with one or more other amino acid residues.

31. The mutated VP of claim 29, wherein the VP is VP3.

32. The mutated VP of claim 30, wherein the mutation is contained in Loop-8.

33. The mutated VP of claim 29, which does not have any one of the amino acid residues at positions 589 and 590, 590 and 591, 591 and 592, and 594 and 595 of VP1, or any combination of amino acid residues at positions corresponding to these.

34. The mutated VP of claim 29, which does not have one or more amino acid residues at positions 496, 497, 498, 499, 502, 504, 591, 592, 593, 594, and 595 of VP1 or their corresponding positions.

35. The rAAV particle of claim 28, which does not have one or more amino acid residues at or corresponding to amino acid residues 496, 497, 498, 499, 502, 504, 591, 592, 593, 594, and 595 of VP1.

36. The rAAV particle of claim 28, which does not have any one of the amino acid residues at positions 589 and 590, 590 and 591, 591 and 592, and 594 and 595 of VP1, or any combination of amino acid residues at positions corresponding thereto.

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