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WO2025230000A1 - Particules d'aav modifiées, procédé de production, médicament et production associés - Google Patents

Particules d'aav modifiées, procédé de production, médicament et production associés

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Publication number
WO2025230000A1
WO2025230000A1 PCT/JP2025/016439 JP2025016439W WO2025230000A1 WO 2025230000 A1 WO2025230000 A1 WO 2025230000A1 JP 2025016439 W JP2025016439 W JP 2025016439W WO 2025230000 A1 WO2025230000 A1 WO 2025230000A1
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WIPO (PCT)
Prior art keywords
protein
amino acid
nucleic acid
ligand
acid sequence
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Pending
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PCT/JP2025/016439
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English (en)
Japanese (ja)
Inventor
勇平 蘆田
理央 鈴木
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JCR Pharmaceuticals Co Ltd
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JCR Pharmaceuticals Co Ltd
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Publication of WO2025230000A1 publication Critical patent/WO2025230000A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/015—Parvoviridae, e.g. feline panleukopenia virus, human parvovirus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • This disclosure relates to improved rAAV particles and methods for producing them.
  • the adeno-associated virus is a linear, single-stranded DNA virus belonging to the Parvoviridae family, one of the smallest naturally occurring viruses, with a viral genome of approximately 4.7 kb. It is non-enveloped and forms icosahedral particles with a diameter of approximately 22 nm.
  • the viral genome When wild-type AAV infects a human host cell alone, the viral genome is integrated site-specifically into chromosome 19 via the inverted terminal repeats (ITRs) present at both ends of the viral genome. Most of the genes of the viral genome integrated into the host cell genome are not expressed, but when the cell is infected with a helper virus, AAV is excised from the host genome and infectious viral replication begins.
  • the helper virus is an adenovirus
  • the genes responsible for the helper function are E1A, E1B, E2A, VA1, and E4.
  • 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.
  • 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 the chromosome.
  • the Cap gene is responsible for the production of three capsid proteins (VP1, VP2, and VP3).
  • the wild-type AAV genome has ITRs at both ends, with the Rep and Cap genes located between the ITRs.
  • Recombinant AAV particles rAAV particles
  • rAAV particles have the regions containing the Rep and Cap genes replaced with genes encoding foreign proteins, and these are then encapsulated in a capsid.
  • plasmids 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).
  • these three types of plasmids are introduced into host cells such as HEK293 cells. rAAV particles are synthesized within the host cells into which the three types of plasmids have been introduced.
  • AAV has many variants with different serum types, called serotypes, and 11 of these serotypes are known to infect human cells.
  • Each AAV serotype has a specific organ tropism.
  • AAV9 is known to infect the central nervous system (CNS), heart, lungs, liver, and skeletal muscle.
  • AAV infects a variety of human organs, it is not thought to be pathogenic. Therefore, attempts are being made to use AAV as a material for producing recombinant viruses for gene therapy.
  • Each AAV serotype has a specific tropism for the organ it infects, so attempts are being made to use different serotypes depending on the organ to which the gene should be introduced via gene therapy.
  • AAV9 has a tropism for the central nervous system (CNS), heart, lungs, liver, and skeletal muscle, so attempts are being made to use it as a material for producing recombinant viruses for introducing genes into these organs and tissues.
  • a recombinant AAV genome in which 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.
  • rAAV particles are produced in a test tube by carrying out the process in which the wild-type AAV genome is encapsulated in capsid protein to form wild-type AAV.
  • Direct recombinant targeting and indirect recombinant targeting are well-known methods for increasing the organ tropism of rAAV particles.
  • Direct recombinant targeting for example, uses rAAV particles containing capsids modified to have affinity for proteins expressed on the surface of target cells.
  • Indirect recombinant targeting involves creating rAAV particles containing capsids with scaffolds that can bind to other molecules, and then attaching, via the scaffolds, substances that have affinity for proteins expressed on the surface of target cells, thereby conferring tropism to the rAAV particles.
  • Patent Documents 4 and 5 Many reports have been published on these direct and indirect recombinant targeting methods.
  • 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.
  • 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.
  • a method for producing recombinant adeno-associated virus particles having a ligand on their surface comprising: A method comprising the steps of: (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, and 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 particles are produced.
  • [Item 2A] The method according to any one of the above items, wherein the ligand is present in a single or multiple form (which may be the same or different types).
  • [Item 3] The method according to any one of the preceding items, wherein the host cell contains the elements necessary for producing adeno-associated virus particles.
  • [Item 4] The method of any one of the preceding items, wherein the necessary elements include a nucleic acid sequence encoding a Rep protein.
  • the nucleic acid sequence encoding the Rep protein and the VP nucleic acid sequence are located, individually or together, between at least two inverted terminal repeats (ITRs).
  • [Item 6] The method according to any one of the preceding items, wherein the necessary elements further comprise a nucleic acid sequence encoding a protein that performs a helper function.
  • the proteins responsible for the helper action include at least one, two, three, four, or all five selected from the group consisting of E1A, E1B, E2A, VA1, and E4.
  • each protein or all proteins are located between at least two inverted terminal repeats (ITRs).
  • nucleic acid sequence encoding the desired protein is located between at least two inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • the desired protein includes a therapeutic protein, a protein for genome editing, a test protein, etc.
  • the nucleic acid sequence encoding the desired protein is incorporated into the recombinant adeno-associated virus particle under conditions in which the particle is produced, so that the nucleic acid sequence is subsequently incorporated into the particle in an expressible state.
  • [Item 16] The method according to any one of the preceding items, wherein, under conditions in which the recombinant adeno-associated virus particles are produced, 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 is 1% to 15%.
  • the ligand is a polypeptide having a length of 41 amino acids or more.
  • the ligand is a polypeptide having a size of 4.5 kDa or more.
  • 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.
  • a host cell that produces recombinant adeno-associated virus particles having VP3 modified with a ligand on the surface.
  • [Item 24A] The host cell according to any one of the preceding items, wherein the ligand is present singly or in plural (which may be the same or different types).
  • [Item 25] The cell of any one of the preceding items, wherein the host cell contains the elements necessary for producing adeno-associated virus particles.
  • 26. The host cell of any one of the preceding claims, wherein the necessary elements include a nucleic acid sequence encoding a Rep protein.
  • the nucleic acid sequence encoding the Rep protein and the VP nucleic acid sequence, respectively or together are located between at least two inverted terminal repeats (ITRs).
  • the proteins responsible for the helper function include at least one, two, three, four, or all five selected from the group consisting of E1A, E1B, E2A, VA1, and E4.
  • the proteins responsible for the helper function when there are two or more, are located between at least two inverted terminal repeats (ITRs), either individually or together.
  • nucleic acid sequence encoding the desired protein is located between at least two inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • the desired protein includes a therapeutic protein, a protein for genome editing, a test protein, etc.
  • nucleic acid sequence encoding the desired protein is incorporated into the recombinant adeno-associated virus particle under conditions in which the particle is produced, so that the nucleic acid sequence is subsequently incorporated into the particle in an expressible state.
  • first nucleic acid sequence and the second nucleic acid sequence are exogenous and have been genetically introduced so that the ratio of the number of molecules of the second nucleic acid molecule 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 37] The host cell according to any one of the preceding items, wherein the first nucleic acid sequence and the second nucleic acid sequence are exogenous and have been genetically introduced so that the ratio of the number of molecules of the second nucleic acid molecule 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 5% to 20%.
  • [Item 38] The host cell according to any one of Items 2 to 2A2, 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 0.5% to 20%.
  • [Item 39] The host cell according to 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%.
  • the ligand is a polypeptide having a length of 41 amino acids or more.
  • the ligand is a polypeptide having a size of 4.5 kDa or more.
  • the ligand is a VHH.
  • [Item 43] The host cell according to any one of the preceding items, wherein the ligand has a specific affinity for a protein present on the surface of a vascular endothelial cell.
  • the host cell according to any one of the preceding items which does not express ligand-modified VP1 and ligand-modified VP2.
  • the host cell according to any one of the preceding items which is substantially free of ligand-modified VP1 and/or ligand-modified VP2.
  • [Item 46] 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 50.
  • 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.
  • serotypes e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03
  • the ligand is a polypeptide having a length of 41 amino acids or more.
  • the ligand is a VHH.
  • the ligand has specific affinity for a protein present on the surface of a vascular endothelial cell.
  • the rAAV particle or rAAV vector according to any one of the preceding items which is substantially free of ligand-modified VP1 and ligand-modified VP2.
  • the rAAV particle or rAAV vector according to any one of the preceding items wherein the number of molecules of the ligand per rAAV particle is 1 to 50.
  • 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.
  • serotype 9 adeno-associated virus amino acid residues
  • the ligand comprises a first linker having an amino acid sequence set forth in any one or more of SEQ ID NOS: 79 to 91, an amino acid sequence set forth in any one of SEQ ID NOS: 5 to 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,
  • (A1) it comprises CDR1 comprising the amino acid sequence shown in SEQ ID NO: 14 or 15, CDR
  • [Item 73] A pharmaceutical composition comprising the rAAV particle or rAAV vector described in any one of the above items.
  • [Item 73A] A method for preventing or treating a disease, disorder, or symptom that is prevented or treated by a desired protein encoded by a nucleic acid molecule contained in an rAAV particle or rAAV vector, comprising administering an effective amount of the rAAV particle or rAAV vector described in any one of the above items to a subject in need thereof.
  • [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 the 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 the 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 the modification with the ligand is 1 (number/particle) or more.
  • composition according to any one of the preceding items, wherein the actual modification rate of the modification with the ligand is 1 to 50 (number/particle).
  • composition according to any one of the preceding items, wherein the actual modification rate of the modification with the ligand is 1 to 22 (number/particle) or more.
  • composition according to any one of the preceding items, wherein the actual modification rate of the modification with the ligand is 2 to 16 (number/particle) or more.
  • composition according to any one of the preceding items, wherein the actual modification rate of the modification with the ligand is 2 to 13 (number/particle) or more.
  • a pharmaceutical composition comprising the VLP of any one of the preceding items, which comprises a nucleic acid sequence encoding a therapeutic or prophylactic protein.
  • 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.
  • serotype 9 adeno-associated virus amino acid residues
  • 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.
  • serotype 9 adeno-associated virus amino acid residues
  • 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.
  • rAAV adeno-associated virus
  • 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.
  • serotype 9 adeno-associated virus amino acid residues
  • 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.
  • 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 amino acid residues of adeno-associated viruses of other serotypes when aligned with serotype 9 adeno-associated virus amino acids.
  • serotype 9 adeno-associated virus amino acid residues
  • VLP virus-like particle
  • the mutated VP according to any one of the preceding items, wherein the VP is VP3.
  • 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 at positions corresponding thereto.
  • the rAAV particle 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 at positions corresponding thereto.
  • the present disclosure also provides: 1.
  • a method for producing recombinant adeno-associated virus particles (rAAV particles) containing 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) in the capsid comprising: A production method comprising introducing into a host cell a first nucleic acid molecule encoding the CAP protein and a second nucleic acid molecule encoding a fusion protein of the CAP protein and another protein (A). 2.
  • the first nucleic acid molecule when introduced into a host cell, is capable of expressing VP1, VP2, and VP3
  • the second nucleic acid molecule when introduced into a host cell, is capable of expressing at least one of a fusion protein of VP1 and another protein (A), a fusion protein of VP2 and another protein (A), and a fusion protein of VP2 and another protein (A).
  • 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.
  • 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 amino acid residues 582 to 604 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 are deleted; (3)
  • the CAP protein encoded by the first nucleic acid molecule is a CAP protein other than that of AAV8, in which one or more amino acid residues corresponding to amino acid residues 582 to 604 from the N-terminus of VP1 of AAV8 have been deleted; (4) The CAP protein encode
  • the protein present on the surface of the 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 a membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor.
  • the other protein (A) comprises a single-chain antibody or a single-domain antibody having specific affinity for the transferrin receptor.
  • the other protein (A) is at least one of fusion proteins of VP1, VP2 and VP3, which is capable of specifically binding to another molecule.
  • the other protein (A) comprises an antibody having affinity for the other molecule.
  • the other molecule is an antibody having affinity for the other protein (A).
  • the other molecule is a fusion protein of an antibody having affinity for the other protein (A) and the other protein (B).
  • the other protein (B) has a specific affinity for a protein present on the surface of vascular endothelial cells.
  • the protein present on the surface of the 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 a membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor.
  • the other protein (B) is a single-chain antibody or a single-domain antibody having specific affinity for the transferrin receptor. 17.
  • An rAAV particle 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 the ratio of the total number of molecules of the VP1, 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) The capsid contains VP1, VP2, and VP3, as well as fusion proteins 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 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.
  • the rAAV particle according to 18 or 19 above which is selected from the following (1) to (8):
  • the fusion protein is one in which 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 450-465, 584-602, 455, 457, 462, 501, 588, or 599 amino acid residues from the N-terminus of VP1, or the amino acid sequence corresponding to the 456-462 or 455-460 amino acid sequence from the N-terminus of VP1 is replaced with the amino acid sequence of the other protein (A);
  • 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, 4
  • the fusion protein is one of the above (3) or (4) further lacking one or more amino acid residues corresponding to the 591st, 592nd, 593rd, or 593rd amino acid residues from the N-terminus of VP1.
  • the rAAV particle according to any one of 18 to 20 above, which is selected from the following (1) or (2): (1) All CAP proteins lack one or more amino acid residues corresponding to amino acid residues 582 to 604 from the N-terminus of VP1; (2) All CAP proteins lack one or more amino acid residues corresponding to the 591st, 592nd, 593rd, or 593rd amino acid residue from the N-terminus of VP1. 22.
  • the other protein (A) has a specific affinity for a protein present on the surface of a vascular endothelial cell.
  • the other protein (A) comprises a single-chain antibody or a single-domain antibody having specific affinity for a transferrin receptor. 25.
  • the other protein (A) comprises an antibody having affinity for the other molecule.
  • the other molecule is an antibody having affinity for the other protein (A).
  • 28. The rAAV particle according to claim 25, wherein the other molecule is a fusion protein of an antibody having affinity for the other protein (A) and the other protein (B). 29.
  • the other protein (B) is a single-chain antibody or a single-domain antibody having specific affinity for the transferrin receptor.
  • a pharmaceutical composition comprising the rAAV particles according to any one of 18 to 30 above. 32.
  • the CAP protein is an AAV8 CAP protein in which one or more amino acid residues corresponding to amino acid residues 582 to 604 from the N-terminus of VP1 have been deleted; (2) The CAP protein 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 is a CAP protein other than that of AAV8, in which one or more amino acid residues corresponding to amino acid residues 582 to 604 from the N-terminus of VP1 of AAV8 have been deleted; (4) The CAP protein is a CAP protein other than that of AAV8, in which one or more amino acid residues corresponding to the 591st, 592nd, 593rd, or 593rd amino acid residue from the N-terminus of AAV8 have been deleted. 33
  • rAAV particles that exhibit high tropism for specific organs in high yields.
  • packaging a nucleotide sequence encoding a physiologically active protein or a nucleic acid molecule containing said nucleotide sequence into such rAAV particles it is possible to produce rAAV particles that can express the protein in a desired organ.
  • the desired protein that is expected to have a therapeutic effect can be expressed in the organ, thereby alleviating the patient's symptoms.
  • 1 shows the yield of rAAV particles produced using mixed vectors prepared by mixing pR2C8(VHH456-462) and pR2C8 so that the ratio (molar ratio) of pR2C8(VHH456-462) to the total was 10%, 30%, and 50%.
  • 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.
  • This figure shows the yield of rAAV particles produced using mixed vectors prepared by mixing pR2C9 (VHH455 Hinge-Hinge) and pR2C9 so that the 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 when the yield when rAAV was produced using only pR2C9 is set to 100%.
  • Figure 1 shows the results of immunohistochemical staining of the brains of hTfRKI mice 2 weeks after administration of rAAV particles prepared using a mixed vector containing pR2C9(VHH455) and pR2C9, with the proportion (molar ratio) of pR2C9(VHH455) adjusted to 5% and 10% of the total.
  • Figure 1 shows 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.
  • the vertical axis shows the relative yield of each rAAV particle, with the yield when rAAV particles were produced using only pR2C8 set to 1.
  • mod3 indicates rAAV particles produced using only pR2C8.
  • Asterisks indicate particles with significantly reduced infectivity.
  • Figure 1 shows the yield of each rAAV particle when rAAV particles were produced using pR2C8 plasmid in which a gene encoding VHH was introduced into the Cap region so that a linker was located at each of the N- and C-termini of VHH.
  • the vertical axis shows the relative yield of each rAAV particle when the yield when rAAV particles were produced using pR2C8 alone is set to 1.
  • the table on the left side of each figure shows the combinations of linkers on the N- and C-termini. Same as Figure 5. Same as Figure 5. Same as Figure 5. Same as Figure 5.
  • Figure 1 shows the yield of rAAV particles produced using the pR2C8 plasmid in which a VHH-encoding gene was introduced into the Cap region so that linkers were located at the N- and C-termini of the VHH.
  • the vertical axis shows the rAAV genome amount (vg).
  • FIG. 1 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.
  • (a), (b), and (c) show the levels of GFP expression in the brain, spinal cord, and liver of mice infected with rAAV particles.
  • the vertical bars represent standard errors.
  • (b) shows the results of measurement of GFP expression levels in the brains of mice infected with rAAV particles, with the vertical axis representing the GFP concentration ( ⁇ g/g wet tissue weight). The vertical bars represent standard errors.
  • Immunohistochemical staining of the whole brain of mice infected with rAAV particles (a) uninfected mice, (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, and (f) anti-TfR VHH(PGK-GFP-WPRE)-AAV9).
  • Figure showing the results of immunohistochemical staining of the cerebrum of mice infected with rAAV particles (a) uninfected mice, (b) AAV9-WT, (c) anti-TfR VHH(CMV-GFP-WPRE)-AAV9, (d) anti-TfR VHH(CAG-GFP-WPRE)-AAV9, (e) anti-
  • Immunohistochemical staining of the cerebellum of mice infected with rAAV particles (a) uninfected mice, (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, and (f) anti-TfR VHH(PGK-GFP-WPRE)-AAV9).
  • Figure showing the results of immunohistochemical staining of the whole brain of mice infected with rAAV particles (a) uninfected mice, (b) AAV9-WT, (c) anti-TfR VHH(CMV-GFP-WPRE)-AAV9, (d) anti-TfR VHH(CAG-GFP-WPRE)-AAV9, (e) anti-
  • 1 shows the results of a pharmacological test of rAAV particles using MPS-II model mice, with the vertical axis showing the quantitative value of rAAV genome (vg/gDNA) in the brain of MPS-II model mice administered with rAAV particles.
  • 1 shows the results of a pharmacological test of rAAV particles using GM-1 gangliosidosis model mice, with the vertical axis showing the concentration of Lyso-GM1 ( ⁇ g/dry tissue weight) in the brain of GM-1 gangliosidosis model mice administered with rAAV particles.
  • FIG. 1 shows the results of a pharmacological test of rAAV particles using GM-1 gangliosidosis model mice, with the vertical axis representing the GLB1 concentration ( ⁇ g/wet tissue weight) in the brain 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
  • scAAV indicates anti-TfR VHH(CBh-ASPA)-scAAV9.
  • Figure 1 shows the results of evaluating the motor function of demyelinating disease model mice administered with rAAV particles.
  • (a) shows the results of a mouse grip strength test.
  • the vertical axis shows the measured grip strength (kfg).
  • (b) shows the results of a rotarod test.
  • the vertical axis shows the duration of exercise (seconds).
  • 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 1 shows the ratio of capsid protein to VHH fusion protein in the capsid protein of rAAV particles, where the white bar indicates the ratio of VP2 and VHH fusion protein (VP2-VHH), and the black bar indicates the ratio of VP3 and VHH fusion protein (VP3-VHH).
  • FIG. 1 Figure showing the yield of rAAV particles with point mutations 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) ⁇ 586S-AAV9[9 ]
  • (d) is anti-TfR VHH (455-460) ⁇ 587A-AAV9[10]
  • (e) is anti-TfR VHH (455-460) ⁇ 588Q-AA V9 [11]
  • (f) is anti-TfR VHH (4
  • Figure 1 shows the level of GFP expression in the brains of mice infected with rAAV particles containing point mutations in the Loop-8 region.
  • the vertical axis indicates the relative yield, with the level of GFP expression after 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) ⁇ 586S-AAV9[9].
  • (d) is anti-TfR VHH (455-460) ⁇ 587A-AAV9 [10]
  • (e) is anti-TfR VHH (455-460) ⁇ 588Q-AAV 9[11]
  • (f) is anti-TfR VHH (455-460) ⁇ 589A-AAV9[12]
  • (g) is anti-TfR VHH (455-460) ⁇ 591A -AAV9[13]
  • (h) is anti-TfR VHH (455-460) ⁇ 592Q-AAV9[14]
  • (i) is anti-TfR VHH (455-460) ⁇ 593T-AAV9 [19]
  • (k) is anti-TfR VHH (455-460) ⁇ 594G-AAV9 [9]
  • (l) is anti-TfR VH H (455- (m) shows the relative yield of anti-TfR VHH(455-460) ⁇ 595W-AAV
  • Figure 1 shows the level of GFP expression 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 level of GFP expression after 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) ⁇ 586S-AAV9[9].
  • (d) is anti-TfR VHH (455-460) ⁇ 587A-AAV9 [10]
  • (e) is anti-TfR VHH (455-460) ⁇ 588Q-AAV 9[11]
  • (f) is anti-TfR VHH (455-460) ⁇ 589A-AAV9[12]
  • (g) is anti-TfR VHH (455-460) ⁇ 591A -AAV9[13]
  • (h) is anti-TfR VHH (455-460) ⁇ 592Q-AAV9[14]
  • (i) is anti-TfR VHH (455-460) ⁇ 593T-AAV9 [19]
  • (k) is anti-TfR VHH (455-460) ⁇ 594G-AAV9 [9]
  • (l) is anti-TfR VHH (455-460)
  • (m) shows the relative yield of anti-TfR VHH(455-460) ⁇ 595W
  • This diagram shows the relationship between the ratio (horizontal axis) of the plasmid having a nucleic acid sequence encoding the ligand-modified (VP1/VP2/VP3) to the total amount of said plasmid and the plasmid having a nucleic acid sequence encoding VP1/VP2/VP3, and the ratio (vertical axis) of the mRNA encoding the ligand-modified VP3 to the total amount of said mRNA and the mRNA encoding VP1/VP2/VP3, during transfection into host cells in the manufacturing process of a ligand-modified AAV vector.
  • the square, circle, and triangle marker systems represent anti-TfR VHH, anti-TfR VHH (VP1/2 unmodified), and FMDV peptide modification, respectively.
  • Plasmid A encodes VP1/2/3 in which amino acid residues 456 to 462 from the N-terminus of VP1 have been replaced with a ligand, and the start codon of VP1/2 has been mutated to make it nonfunctional.
  • Plasmid B encodes VP1/2/3.
  • FIG. 1 is a diagram showing the relationship between the ratio (horizontal axis) of a plasmid having a nucleic acid sequence encoding a ligand-modified VP3 to the total amount of the plasmid and the plasmids having nucleic acid sequences encoding VP1/VP2/VP3 during transfection into host cells in the production process of a ligand-modified AAV vector, and the productivity of the AAV vector (vertical axis), where the vertical axis represents the total amount of viral genome contained in the culture supernatant.
  • This figure shows SDS-PAGE of culture supernatants obtained by varying the ratio of a plasmid having a nucleic acid sequence encoding a ligand-modified VP3 to the total amount of said plasmid and plasmids having nucleic acid sequences 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 ratio of the plasmid having a nucleic acid sequence encoding a ligand-modified VP3 was 0, 1, 3, 5, 10, 20, 30, and 50%, respectively.
  • the arrows indicate the band of ligand-modified VP3.
  • 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).
  • the vertical axis shows the relative amount of GFP expression, with the amount of GFP expression in the brain and liver following infection with anti-TfR VHH(455-460)-AAV9 set to 1.
  • Figure 33A Same as Figure 33A.
  • Figure 1 shows the amount of viral mRNA detected in the liver (left) and brain (right) of mice infected with rAAV particles containing point mutations in 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.
  • Figure 34 shows the amount of viral mRNA detected in the liver (left) and brain (right) of mice infected with rAAV particles containing point mutations in 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.
  • (Left) Graph showing the amount of viral genome detected in the liver and (right) the amount of viral mRNA detected in the brain of mice infected with rAAV particles containing point mutations in the loop region.
  • the vertical axis shows the relative amount, with the amount after infection with anti-TfR VHH(455-460)-AAV9 set to 1.
  • Figure 37 Same as Figure 37.
  • Figure 1 shows the amount of viral mRNA detected in the brain (left) and the amount of viral genome detected in the liver (right) of mice infected with rAAV particles containing point mutations in the loop region.
  • the vertical axis shows the relative amount, with the amount after infection with anti-TfR VHH(455-460)-AAV9 set to 1.
  • Same as Figure 40. 1 shows the amount of viral genome detected in the plasma of mice infected with POD peptide-modified rAAV particles in which a point mutation was introduced into the loop region, with the vertical axis representing the amount of viral genome contained in mL of plasma.
  • 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).
  • FIG. 1 shows the viral genome load detected in the (left) heart, (center) quadriceps, and (right) liver of mice infected with FMDV peptide-modified rAAV particles containing point mutations in the loop region.
  • the vertical axis shows the relative amount of viral genome, with the viral genome load after infection with anti-TfR VHH(455-460)-AAV9 set at 1.
  • Figure 1 shows the amount of viral genome detected in the plasma of mice infected with anti-TfR VHH-modified rAAV particles with point mutations introduced into the loop region.
  • the vertical axis shows the amount of viral genome contained in mL of plasma.
  • 1 is a table showing the test configuration for the infection efficiency test using monkeys. This figure shows the amount of GFP and mCherry mRNA in each tissue of monkeys administered each AAV vector.
  • the vertical axis shows the relative amount of mRNA when the amount of mRNA in the cerebrum cortex of AN1 is set to 1.
  • the black and white bars show the amount of GFP and mCherry mRNA, respectively.
  • Figure 46 Same as Figure 46.
  • Figure 46 Same as Figure 46.
  • Figure 1 shows 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 the viral genome encoding GFP and mCherry, respectively.
  • the term “about” refers to ⁇ 10% of the numerical value that follows, or to significant figures. For example, “about 20” includes “18 to 22.” A range of numerical values includes all values between and at the endpoints. When “about” is used in reference 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 are intended to include significant figures.
  • the term "surface” refers to being present on the surface of a cell when expressed.
  • 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, glycans, lipids, and small molecules.
  • the ligands described herein are displayed 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.
  • rAAV adeno-associated virus
  • Ligands used in the present disclosure desirably have specific binding ability to target cells.
  • 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.
  • 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 molecules), cytokines and their receptor-binding domains (e.g., interleukin-2, interferon- ⁇ ), 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).
  • the recombinant adeno-associated virus vectors of the present invention enable efficient and specific gene transfer into target cells.
  • AAV adeno-associated virus
  • viruses belonging to the genus Dependovirus in the family Parvoviridae.
  • virus is used in the same sense as commonly used in the art, and will be understood by those of skill in the art depending on the context.
  • AAV is generally a non-pathogenic virus approximately 20-25 nm in diameter that contains a single-stranded DNA genome within a capsid. AAV cannot replicate efficiently on its own, and can only replicate efficiently in the presence of a helper virus such as an adenovirus or herpesvirus.
  • adeno-associated virus or “AAV” includes wild-type AAV and its derivatives and mutants, as well as recombinant adeno-associated virus (rAAV).
  • rAAVs 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).
  • 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 interpreted as encompassing these viruses and their recombinant forms, unless otherwise specified by the context.
  • recombinant adeno-associated virus As used herein, the terms “recombinant adeno-associated virus,” “recombinant AAV,” and “rAAV” are used interchangeably and refer to a viral vector for gene transfer constructed by artificially modifying part or all of the genomic sequence of an adeno-associated virus (AAV) to include a foreign gene (therapeutic gene, reporter gene, target sequence, etc.).
  • adeno-associated virus rAAV
  • rAAV recombinant AAV
  • rAAV is constructed by removing from the viral genome both or either the Rep gene required for replication and the Cap gene required for capsid formation, and inserting instead a cassette for the foreign gene to be introduced. This modification makes rAAV non-pathogenic while retaining its infectious properties, allowing it to be used as a safe and efficient means of gene delivery.
  • virus-like particle or "VLP (Virus-Like Particle) refers to a structure that mimics the structural characteristics of a virus but does not contain the viral genome or lacks replication capability. It is defined as a safe nanoparticle that is non-infectious.
  • VLPs are particles formed by the self-assembly of natural viral capsid proteins (e.g., VP1, VP2, and VP3 in adeno-associated viruses). Externally, they have nearly identical size, shape, and symmetry to viruses, but differ in that they lack pathogenicity and replication capability. Because VLPs mimic the three-dimensional structure of virus particles, they exhibit biological behavior similar to that of natural viruses in the host's immune system and cellular receptors.
  • 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.
  • AAV adeno-associated virus
  • VLPs are often prepared as rAAV particles carrying therapeutic recombinant genes and may be configured with modifications (e.g., ligand modification or mutation) to the surface loop structure of VP3. This allows VLPs to function as targeting drug delivery particles or vaccine carriers responsible for antigen presentation.
  • virus-like particles include structures that meet any of the following requirements: they contain viral capsid proteins and form a particle structure through self-assembly; they are morphologically similar to infectious viruses but lack the ability to replicate or be pathogenic; 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 a variety of 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 this disclosure is recombinant adeno-associated virus particles (rAAV particles), and unless specifically mentioned with respect to AAV, both refer to the same subject.
  • rAAV particle refers to an artificially constructed particle that has a capsid structure derived from an adeno-associated virus (AAV), but, unlike natural AAV viruses, lacks viral replication ability and encapsulates a gene of interest for therapeutic or experimental use.
  • 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.
  • rAAV particles may be referred to as "recombinant adeno-associated virus vectors" or "rAAV vectors.” Where applicable, descriptions of rAAV particles may also apply to rAAV vectors, and those skilled in the art will understand their applicability depending on the context.
  • 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.
  • 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.
  • capsid capsid-constituting proteins
  • 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:
  • rAAV particles can be modified with appropriate serotypes and structural modifications depending on the type of disease or therapeutic target, such as nerve cells, muscle cells, liver cells, eye 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.
  • rAAV particles also includes so-called “empty capsid” particles that do not carry a gene of interest, as well as particles used in comparative experiments, target recognition ability evaluation, manufacturing process verification, etc. Therefore, "recombinant adeno-associated virus particles” or “rAAV particles” are defined in this specification 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 that have extremely high applicability as gene transfer vehicles.
  • “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.”
  • the “serotype” depends primarily on the amino acid sequence of the capsid proteins (VP1, VP2, VP3) on the surface of the virus particle, and shows differences in reactivity to different neutralizing antibodies, tissue tropism, cell entry route, etc.
  • “serotype X AAV” refers to a specific type that corresponds to X among AAVs classified based on the structure and antigenicity of the capsid protein.
  • 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.
  • AAV2 is a classic human-derived AAV with high neuronal infection efficiency.
  • AAV9 can cross the blood-brain barrier and be delivered to the central nervous system.
  • AAV8 and AAVrh10 have excellent liver tropism, while AAV5 has the ability to efficiently infect respiratory tract and brain neurons.
  • 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, "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.
  • 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 nucleic acid sequences such as nucleic acid sequences encoding genes, in which case the vector itself is often composed of nucleic acids.
  • other factors e.g., proteins
  • rAAV vectors can also be used as component factors, such as rAAV vectors.
  • the term can also refer to nucleic acid molecules or complexes thereof used to introduce and express foreign gene sequences (e.g., therapeutic genes, reporter genes, regulatory sequences, RNA interference sequences, etc.) into cells using genetic engineering techniques, such as viral vectors.
  • a vector stably or transiently delivers a target gene to a target such as a host cell and controls gene expression within the cell.
  • 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.
  • non-viral vectors examples 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.
  • the rAAV vector is advantageous in that it provides specific target cell targeting, low immunogenicity, and stable gene expression.
  • recombinant adeno-associated virus vector As used herein, the terms “recombinant adeno-associated virus vector,” “recombinant AAV vector,” and “rAAV vector” are used interchangeably and refer to a virus-derived vector in which the adeno-associated virus (AAV) genome has been genetically modified to incorporate a foreign gene sequence (such as a therapeutic gene, a reporter gene, or a regulatory sequence). These vectors are constructed for the purpose of gene delivery to target cells. Specific examples include, but are not limited to, recombinant AAV vectors constructed by removing or replacing the Rep and Cap gene regions present in the wild-type AAV genome and instead introducing a desired foreign gene cassette. This allows for safe gene transfer without the vector itself autonomously replicating.
  • an rAAV vector transports a desired foreign protein, and can also be interpreted as referring to other parts (in the narrow sense).
  • the term generally refers to the entire vector, including foreign components. Therefore, when foreign components are included, the term is essentially synonymous with recombinant adeno-associated virus particle or rAAV particle.
  • “Introduction” refers to the act of artificially introducing nucleic acid molecules, proteins, small molecules, or carriers containing them (e.g., plasmids, vectors, liposomes, nanoparticles, etc.) from the outside into target cells (e.g., host cells, target cells, primary cells, cell lines, etc.).
  • target cells e.g., host cells, target cells, primary cells, cell lines, etc.
  • introduction is used as a broader concept encompassing any process of introducing any biological molecule or construct 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 introduction.
  • viral vectors e.g., adenovirus, adeno-associated virus (AAV), lentivirus, etc.
  • non-viral methods e.g., electroporation, lipofection, microinjection, nanoparticle carriers, peptide-based transfection, etc.
  • the "transduction" of particular interest herein refers to a technique for efficiently and selectively delivering a target exogenous gene or regulatory molecule into cells, with viral-based transduction methods using recombinant adeno-associated virus (rAAV) being a particularly preferred embodiment.
  • rAAV recombinant adeno-associated virus
  • transduction refers to a broad transduction procedure, including both viral and non-viral methods, as described above.
  • transduction target is a nucleic acid molecule, it is understood to be a form of "gene transduction.”
  • gene transfer refers to the artificial introduction of a foreign gene sequence of interest (such as a therapeutic gene, reporter gene, regulatory sequence, or RNA interference sequence) into a host cell and the expression of that gene within the host cell.
  • a foreign gene sequence of interest such as a therapeutic gene, reporter gene, regulatory sequence, or RNA interference sequence
  • the host cell transiently or sustainably produces a protein or RNA molecule encoded by the gene of interest.
  • 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).
  • AAV adeno-associated virus
  • a gene transfer method of particular interest herein uses a recombinant adeno-associated virus (rAAV) vector, which is characterized by enabling specific and efficient gene transfer into target cells.
  • rAAV recombinant adeno-associated virus
  • VP is an abbreviation for "virion protein” and refers to the proteins that make up the capsid of adeno-associated virus (AAV).
  • AAV capsid is primarily composed of three structural proteins: VP1, VP2, and VP3.
  • VP1 has the largest molecular weight, followed by VP2 and the smallest by VP3.
  • VP3 approximately 1:1:10.
  • VP3 is the major component of the capsid and plays an important role in vector particle formation, target cell binding, intracellular entry, and immunogenicity. Furthermore, in the present invention, by genetically modifying the VP protein and introducing or presenting a specific ligand, it is possible to improve the targeting and infection specificity of AAV vectors. Therefore, when referred to in this specification, “VP” refers to the capsid-constituting proteins of adeno-associated viruses (VP1, VP2, VP3, etc.), unless otherwise specified.
  • 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 methods.
  • 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 form of substitution, insertion, addition, etc. Modification makes it possible to present a ligand that has binding affinity for a target cell receptor or a specific molecule on the surface of the AAV capsid containing the VP3 protein.
  • Modification methods include: (1) genetic engineering fusion, in which a gene sequence encoding a ligand is directly fused to the VP3 gene sequence; (2) chemical modification, 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) tag-mediated modification, 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).
  • a specific affinity tag such as a His tag, a FLAG tag, or a SpyTag
  • 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.
  • nucleic acid molecule includes both the singular and plural, and encompasses single-stranded or double-stranded DNA molecules, RNA molecules, DNA-RNA hybrid molecules, as well as mutants, derivatives, or modifications thereof. Specifically, “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.
  • nucleic acid molecule includes artificial or naturally occurring nucleic acids obtained by genetic engineering techniques, chemical synthesis techniques, enzymatic techniques, or combinations thereof. It 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 interpreted 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.
  • 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 this 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.), etc.
  • 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., Sac
  • host cells used herein is intended to broadly encompass packaging cells used in the production of recombinant adeno-associated virus (rAAV) vectors, cells that produce target gene products, cells used in gene function analysis, and the like.
  • rAAV adeno-associated virus
  • condition for expressing 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 host cells to produce a functional gene product (protein or functional RNA (including mRNA)).
  • a gene sequence e.g., a therapeutic gene, a reporter gene, a regulatory RNA, etc.
  • a functional gene product protein or functional RNA (including mRNA)
  • nucleic acid molecule expression conditions include a temperature suitable for the host cells (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).
  • expression conditions for nucleic acid molecules 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.
  • condition for expressing a nucleic acid molecule is used in a broad sense to encompass a series 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.
  • condition 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 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 are appropriately introduced into the host cell.
  • 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).
  • 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.).
  • nucleic acid sequence to be expressed and “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 functional RNA molecule.
  • expression regulators such as an appropriate promoter or regulatory sequence
  • stimuli such as chemicals
  • 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).
  • the nucleic acid sequence must generally 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.
  • the nucleic acid sequence encodes a therapeutic gene (e.g., an enzyme, receptor, or cytokine) or a reporter gene (e.g., GFP, luciferase, or ⁇ -galactosidase), it is expected that the gene will be appropriately expressed in the cells 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 that have a structure that allows for expression in a host cell, unless otherwise specified.
  • a therapeutic gene e.g., an enzyme, receptor, or cytokine
  • a reporter gene e.g., GFP, luciferase, or ⁇ -galactosidase
  • polypeptide refers to a molecule formed by linearly polymerizing amino acids via peptide bonds, and is used to refer to proteins or fragments thereof with specific functions or structures.
  • polypeptide includes naturally occurring full-length proteins, recombinant proteins, artificially synthesized proteins, as well as subsequences, fragments, derivatives, or variants thereof.
  • polypeptides often possess specific biological functions (e.g., enzymatic activity, receptor-binding activity, antibody activity, signal transduction ability, etc.), and these functions are used for therapeutic, diagnostic, and targeting applications.
  • polypeptides also include those with post-translational modifications (e.g., phosphorylation, glycosylation, methylation, etc.) or tag sequences (e.g., His tag, FLAG tag, etc.).
  • post-translational modifications e.g., phosphorylation, glycosylation, methylation, etc.
  • tag sequences e.g., His tag, FLAG tag, etc.
  • polypeptide length there are no particular limitations on peptide length, and the term broadly encompasses everything from short oligopeptides of approximately 10 residues to long polypeptides of 100 or more residues, and even multimeric proteins composed of multiple subunits. Therefore, unless otherwise specified, the term "polypeptide" used herein should be interpreted in a broad sense to include naturally or artificially derived functional proteins, protein fragments, peptide sequences, and modifications thereof.
  • the identity between the amino acid sequence of the original protein and the amino acid sequence resulting from the mutation can be easily calculated using well-known homology calculation algorithms.
  • homology calculation 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)).
  • reference to identity refers to identity calculated using these algorithms. Note that the terms amino acid sequence homology and amino acid sequence identity are used interchangeably throughout this specification.
  • the number of amino acids substituted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
  • the number of amino acids 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.
  • 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 the N-terminus or C-terminus of the wild-type protein.
  • the amino acid sequence of the mutated protein preferably exhibits 85% or more identity with the amino acid sequence of the corresponding wild-type protein, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.
  • amino acid families for which "conservative amino acid substitutions" are concerned include, for example, the following: (1) The acidic amino acids aspartic acid and glutamic acid, (2) the basic amino acids histidine, lysine, and arginine; (3) the aromatic amino acids phenylalanine, tyrosine, and tryptophan; (4) Serine and threonine, which are amino acids having a hydroxyl group (hydroxyamino acids), (6) the neutral hydrophilic amino acids cysteine, serine, threonine, asparagine, and glutamine; (7) glycine and proline, amino acids that affect the orientation of peptide chains; (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, such as alanine, glycine, serine, and threonine; (11) Alanine and g
  • 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 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.
  • 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 binding).
  • binding affinity e.g., Kd value
  • “specific affinity” can be evaluated quantitatively or qualitatively using parameters such as affinity constants (Ka, Kd), binding ratio, and selectivity through experimental techniques such as ELISA, flow cytometry, and surface plasmon resonance (SPR). Therefore, when used herein, the term “specific affinity” refers to the property of preferentially and selectively binding to a target molecule, unless otherwise specified, and is interpreted as including affinity binding that can be clearly distinguished from binding to non-target molecules.
  • capsid protein As used herein, “capsid protein” (also written as “kapusido” in Japanese, but both are written as “capsid” in English) or “CAP protein” are used interchangeably and refer to the constituent proteins that form the outer shell structure of adeno-associated viruses (AAV) and are the main components that make up the capsid of virus particles (virions).
  • CAP proteins protect the viral genome and play central roles in the entire infection process, including 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.
  • 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.
  • the CAP protein contains regions that are displayed on the capsid surface, and in the present invention, fusing ligands or tags to these regions makes it possible to adjust the targeting and in vivo dynamics of recombinant adeno-associated virus vectors. Modifying the amino acid sequence of specific sites in the CAP protein can also improve functionality, such as changing serotype specificity, reducing immunogenicity, and enhancing tissue tropism.
  • capsid protein used herein is intended to be interpreted in a broad sense as including the group of proteins that comprise the AAV capsid, including VP1, VP2, and VP3, as well as their mutants, derivatives, fusion products, and the like.
  • 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.
  • PUA2 phospholipase A2
  • 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.
  • ACG upstream non-conventional ATG
  • 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 Ca2 + -dependent, 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.
  • NLS nuclear localization signal
  • 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.
  • AAV adeno-associated virus
  • VP2 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 like VP1, it tends to be translated at a low rate. Recent research has 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 dynamics of rAAV vectors by adjusting the ratio of VP1 and VP3.
  • VP3 refers to the smallest and most abundant structural protein constituting the capsid structure of adeno-associated virus (AAV), translated (by alternative splicing) from the downstream ATG initiation codon in the cap gene. VP3 accounts for approximately 80-90% of the capsid protein, with approximately 45-50 molecules of the total 60-mer 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.
  • AAV adeno-associated virus
  • VP3 While VP3 primarily plays a structural role, it is also believed to contribute to resistance to the external environment (e.g., acidic pH and proteases) and to forming binding sites with 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.
  • VP3 also contains a region that functions as an epitope for neutralizing antibodies, making it an important target in the design of immune-evasive vectors. 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.
  • 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 commonly referred to as a "nanobody.”
  • VHHs are significantly smaller (approximately 12-15 kDa) than conventional antibodies and consist of a single domain structure. Despite possessing antigen-binding ability, they are characterized by functioning solely through the variable domain of the heavy chain (VH), without requiring a light chain. Furthermore, due to their excellent thermal stability and solubility, as well as their high permeability into cells and deep tissues, they are attracting attention for a wide range of applications, including diagnosis, therapy, and targeted carriers.
  • VHH refers to a single-domain antibody fragment as described above, and unless otherwise specified, should be interpreted in a broad sense to include naturally or artificially obtained mutants, fusion products, modified products, etc.
  • 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 vascular lumen 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 transport of substances, cell-cell adhesion, signal transduction, and regulation of immune responses, and play a central role in maintaining vascular homeostasis and in the response mechanisms in pathological conditions. The identification and confirmation of the 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.
  • flow cytometry analysis using fluorescently labeled antibodies allows for quantitative and highly sensitive evaluation of the amount or presence of expression on the cell membrane.
  • 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.
  • 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.
  • 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 transporters, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 8, and membrane-bound heparin-binding epidermal growth factor-like growth factor.
  • 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 transporters, low-density lipoprotein
  • PECAM-1 Platinum Endothelial Cell Adhesion Molecule-1
  • VAGFR2 vascular endothelial growth factor receptor 2
  • ICAM-1 Intercellular Adhesion Molecule-1
  • VCAM-1 Vascular Cell Adhesion Molecule-1
  • a protein with 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 through ligand-receptor interactions, antigen-antibody recognition, or non-covalent 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.
  • proteins exhibit binding activity for 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 using SPR (surface plasmon resonance), ELISA (enzyme-linked immunosorbent assay), flow cytometry, cell immunostaining, or co-immunoprecipitation.
  • 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.
  • 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.
  • transferrin receptor transferrin or anti-transferrin receptor antibodies are used.
  • transferrin or anti-transferrin receptor 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 anti-VEGFR2 antibodies that exhibit high affinity for VEGFR2, which is selectively expressed at sites of angiogenesis, and anti-ICAM-1 scFvs that specifically bind 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 selectively act on targets on the surface of vascular endothelial cells.
  • loop refers to a variable region in the primary structure of a polypeptide or protein that does not correspond to the secondary structures of alpha helices or beta sheets. It is a structural moiety that is primarily flexible and connects 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 the amino acid sequence, are highly flexible in molecular dynamics, and often form spatially protruding structures. In many cases, three-dimensional structural analyses (X-ray crystallography, NMR, or cryo-EM) identify loops as regions sandwiched between adjacent stable secondary structural elements (e.g., beta strands).
  • NMR nuclear magnetic resonance
  • cryo-EM three-dimensional structural analyses
  • Loops can be identified using protein structure prediction algorithms, structural modeling based on the PDB database, or three-dimensional structural analysis methods such as Ramachandran plots. Furthermore, the functional role of loop regions is evaluated by mutagenesis experiments, antibody epitope mapping, ligand binding assays, and the like. In this 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, "loops" in this specification include 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.
  • CDR Complementary-Determining Region
  • 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-forming protein VP in adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • VP is a major component of the AAV capsid surface 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 beta strands that make up the beta 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 cell 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.
  • Loop-4 was performed by structural analysis based on known AAV capsid 3D structures (e.g., PDB IDs: 1LP3, 3NG9, 7KNP, etc.).
  • Various modeling software e.g., PyMOL, SWISS-MODEL
  • Functional analysis can also be performed to verify its biological function and associated regions through site-directed mutations, antibody epitope mapping, cell infection assays, receptor binding assays, and other methods.
  • VP Loop-4" encompasses not only the native sequence but also mutant Loop-4 regions that have been artificially mutated, inserted, substituted, or otherwise modified, which are designed for the purposes of altering tissue tropism, improving immune evasion, or conferring novel targeting. Therefore, "VP Loop-4" 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 represents an important site that can be targeted for structural design and functional modification.
  • loop-5" of VP refers to a highly flexible region that protrudes from the surface of the VP, sandwiched between the ⁇ -barrel structures, and has a different sequence, length, and configuration for each serotype. It differs depending on the serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03), but is the same for AAV9 or other serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03).
  • AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03 e
  • Loop-5 corresponds to approximately amino acid residue position 500 (e.g., 489-510 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 alters the virus's ability to escape neutralizing antibodies, its ability to bind to target cells, and its infection efficiency.
  • 500 e.g., 489-510 of SEQ ID NO: 3
  • amino acid substitution, insertion, or deletion in this region significantly alters the virus's ability to escape neutralizing antibodies, its ability to bind to target cells, and its infection efficiency.
  • Loop-5 can be achieved through 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 the ⁇ -strands and distinguished it from other loops (Loop-1 to Loop-4). These analyses utilize 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, and receptor binding assays.
  • 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 the ⁇ -strands and distinguished it from other loops (Loop-1 to Loop-4). These analyses utilize structural visualization and modeling tools such as PyMOL, UCSF
  • Loop-5 is known to be involved in interactions with heparan sulfate proteoglycans (HSPGs) and other cell surface glycans in AAV2 and AAV9.
  • HSPGs heparan sulfate proteoglycans
  • VP Loop-5" includes not only sequences derived from natural AAV serotypes, but also artificially designed mutant Loop-5 sequences modified by, for example, amino acid substitution, insertion, deletion, or chimeric structures with different serotypes. This can confer new immune evasion properties, target cell selectivity, or tissue tropism.
  • VP Loop-5" refers to a structurally flexible and functionally important variable region present on the surface of the AAV capsid, and represents a potential modification site for regulating the infectivity and immunological properties of the virus.
  • Loop-8 of VP refers to the eighth variable region among the loop structures present in the three-dimensional structure of the capsid-forming protein VP of adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • VP is a major component forming the outer shell of AAV particles, and is based on a " ⁇ -barrel" structure in which multiple ⁇ -strands are folded into a barrel shape. The loop structures that protrude 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.
  • 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).
  • serotype e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11.
  • 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.
  • Loop-8 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 evaluation tests, and antibody neutralization tests. For example, introducing specific amino acid substitutions into Loop-8 can avoid the binding of neutralizing antibodies or confer the ability to infect new cell types.
  • VP Loop-8 includes not only sequences contained in natural AAV serotypes, but also artificially modified sequences, such as chimerization with other types of Loop-8, or regions constructed by amino acid sequence substitution, insertion, or deletion. 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 properties, immune responses, drug delivery efficiency, etc. Structural modifications targeting Loop-8 are useful for controlling the tissue tropism and immune evasion ability of AAV, and are particularly useful in the development of AAV vectors aimed at improving neural delivery efficiency.
  • AAV-PHP.B an AAV9-based mutant, and its improved version AAV-PHP.eB, can efficiently cross the mouse blood-brain barrier (BBB) and enable highly efficient gene delivery to the central nervous system by introducing specific amino acid substitutions into the Loop-8 region.
  • BBB mouse blood-brain barrier
  • enhanced neuronal tropism via the Ly6a receptor has been achieved by modifying the capsid surface residues located in Loop-8.
  • substitution deletion of the amino acid residues located in Loop-8 in AAV9 has the effect of improving selective delivery and immune resistance without significantly impairing the capsid structure.
  • AAV-PHP.eB can be used to efficiently cross the mouse blood-brain barrier (BBB) and enable highly efficient gene delivery to the central nervous system.
  • Loop-8 is a key region for controlling tissue selectivity.
  • inserting peptide sequences against integrins or EGFR into the Loop-8 region can be used to artificially design targeting of tumor cells or specific tissues.
  • These modified AAV vectors are also being used in ongoing preclinical and clinical trials and constitute an important technical platform for centrally targeted gene therapy, particularly for neurodegenerative diseases (e.g., ALS, SMA, Alzheimer's disease) and retinal diseases.
  • 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 high-precision delivery to therapeutic targets.
  • novel AAV vectors with desirable tissue tropism or immunological properties can be provided by introducing specific mutations into the capsid region containing Loop-8. Preferred mutations in various LOOPs are described elsewhere in this specification and demonstrated in the Examples.
  • 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, and stability 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 and serine, such as repeats of Gly-Gly-Gly-Gly-Ser, are preferred to provide flexibility and mobility. Specific examples are given in SEQ ID NOS: 79-91 herein, but are not limited to these.
  • linkers may also 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).
  • 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).
  • ADCs protein-drug conjugates
  • 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.
  • anchor refers to a structure or sequence that physically or functionally fixes or localizes a specific molecule, component, or complex to a predetermined location or structure.
  • the term refers to the other protein (A) used in a fusion protein, where the function of the other protein (A) must be fulfilled.
  • Anchors are used for purposes such as membrane localization of biomolecules, targeting to intracellular organelles, fixation to substrate surfaces, or positional control in higher-order complex formation.
  • 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.
  • membrane anchors with transmembrane domains are used as a means of immobilizing proteins on cell membranes.
  • 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.
  • GPI glycosylphosphatidylinositol
  • 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.
  • anchors 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.
  • 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. It is understood that anchors known in the art that are not explicitly described in the specification can also be used as appropriate.
  • 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. That is, “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 typically consist of two heavy chains (H chains) and two light chains (L chains), each of which has a variable region and a constant region.
  • H chains heavy chains
  • L chains light chains
  • variable regions contain complementarity-determining regions (CDRs) involved in antigen binding, and antigen specificity is determined by the amino acid sequence of these regions.
  • antibody includes antibody fragments, i.e., Fab (antigen-binding fragment), Fab', F(ab') 2 , and Fv (variable fragment), as well as single-chain variable fragments (scFv) in which these fragments are linked together in a single chain, and multivalent antibody structures such as diabodies, trimerbodies, and tandem scFv (taFv) in which these fragments are dimerized for stabilization.
  • VHH variable domain of heavy chain antibodies
  • nanobodies single-chain antibody domains derived from camelids
  • VHH variable domain of heavy chain antibodies
  • humanized VHH antibodies engineered based on these 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.
  • 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
  • fully human antibodies e.g., derived from human antibody libraries or transgenic animals
  • 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.
  • ADCs antibody-drug conjugates
  • 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.
  • anti-EGFR scFvs designed as scFvs, anti-PD-L1 VHHs, humanized anti-CD3 ⁇ antibodies, fully human anti-IL-6R antibodies, Fc-silenced anti-CD47 antibodies, the HER2-targeting ADC trastuzumab emtansine (T-DM1), and the bispecific antibody blinatumomab (CD3 x CD19).
  • T-DM1 trastuzumab emtansine
  • CD3 x CD19 bispecific antibody blinatumomab
  • 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 immunoglobulin-like domains structurally or sequentially and can specifically bind to a desired antigen.
  • anti-XXX antibody such as “anti-transferrin receptor antibody” refers to any antibody identified with XXX as an antigen, and its description is provided herein and can be appropriately recognized by those skilled in the art using information known in the art.
  • YYY e.g., VHH
  • anti-XXXYYY a specific example is “anti-transferrin receptor VHH.”
  • 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 multiple nucleic acid molecules.
  • This "ratio" is typically used when defining 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 of each nucleic acid, translation efficiency, or the expression of the desired biological function.
  • the ratio is expressed as a copy number ratio, molar ratio, or weight ratio; unless otherwise specified, the ratio is interpreted as a molar ratio.
  • Measurement and confirmation of the 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.
  • the ratio may be controlled 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 target polypeptide, co-introduction of the first and second nucleic acid sequences at a 1:5 ratio means that one molecule of the first sequence is present and five molecules of the second sequence are present.
  • a 1:1 ratio is intended to achieve equal expression balance between the two.
  • a specific ratio may be an important parameter for achieving a desired biological activity (e.g., induction of cell function, optimization of protein expression, differentiation control, 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.
  • the "molecular number ratio" of mRNA refers to the relative relationship between the number of molecules of each type of messenger RNA (mRNA) molecule among multiple types of messenger RNA (mRNA).
  • the “molecular number” here refers to the quantitatively measured number of mRNA copies, or the equivalent molar amount (mol), and indicates the amount of one mRNA and another mRNA present at a specific time point or state.
  • This ratio can be determined as the mixture ratio when mRNA is introduced into cells, or evaluated by measuring the amount of mRNA actually expressed in cells. In the former case, it 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.
  • qRT-PCR real-time RT-PCR
  • digital PCR digital PCR
  • RNA-Seq quantitative molecular biology techniques
  • the molecular ratio of the two mRNAs can be an important parameter for achieving optimal immune induction. It is known that combining 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, leading to the efficient formation of functional multiprotein complexes and the expression of downstream physiological activities.
  • an immunostimulatory factor e.g., CD80, CD86, GM-CSF, etc.
  • the term "length" refers to the number of amino acid residues that make up a polypeptide or protein, and is defined as the total number of consecutive amino acids contained in a specific amino acid sequence. In other words, 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 evaluated by simply counting the number of residues based on sequence information; if the amino acid sequence is explicitly stated, the length of that sequence immediately corresponds to the "length.”
  • length refers to the total number of residues comprising the translated contiguous sequence, from the initial methionine residue at the N-terminus to the final residue at the C-terminus. For example, if a protein is 300 residues in total, its length is considered to be 300 amino acids. Furthermore, when a peptide fragment is described as being “15 residues in length,” 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 when designing antigen peptides, linker sequences, excising structural domains, adding tag sequences, and so on.
  • epitope peptides that induce immune responses have lengths of 8 to 11 amino acids suitable for MHC class I and 13 to 25 amino acids suitable for MHC class II.
  • lengths of approximately 10 to 30 amino acids are sometimes preferred to ensure flexibility.
  • shorter (3 to 5 residues) or longer (50 or more residues) linkers may be used to control the three-dimensional configuration of binding sites or for functional separation. Therefore, "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 fundamental design element for functionally constructing and optimizing a target polypeptide.
  • the "average number of ligands in an AAV particle population” refers to the number average, and is a value measured by the following method. However, when the ligands are the same or similar types, the molecular weights are almost similar, so the weight average may also be used. In other words, it means the value obtained by tallying the number of ligands attached to each particle among all AAV particles present in a particle population and dividing this total by the number of particles. The number average is useful as an index for quantitatively evaluating the attachment state of the population as a whole, even when ligands are not attached uniformly to individual particles.
  • the average number of ligands can be determined by the following method: 1) A method in which AAV particles whose mobility has changed due to the addition of a ligand are separated using electrophoresis (e.g., SDS-PAGE or isoelectric focusing), and the degree of ligand addition is estimated based on the area ratio of each resulting 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, which makes it possible to derive the average amount of ligand added per particle.
  • electrophoresis e.g., SDS-PAGE or isoelectric focusing
  • the percentage of particles to which ligands have been added is calculated separately by bead pull-down or immunoprecipitation, and the average number of added particles among all particles is corrected based on this.
  • a method in which whether or not a ligand is attached to each particle is visually confirmed by observation using a transmission electron microscope (TEM) or a cryo-electron microscope (Cryo-EM), and the average number of attached ligands is estimated by statistically processing the frequency.
  • TEM transmission electron microscope
  • cryo-EM cryo-electron microscope
  • These measurement methods can be used alone, or by combining multiple methods, more accurate estimation of the average number of ligands is possible.
  • the method selected depends on the sample properties, the purpose of the analysis, the required accuracy, and the constraints of the experimental system. Note that the "number average" in the definition is a statistical quantity distinct from the weight average and area average, and is an important index in analyses that take into account variations in physicochemical properties.
  • a recombinant AAV 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 containing 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.
  • 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.”
  • capsid protein refers to the protein that constitutes the capsid of the rAAV particle.
  • CAP protein includes VP1, VP2, and VP3, as well as fusion proteins of these proteins with another protein (A).
  • 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.
  • 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.
  • 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).
  • 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.
  • the host cell used in the methods of the present disclosure contains the elements necessary for producing adeno-associated virus particles.
  • 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.
  • the necessary elements used in the present disclosure further include a nucleic acid sequence encoding a protein responsible for helper function.
  • 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.
  • 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.
  • the nucleic acid sequence encoding a desired protein is located between at least two inverted terminal repeats (ITRs).
  • desired proteins include therapeutic proteins, proteins for genome editing, experimental proteins, etc.
  • nucleic acid sequence encoding the desired protein used in the present disclosure is incorporated into the recombinant adeno-associated virus particle under conditions that allow the particle to be produced, so that it is subsequently incorporated into the particle in an expressible state.
  • plasmid 1 having a structure containing a base sequence including a first inverted terminal repeat (ITR) and a base 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;
  • plasmid 2 containing an AAV Rep gene (Rep region) that has the functions necessary to integrate the base sequence of the region sandwiched between the ITR sequences (including the ITR sequence) into the genome of the host cell, and a gene encoding the 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.
  • ITR inverted terminal repeat
  • ITR second inverted terminal repeat
  • the host cells of the present disclosure may contain or be introduced with these plasmids.
  • the Cap (capsid) gene refers to a single gene region that encodes the capsid-constituting proteins VP1, VP2, and VP3 (virion proteins, VP).
  • Three types of VP are produced from the Cap gene due to differences in splicing and translation start sites, and therefore the characteristics of this disclosure can be said to be characteristics of Plasmid 2.
  • the basic principle is to manipulate the Cap gene.
  • adeno-associated virus adeno-associated virus
  • these three types of plasmids are first introduced into host cells, such as HEK293 cells, whose genomes contain adenovirus E1a and E1b genes. This results in 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, being 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.
  • ITR inverted terminal repeat
  • ITR second inverted terminal repeat
  • rAAV adeno-associated virus
  • rAAV particles are produced by introducing the three types of plasmids 1 to 3 described above into a host cell.
  • a plasmid in which two of the above plasmids 1 to 3 are linked together is used.
  • the linked 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 linked plasmid and the remaining two types of plasmids into a host cell.
  • a plasmid in which three of the above plasmids 1 to 3 are linked together is used. In this case, rAAV particles can be produced by introducing only this linked plasmid into a host cell.
  • a nucleic acid molecule containing the following is introduced into the host cell: (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 including a first inverted terminal repeat (ITR); (d) a nucleotide sequence including 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 adenovirus E2A protein or a functional equivalent thereof; (g) a nucleotide sequence encoding an adenovirus E4 protein or a functional equivalent thereof; and (h) a nucleotide sequence encoding an adenovirus VA1 RNA or a functional equivalent thereof.
  • ITR in
  • 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.
  • the Rep protein of adeno-associated virus is encoded by the Rep gene of AAV.
  • the Rep protein has the function required for integrating the AAV genome into the genome of a host cell, for example, via the ITRs present in the genome.
  • Rep68 and Rep78 are required for integration of the AAV genome into the genome of a host cell.
  • Rep68 and Rep78 are the translation products of two types of mRNA transcribed by alternative splicing from the same gene.
  • the term "AAV Rep protein" includes at least the two types of proteins, Rep68 and Rep78.
  • 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 with a mutation added thereto.
  • the Rep proteins are preferably those of AAV serotype 2, but are not limited to this and may be those of any of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • Rep68 exerts its function, it may be modified by substitution, deletion, addition, or other such changes to 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.
  • the number of amino acids substituted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
  • the number of amino acids deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
  • Rep68 to which mutations that combine these amino acid substitutions and deletions have been added is also Rep68.
  • 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 to which mutations that combine these amino acid additions, substitutions, and deletions have been added is also included in Rep68.
  • the amino acid sequence of the mutated Rep68 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 wild-type Rep68.
  • Rep78 exerts its function, it may be modified by substitution, deletion, addition, or other such changes to 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.
  • the number of amino acids substituted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
  • the number of amino acids deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
  • Rep78 to which mutations that combine these amino acid substitutions and deletions have been added is also Rep78.
  • 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 to which mutations that combine these amino acid additions, substitutions, and deletions have been added is also included in Rep78.
  • the amino acid sequence of the mutated Rep78 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 wild-type Rep78.
  • Substitutions of amino acids in the amino acid sequence of Rep68 with other amino acids occur within a family of amino acids that are related, for example, by their side chains and chemical properties. Substitutions within such amino acid families are not expected to significantly alter the function of anti-hRep68 (i.e., are conservative amino acid substitutions).
  • a functional equivalent of an AAV REP protein refers to a substance that can be used functionally in place of Rep68, and to a substance that can be used functionally in place of Rep78.
  • a nucleotide sequence encoding an adeno-associated virus CAP protein refers to a nucleotide sequence encoding at least VP1, a protein that constitutes 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.
  • 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.
  • the positions are those aligned with the serotype 9 sequence as a reference.
  • the term "adeno-associated virus inverted terminal repeat (ITR)” refers to a base sequence essential for the integration of an adeno-associated virus gene into the genomic sequence of a host cell by non-homologous recombination.
  • two adeno-associated virus inverted terminal repeats (ITR) are present in the nucleic acid molecule, and are referred to as the first adeno-associated virus inverted terminal repeat (ITR) and the second adeno-associated virus inverted terminal repeat (ITR), respectively.
  • 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).
  • ITR first adeno-associated virus inverted terminal repeat
  • ITR located on the 3' side is referred to as the second adeno-associated virus inverted terminal repeat (ITR).
  • the inverted terminal repeats are preferably those of AAV serotype 2, but are not limited to this and may be those of any of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • ITRs inverted terminal repeats
  • they may be modifications such as substitutions, deletions, or additions to the base sequence of the wild-type ITR of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. ITRs with these mutations are also included in the ITR category.
  • the number of bases replaced is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
  • the number of bases deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
  • ITRs with mutations that combine these base substitutions and deletions are also considered ITRs.
  • 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.
  • ITRs with mutations that combine these base additions, substitutions, and deletions are also included in the ITR.
  • the base sequence of a mutated ITR preferably exhibits 85% or more identity to the base sequence of a wild-type ITR, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.
  • a functional equivalent of an AAV ITR is one that can be used functionally in place of an AAV ITR.
  • an ITR artificially constructed based on an AAV ITR is also a functional equivalent of an AAV ITR as long as it can replace an AAV ITR.
  • 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.
  • a base sequence for inserting a base sequence encoding a foreign protein refers to a base sequence that contains a base sequence that can be specifically cleaved with a restriction enzyme. This also includes so-called multicloning sites. This base sequence can be cleaved with a restriction enzyme, and a nucleic acid molecule encoding the desired foreign protein can be inserted at the cleavage site.
  • the foreign protein there are no particular limitations on the foreign protein that can be encoded in a nucleic acid molecule.
  • the foreign protein is derived from a specific biological species, there are no particular limitations on the biological species, and it may be a protein encoded in the genome of a prokaryotic or eukaryotic cell.
  • eukaryotic cells include, for example, fungi, yeast, insects, protozoa, amphibians, reptiles, birds, mammals, and plants.
  • examples include humans, non-human primates, livestock such as cows, horses, pigs, and sheep, and pets such as cats and dogs.
  • the foreign protein may be a protein obtained by adding mutations such as substitutions, deletions, and additions to the amino acid sequence of a wild-type protein derived from a specific biological species.
  • the foreign protein may be an artificial protein containing an amino acid sequence that does not exist in nature.
  • the number of amino acids substituted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
  • the number of amino acids 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.
  • 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 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 with the amino acid sequence of the corresponding wild-type protein, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.
  • the foreign protein is not particularly limited, but examples include proteins that are partially or completely functionally deficient in genetic diseases.
  • 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
  • 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- ⁇ antibodies.
  • the gene for the foreign protein may be ⁇ -L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, ⁇ -galactosidase, GM2 activator protein, ⁇ -hexosaminidase A, ⁇ -hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, ⁇ -mannosidase, ⁇ -mannosidase, galactosylceramidase, saposin C, arylsulfatase A, ⁇ -L-fucosidase, aspartylglucosaminidase, ⁇ - Examples include N-acetylgalactosaminidase, acid sphingomyelinase, ⁇ -galactosidase A, ⁇ -glucuronidase, heparan N-sulfatas
  • the foreign protein may be a fusion protein of an antibody and another protein.
  • fusion proteins include a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, or a human antibody, and 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, darbepoetin, tissue plasminogen activator (t-PA), thrombomodulin, follicle-stimulating hormone, DNase I, thyroid-stimulating hormone (TSH), and neurotransmitter.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • Examples 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 ⁇ , interferon ⁇ , interferon ⁇ , interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor ⁇ receptor (TNF- ⁇ receptor), and enzymes with beta-amyloid degrading activity.
  • NGF growth factor
  • CNTF ciliary neurotrophic factor
  • GDNF glial cell line neurotrophic factor
  • neurotrophin 3 neurotrophin 3
  • neurotrophin 4/5 neurotrophin 6 neuregulin 1
  • EGF epidermal growth factor
  • VEGF vascular endothelial growth factor
  • interferon ⁇ interferon ⁇
  • the foreign protein may be a fusion protein of an antibody and a lysosomal enzyme.
  • the antibody is any of a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, and a human antibody
  • the lysosomal enzyme is any of ⁇ -L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, ⁇ -galactosidase, GM2 activator protein, ⁇ -hexosaminidase A, ⁇ -hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, ⁇ -mannosidase, ⁇ -mannosidase, galactosylceramidase, saposin C, arylsulfatase A, ⁇ -L-fucosidase, and aspartylglucosidase.
  • Examples include ceramidase, ⁇ -N-acetylgalactosaminidase, acid sphingomyelinase, ⁇ -galactosidase A, ⁇ -glucuronidase, heparan N-sulfatase, ⁇ -N-acetylglucosaminidase, acetyl-CoA ⁇ -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.
  • 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, for example.
  • 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.
  • an organic anion transporter is OATP-F
  • an example of a monocarboxylate transporter is MCT-8.
  • 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.
  • ITR inverted terminal repeats
  • ITR inverted terminal repeats
  • CBh promoter 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.
  • Adenovirus provides the functions necessary for the AAV genome to replicate in host cells, be packaged into capsids, and form viral virions. This function is performed by the E1, E2A, E4, and VA1 RNA regions of the adenovirus genome.
  • the term "recombinant AAV particle” refers to an AAV capsid protein (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.
  • rAAV genome refers to a nucleic acid molecule in which a wild-type AAV genome has been modified.
  • the rAAV genome packaged in the rAAV particle is single-stranded DNA.
  • nucleic acid molecules examples include single-stranded DNA that, from the 5' end, contain a base sequence containing the 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 the inverted terminal repeat (ITR) of a second adeno-associated virus.
  • ITR inverted terminal repeat
  • rAAV genomes such nucleic acid molecules not packaged in the rAAV particle can also be referred to as rAAV genomes. Therefore, the rAAV genome may be either single-stranded or double-stranded DNA.
  • rAAV particles in host cells require the functions of the adenovirus E1 region. rAAV particles are generally produced using host cells that contain all or part of the E1 region. HEK293 cells are known as such cells. The genome of HEK293 cells contains at least the coding regions for E1A and E1B.
  • the regions of the adenovirus genome necessary for the AAV genome to replicate and be packaged into capsids to form viral virions are the E2A region, E4 region, and VA1 RNA region. These regions encode proteins and RNA required for AAV replication. With regard to 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.
  • ORF6 open reading frame 6
  • the E2A region may be that of any of the adenoviruses whose serotypes are 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 Ad3, canine Ad2, ovine Ad, or porcine Ad3, 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.
  • E2A region exhibits the original function of the region
  • E2A regions with these mutations are also included in the E2A region.
  • the number of bases replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
  • the number of bases deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
  • E2A regions that have been mutated by a combination of these base substitutions and deletions are also included in the E2A region.
  • 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 a combination of these base additions, substitutions, and deletions are also included in the E2A region.
  • the nucleotide sequence of the mutated E2A region preferably exhibits 85% or more identity to the wild-type E2A nucleotide sequence, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.
  • 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, 24-30, 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.
  • the E4 region can be a wild-type adenovirus E4 region that has been modified by substitution, deletion, addition, or other means, as long as the E4 region still exhibits its inherent function.
  • E4 regions with these mutations are also included in the E4 region.
  • the number of bases replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
  • the number of bases deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
  • E4 regions with mutations that combine these base substitutions and deletions are also included.
  • 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.
  • E4s with mutations that combine these base additions, substitutions, and deletions are also included in E4.
  • the base sequence of mutated E4 preferably exhibits 85% or more identity to the wild-type E4 base sequence, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.
  • the VA1 RNA region may be used in any of the following serotypes, as long as it exhibits the original functions required for AAV replication: 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,
  • the adenovirus may be any of 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.
  • the VA1 RNA region of serotype 2 adenovirus is one of the preferred adenoviruses in the present invention.
  • the VA1 RNA region may be a wild-type adenovirus VA1 RNA region that has been modified by substitution, deletion, addition, or other means, as long as the region still exhibits its inherent function.
  • VA1 RNA regions with these mutations are also included in the VA1 RNA region.
  • the number of bases replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
  • the number of bases deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
  • VA1 RNA regions with mutations that combine these base substitutions and deletions are also VA1 RNA regions.
  • 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 with mutations that combine 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 to the base sequence of the wild-type VA1 RNA region, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.
  • the E2A region, E4 region, and VA1 RNA region may be located in any order.
  • a base sequence containing the E2A region, E4 region, and VA1 RNA region is called a helper region, and a plasmid containing such a helper region is called a helper plasmid.
  • helper plasmid is one that contains a base sequence in which the E4 region is located downstream of the E2A region and the VA1 RNA region is located further downstream. Modifications to this base sequence, such as substitutions, deletions, or additions, can also be used as helper plasmids, as long as the E2A region, E4 region, and VA1 RNA region each exhibit their original functions.
  • the base sequence containing the first gene expression control site that controls the expression of the REP protein there are no particular limitations on the base sequence that can be used as the base sequence containing the first gene expression control site that controls the expression of the REP protein, as long as it is controlled by the protein and RNA encoded in a region including the E2A region, E4 region, and VA1 RNA region, but it is preferably the AAV p5 promoter.
  • the first gene expression control site is an AAV p5 promoter, it is preferably the p5 promoter of AAV serotype 2, but is not limited to this and may be any of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • the AAV p5 promoter may be one in which the base 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.
  • p5 promoters with these mutations are also included in the p5 promoter.
  • a region containing the p5 promoter or a functional equivalent thereof and a base sequence encoding the adeno-associated virus REP protein or a functional equivalent thereof is referred to as the Rep region.
  • the base sequence containing the second gene expression control site that controls the expression of the CAP protein there are no particular limitations on the base sequence that can be used as the base sequence containing the second gene expression control site that controls the expression of the CAP protein, 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 it is preferably the AAV p40 promoter.
  • the p40 promoter it is preferably the p40 promoter of AAV serotype 2, but is not limited to this and may be any of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • the AAV p40 promoter may be one in which the base sequence of the wild-type p40 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.
  • p40 promoters with these mutations are also included in the p40 promoter.
  • the number of substituted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
  • the number of deleted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
  • p40 promoters with mutations that combine these base substitutions and deletions are also p40 promoters.
  • bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added within the base sequence or to the 5' end or 3' end of the wild-type p40 promoter.
  • p40 promoters that have been mutated by combining 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.
  • the p40 promoter or its functional equivalent is usually located upstream of the base sequence encoding the adeno-associated virus CAP protein or its functional equivalent.
  • the region containing the p40 promoter or its functional equivalent and the base sequence encoding the adeno-associated virus CAP protein or its functional equivalent is called the Cap region.
  • the serotype of AAV from which the Cap region is derived there is no particular limitation on the serotype of AAV from which the Cap region is derived, and the serotype may be any of 1, 3, 4, 5, 6, 7, 8, 9, 10, or 11, although Cap regions of serotypes 8 and 9 are preferably used.
  • a Cap region in which the base sequence of the Cap region has been modified by substitution, deletion, addition, or the like can also be used as long as it still exhibits the original function of the Cap region.
  • the number of substituted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
  • the number of deleted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
  • a Cap region can also be used in which mutations that combine these base substitutions and deletions have been added.
  • bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added within the base sequence or to the 5' end or 3' end.
  • a Cap region can also be used in which mutations that combine these base additions, substitutions, and deletions have been added.
  • 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.
  • 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.
  • plasmid 2 contains this Rep-Cap region.
  • 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 base sequence including a first inverted terminal repeat (ITR) and a base 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 that has the functions necessary to integrate the base sequence of the region (including the ITR sequence) sandwiched between 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.
  • ITR inverted terminal repeat
  • Plasmids 1 to 3 can be linked together to form a single plasmid, or all three can be linked together to form 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.
  • plasmid 2 is normally a plasmid containing the AAV Rep gene and a gene encoding an AAV capsid protein, but it is also possible to prepare a plasmid containing the AAV Rep gene and a plasmid containing a gene encoding an AAV capsid protein, and use these for rAAV particles. Note that the plasmid containing the gene encoding the AAV capsid protein is referred to as plasmid 2 in this specification.
  • the rAAV produced in one embodiment of the present invention has a protein with a desired function on its surface.
  • rAAVs There are two types of rAAVs that have a protein with a desired function on their surface: (1) A capsid containing a fusion protein of at least one of VP1, VP2, and VP3, which are proteins constituting the capsid (capsid proteins), with another protein (A), wherein the other protein (A) contains a protein having a desired function; (2) The capsid contains a fusion protein of at least one of the capsid proteins VP1, VP2, and VP3 with another protein (A), and a separately prepared protein with a desired function is bound to the capsid via this other protein (A).
  • the rAAV preferably contains, in its capsid, fusion proteins of all of the capsid proteins VP1, VP2, and VP3 with another protein (A).
  • the rAAV may also contain, in its capsid, only fusion proteins of VP1 and another protein (A), only fusion proteins of VP2 and another protein (A), or only fusion proteins of VP1 and VP2 and another protein (A).
  • the rAAV may contain, in its capsid, only fusion proteins of VP3 and another protein (A), only fusion proteins of VP2 and VP3 and another protein (A), or only fusion proteins of VP1 and VP3 and another protein (A).
  • a plasmid 2 is used in which a base sequence encoding the other protein (A) is added to the base sequence of the Cap region so that it is in-frame with the base sequence encoding the capsid protein.
  • a plasmid 2 can express the fusion protein of the capsid protein and the other protein (A) in a host cell.
  • the site in the fusion protein to which the other protein (A) should be added will be described in detail below, taking as an example the case where the cap region is derived from AAV8.
  • the site is preferably the C-terminal side of any amino acid residue contained in 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.
  • the site is preferably 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, more preferably the C-terminal side of amino acid residues 584 to 602, 586 to 600, or 588 to 600 from the N-terminus of VP1, for example, the C-terminal side of amino acid residue 588 or 599.
  • 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 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).
  • the amino acid sequence 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 amino acid residues 707-717, 707-712, or 707 from the N-terminus of VP1.
  • AAV8 VP1 The amino acid sequence of AAV8 VP1 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 produced by alternative splicing of a single gene.
  • 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.
  • 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 are 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).
  • plasmid 2 a plasmid 2 containing a base 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 introduced into the same host cell; alternatively, a plasmid 2 containing a base 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 introduced into a host cell.
  • a nucleic acid molecule encoding a normal capsid protein is referred to as a first nucleic acid molecule
  • 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.
  • both the fusion protein of the capsid protein and another protein (A) and the normal capsid protein are expressed. Therefore, by using such host cells, rAAV particles in which the capsid protein is composed of the fusion protein and the normal capsid protein can be obtained.
  • the ratio of fusion protein to capsid protein that makes up the rAAV particle is important. This is because an increase in the ratio of 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 of producing them also increases.
  • the ratio of the total number of molecules of the normal VP1, VP2, and VP3 in the capsid proteins of an rAAV particle to the total number of molecules of the 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, 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.
  • 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.
  • a first nucleic acid sequence is introduced as a first nucleic acid molecule, such that VP1, VP2, and VP3 are expressible upon gene transfer
  • a second nucleic acid sequence is introduced as a second nucleic acid molecule, such that VP3 modified with a ligand is expressible upon gene transfer
  • 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 introduced and the number of molecules of the second nucleic acid sequence introduced is, for example, 1-50%
  • 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) when there is no fusion protein of VP1 and another protein (A), when there is no fusion protein of VP2 and another protein (A), and the sum of the number of molecules of these fusion proteins and VP1, VP2, and VP3 corresponds to the
  • the ratio of the total number of molecules of normal VP2 and VP3 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 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, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the ratio of the total number of molecules of the normal VP1, VP2, and VP3 in the capsid protein of an rAAV particle to the total number of molecules of the fusion protein of each of these (i.e., VP1, 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 10
  • 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.
  • 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, or 6 to 12.
  • the number of proteins constituting the capsid of an AAV particle is, for example, 60.
  • 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, 1
  • 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.
  • 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.
  • 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.
  • 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, 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.
  • 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.
  • 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 make up the capsid of an AAV particle is, for example, 60.
  • 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, 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.
  • 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.
  • 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 make up the capsid of an AAV particle is, for example, 60.
  • the capsid contains a fusion protein of at least one of the capsid proteins VP1, VP2, and VP3 with another protein (A), and the other protein (A) contains a protein with a desired function.
  • 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 normal capsid protein is used as the first nucleic acid molecule. Therefore, the resulting rAAV contains normal VP1, VP2, and VP3, as well as the fusion protein of the capsid protein and the other protein (A), in the capsid.
  • AAV serotype of the Cap region used here, but it is preferably derived from AAV8 or AAV9.
  • 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 that order from the N-terminus, or a functional protein and a second linker in that order from the N-terminus, but a linker is not essential.
  • the first linker and second linker either in combination or individually, serve to enable the fusion protein to exhibit the function derived from the other protein (A).
  • a "functional protein” can also be referred to as a "functional region,” "functional region,” “functional protein,” etc.
  • a functional protein is one that itself has a specific physiological activity.
  • the size of the other protein (A) 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.
  • 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 second linker are shown in Tables 1 and 2, respectively.
  • the amino acid sequence of the first linker 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 multiple of these or the amino acid sequences shown in Table 1 are linked together.
  • the amino acid sequence of the second linker may also 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 multiple of the amino acid sequences shown in Table 2 are linked together.
  • Preferred amino acid sequences for the first linker and second linker are shown in Tables 1 and 2, respectively.
  • examples include 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 multiple of these and the amino acid sequences shown in Table 1 are linked together.
  • a first linker is GGGGS x 1 and a second linker is GGGGS x 3; a first linker is GGGGS x 1 and a second linker is cIgG2a hinge; a first linker is cIgG2a hinge and a second linker is GGGGS x 3; a first linker is cIgG2a hinge and a second linker is cIgG2a hinge; a first linker is 2xEAAAK and a second linker is cIgG2a hinge; and a first linker is GGGGS x 3 and a second linker is GGGGS x 3.
  • the functional protein constituting the other protein (A) is one that itself has the desired physiological activity, and there are no particular limitations on its type. 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.
  • transferrin receptor insulin receptor
  • leptin receptor insulin-like growth factor I receptor
  • insulin-like growth factor II 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, particularly transferrin receptor and insulin receptor.
  • the functional protein has specific affinity for the transferrin receptor (TfR)
  • the functional protein is transferrin, a fragment of transferrin containing the TfR-binding domain, or an antibody with affinity for TfR.
  • a single-domain antibody is an antibody that has 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 nanobodies are types of single-domain antibodies.
  • ScFv is a preferred single-chain antibody.
  • VNARs are described in more 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.
  • VHHs are described in more 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 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 made to the amino acid sequence of camelid antibodies (including VHHs) in order to reduce antigenicity when the camelid-derived antibody is administered to humans.
  • the functional protein is a VHH with affinity for human TfR (hTfR)
  • preferred amino acid sequences of the VHH are exemplified in Table 3.
  • the term "human transferrin receptor” or “hTfR” refers to a membrane protein having the amino acid sequence set forth in SEQ ID NO: 4.
  • the anti-hTfR antibody of the present invention specifically binds to the portion of the amino acid sequence set forth in SEQ ID NO: 1, from the 89th cysteine residue from the N-terminus to the C-terminal phenylalanine (the extracellular domain of hTfR).
  • a VHH with affinity for human TfR preferably has a dissociation constant (KD) with hTfR, as measured by the method described in Example 44, of preferably 5 x 10 -8 M or less, more preferably 2 x 10 -8 M or less, for example, 1 x 10 -8 M or less, 5 x 10 -9 M or less, or 1 x 10 -9 M or less.
  • KD dissociation constant
  • preferred ones have a dissociation constant of 5 ⁇ 10 ⁇ 11 M to 1 ⁇ 10 ⁇ 8 M, 2 ⁇ 10 ⁇ 11 M to 1 ⁇ 10 ⁇ 8 M, 1 ⁇ 10 ⁇ 10 M to 1 ⁇ 10 ⁇ 8 M, or 1.0 ⁇ 10 ⁇ 9 to 1.0 ⁇ 10 ⁇ 8 M.
  • Table 4 shows the amino acid sequences of CDR1 to CDR3 of the VHHs shown in Table 3. Regions other than the CDRs are called frame regions (FR). From the N-terminus, VHHs have the sequences FR1, CDR1, FR2, CD2, FR3, CDR3, and FR4.
  • amino acid sequences of the VHHs shown in Table 3 may be mutated, as long as the VHHs can specifically bind to hTfR.
  • the amino acid sequence of the VHHs after mutation preferably has 80% or more identity to the original amino acid sequence, 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.
  • the amino acid sequence of the variable region after making 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.
  • mutations are made to the amino acid sequence of the variable region, it is also possible to make mutations only in the CDR region, without making mutations in the amino acid sequence of the FR.
  • the amino acid sequence of the variable region after making the mutations preferably has an identity of 90% or more, more preferably 95% or more, for example, 98% or more, to the original amino acid sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. It is also possible to make mutations that combine these amino acid substitutions and deletions.
  • 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.
  • VHH humanization When mutations are made 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 the variable region of an IgG-type human antibody.
  • this method is referred to as VHH humanization.
  • VHH humanization Such a method is disclosed, for example, in Vincle C., et al., J. Biol. Chem. 284, 3273-84 (2009).
  • the original antibody is an alpaca antibody, it may be recognized as an antigen when administered to humans.
  • a humanized antibody is expected to have lower antigenicity than the original antibody.
  • humanization is a preferred embodiment.
  • VHH also includes humanized VHH.
  • mutations can be made only in the CDR regions without making mutations in the amino acid sequence of the FR region.
  • 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.
  • 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.
  • 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.
  • 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 produced molecule with a desired function is bound to the capsid via this other protein (A).
  • 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 normal capsid protein is used as the first nucleic acid molecule. Therefore, the resulting rAAV contains normal VP1, VP2, and VP3, as well as a fusion protein of a capsid protein and another protein (A), in the capsid.
  • AAV serotype of the Cap region used here is preferably derived from AAV8 or AAV9.
  • the fusion protein must be able to exert the function of the other protein (A).
  • the function of the other protein (A) is to allow a separately produced molecule with a desired function to bind to the capsid.
  • the other protein (A) binds a separately produced protein with a desired function, it can also be called an "anchor.”
  • the amino acid sequence that can be used as this anchor as long as it can exert the desired function.
  • the number of amino acids that make up the anchor 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.
  • an anchor is the ALFA Tag, which has the amino acid sequence of SEQ ID NO: 52.
  • a linker is not necessarily required. However, the above-mentioned linker may be attached to the N-terminus and/or C-terminus.
  • Separately prepared molecules with desired functions that are bound to rAAV particles via anchors are not limited to proteins. As long as they can specifically bind to the anchor, they may be proteins, conjugates of proteins and non-protein substances, non-protein substances, or conjugates of non-protein substances together.
  • the protein may be, for example, a fusion protein of a protein with the desired function and a protein capable of binding to an anchor sequence.
  • the protein with the desired function and the protein capable of binding to an anchor sequence may be prepared separately and then chemically conjugated together to form a conjugate. It may also be a bifunctional antibody.
  • 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 linker sequence described above.
  • an example of a protein capable of binding to an anchor is an antibody against the anchor.
  • 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.
  • a suitable antibody against the anchor is a nanobody against the ALFA Tag having the amino acid sequence of SEQ ID NO: 53.
  • antibodies against the anchor can also be used that have mutations (substitutions, deletions, additions) in one to three (e.g., one or two) amino acids in the amino acid sequence of SEQ ID NO: 53.
  • molecule with the desired function is a fusion protein of a protein with the desired function and a protein capable of binding to an anchor sequence
  • a fusion protein of a protein with specific affinity for a protein present on the surface of vascular endothelial cells and a protein capable of binding to an anchor sequence is a fusion protein of a protein with specific affinity for a protein present on the surface of vascular endothelial cells and a protein capable of binding to an anchor sequence.
  • the proteins present on the surface of vascular endothelial cells are 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 the membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor.
  • Proteins that have specific affinity for proteins present on the surface of these vascular endothelial cells include ligands or antibodies for these receptors.
  • Suitable antibodies that have affinity for TfR include, but are not limited to, single-domain antibodies, single-chain antibodies such as ScFv, and Fab.
  • Suitable single-domain antibodies include those shown in Tables 3 and 4.
  • Fabs include those whose heavy chain variable region amino acid sequence is SEQ ID NO: 54 and whose light chain variable region amino acid sequence is 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 for mutating VHHs can also be applied to mutating Fabs.
  • AAV includes a fusion protein of at least one of the capsid proteins VP1, VP2, and VP3 with another protein (A).
  • AAV in which a separately prepared molecule with a desired function is bound to the capsid via this other protein (A) can be obtained by first obtaining AAV, mixing it with the molecule with the desired function to bind the two, and then purifying the bound product by means of chromatography or the like.
  • a method for producing rAAV particles in one embodiment of the present invention is described below.
  • a predetermined amount of host cells is cultured.
  • Three types of plasmids are introduced into these host cells: (1) a plasmid (plasmid 1) having a structure containing a base sequence including a first inverted terminal repeat (ITR) and a second inverted terminal repeat (ITR) derived from a virus such as AAV, and a gene encoding a desired protein positioned between these two ITRs; (2) a plasmid (plasmid 2) containing an AAV Rep gene (Rep region) that has the function necessary to integrate the base sequence of the region sandwiched between the ITR sequences (including the ITR sequence) into the genome of the host cell, and a gene encoding the 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, but cells having the E1A and E1B genes are preferred, and cells having the E1A and E1B genes and expressing the SV40 virus large T antigen gene, such as HEK293T cells, a cell line derived from human embryonic kidney cells, are even more preferred.
  • Two types of plasmid 2 are used: one with a normal Rep region and Cap region, and one with 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.
  • 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 this disclosure.
  • the relevant calculation is possible, with 9.9:0.1 roughly corresponding to 1%, and 8.0:2.0 roughly corresponding to 20%.
  • 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 host cells After introducing 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 degrade DNA and RNA contained in the culture solution. Benzonase TM is a suitable endonuclease.
  • 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 the AAV genome. Empty AAV particles are removed from the purified rAAV particles by density gradient centrifugation to obtain a purified rAAV particle product.
  • the process of genetically introducing into a host cell 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 (which may be the same or different) ligands, as well as nucleic acid molecules containing nucleic acid sequences encoding desired proteins, if necessary, can be realized 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).
  • each sequence 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 form in which each molecule is expressed individually, or in combinations thereof.
  • multiple sequences are present, with the sequences encoding VP1, VP2, VP3, and modified VP3 all linked together on a single nucleic acid molecule.
  • VP1, VP2, and VP3 can be expressed by alternative splicing. It is also possible to incorporate a modified version of VP3.
  • each sequence may be separated by an internal ribosome entry site (IRES) or a self-cleaving peptide sequence (2A sequence), and designed as a multi-gene expression cassette. This allows multiple proteins to be simultaneously expressed with a single introduction, which has the advantage of simplifying cell manipulation.
  • IRS internal ribosome entry site
  • 2A sequence self-cleaving peptide sequence
  • a configuration of multiple molecules can also be envisioned.
  • two nucleic acid molecules are introduced separately, each with a different function.
  • One molecule encodes wild-type VP1, VP2, or VP3, typically employing alternative splicing.
  • the other molecule encodes modified VP3, in which a ligand modification site has been added to VP3.
  • alternative splicing can be prevented by disabling the start codon using point mutations or other methods to intentionally suppress translation. This enables selective control, allowing only modified VP3 to function under specific conditions.
  • individual expression forms can also be combined.
  • Individual molecule introduction involves the introduction of independent nucleic acid molecules encoding VP1, VP2, VP3, and modified VP3, either individually or in combination. This makes it possible to individually adjust the expression level, control the timing of introduction, and select and use different promoters, allowing for the design of complex expression patterns.
  • the expression level can be adjusted by constitutively expressing VP1 under a constitutive promoter and regulatedly expressing modified VP3 under an inducible promoter.
  • the first nucleic acid sequence and second nucleic acid sequence of the present disclosure may be introduced as separate nucleic acid molecules.
  • the "step of subjecting host cells to conditions for producing recombinant adeno-associated viral 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 viral 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 viral 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.
  • conditions for producing recombinant adeno-associated viral 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).
  • 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.
  • this step goes beyond simple nucleic acid introduction and includes the setting of conditions and manipulations necessary to induce and promote substantial "expression” of the introduced nucleic acid (production of transcription or translation products).
  • the "expression conditions” in this step include the physical, chemical, culture, and induction conditions exemplified below: (Regarding temperature conditions, incubation at 37°C is generally desirable for mammalian cells.
  • a temporary temperature shift (e.g., 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.
  • time conditions a culture time is ensured after introduction, for example, several hours to several days, until expression is stabilized.
  • an expression inducer when an inducible promoter is used, inducers such as doxycycline, IPTG, and tamoxifen can be added at an appropriate concentration.
  • O2 concentration usually 5%
  • O The partial pressure is appropriately adjusted.
  • the cell density after transfection can be appropriately controlled to optimize expression efficiency. Passage procedures may be performed. For example, this can be done by reseeding cells at high density to maintain 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.
  • cytokines, stress factors, pH fluctuations, and other factors that affect expression can be suppressed.
  • stable gene expression is often confirmed by culturing in a complete medium containing FBS for approximately 72 hours after transfection.
  • the temporal expression of the target gene can be controlled by adjusting the timing and concentration (e.g., 1 ⁇ g/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.
  • a method for producing a recombinant adeno-associated virus vector having a ligand on its surface comprising: (A) the steps of: (1) introducing into a host cell a first nucleic acid sequence that, when introduced, enables the expression of VP1, VP2, and VP3; (2) the steps of introducing into a host cell a second nucleic acid sequence that, when introduced, enables the expression of VP3 modified with a ligand; and (3) the steps of introducing into a host cell a nucleic acid sequence encoding a desired protein; and (B) the steps of subjecting the host cell to conditions under which the recombinant adeno-associated virus particles are produced.
  • (A) the step of genetically introducing into a host cell (1) a first nucleic acid sequence that, upon genetic introduction, enables the expression of VP1, VP2, and VP3, (2) a second nucleic acid sequence that, upon genetic introduction, enables the expression of VP3 modified with a ligand, 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.
  • nucleic acid sequences that enable the expression of multiple virion proteins (VPs) involved in virus particle formation are genetically introduced into the host cell, and here, (1) a first nucleic acid sequence designed to express VP1, VP2, and VP3, and (2) a second nucleic acid sequence designed to express VP3 modified with a ligand ("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.
  • 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.
  • any known method can be used for introduction, including transfection in the form of plasmid DNA, introduction using viral vectors such as lentiviral vectors and adeno-associated viral (AAV) vectors, electroporation, and lipid particle-mediated methods.
  • viral vectors such as lentiviral vectors and adeno-associated viral (AAV) vectors
  • electroporation and lipid particle-mediated methods.
  • 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.
  • 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) is 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 to this. A lower ratio is preferable when productivity is prioritized, and a higher ratio is acceptable when infection efficiency is prioritized.
  • this 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., but is not limited to these.
  • 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 is preferably within a predetermined range.
  • the modified VP3 mRNA ratio is typically 0.2% to 30%, more typically 0.5% to 20%.
  • productivity may decrease, and if it is above the upper limit, a decrease in infection efficiency may be observed.
  • it is 1% to 15%, and more preferably 2% to 10%.
  • 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, or 100 amino acids or more, with an upper limit of 500 amino acids or less, 1000 amino acids or less, etc.
  • the size of the ligand can also be expressed in terms of size, and the ligand used in the present disclosure may be one that is larger than a predetermined molecular weight.
  • 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.
  • upper limits may include, but are not limited to, 100 kDa or less, 50 kDa or less, or 20 kDa or less.
  • ligands that can be used include, but are not limited to, VHHs, VNARs (shark heavy chain antibody variable regions), etc.
  • conservative amino acid substitutions referred to in this specification also apply when substituting an amino acid for another amino acid in the amino acid sequence of another protein, such as a VHH.
  • the ligand used has specific affinity for a protein present on the surface of vascular endothelial cells.
  • the host cells used in the present disclosure do not express ligand-modified VP1 and/or ligand-modified VP2.
  • "not expressing” includes not expressing them at a level lower than that normally expressed in nature, as well as not expressing them at all.
  • 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.
  • the mutated VPs may take 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.
  • 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.
  • serotype 9 adeno-associated virus amino acid residues e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03
  • the ligand has specific affinity for another molecule.
  • the surface has two or more types of ligands.
  • the ligand comprises an anti-transferrin receptor VHH.
  • 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,
  • (A1) it comprises 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, or
  • the present disclosure provides a host cell that produces a recombinant adeno-associated virus particle or a recombinant adeno-associated virus vector having a ligand-modified VP3 on its surface.
  • the host cell used in the present disclosure contains the elements necessary for producing adeno-associated virus particles.
  • the host cell is designed to improve targeting ability and maximize gene transfer efficiency into specific tissues or cells.
  • the host cell is a mammalian cell line, particularly a cell 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 cell in the present disclosure is 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.
  • 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.
  • the present disclosure provides a host cell comprising: (1) a first nucleic acid sequence that, upon expression, enables the expression of VP1, VP2, and VP3; (2) a second nucleic acid sequence that, upon expression, enables the expression of VP3 modified with the ligand; and (3) a nucleic acid sequence encoding a desired protein.
  • the host cell can produce a recombinant adeno-associated virus vector having VP3 modified with a ligand on its surface.
  • the present disclosure provides a host cell having one or more nucleic acid molecules encoding nucleic acid sequences that, when expressed, enable expression of VP1, VP2, VP3, and ligand-modified VP3.
  • the host cell can produce a recombinant adeno-associated virus vector having ligand-modified VP3 on its surface.
  • 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.
  • this ratio is 5%-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.
  • the ligand possessed by the AAV particles or AAV vectors produced by the host cells of 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 ligands used in the present disclosure may be those larger than a predetermined molecular weight.
  • the predetermined molecular weight may be, for example, a polypeptide or other substance having a size of 4.5 kDa or more, or 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.
  • the upper limit can include, but is not limited to, 100 kDa or less, 50 kDa or less, 30 kDa or less, etc.
  • the ligand possessed by the host cell of the present disclosure may be a VHH, a VNAR (shark heavy chain antibody variable region), or the like.
  • the ligand possessed by the host cells of the present disclosure has specific affinity for a protein present on the surface of vascular endothelial cells.
  • the host cell of the present disclosure does not express ligand-modified VP1 and/or ligand-modified VP2.
  • "not expressing” includes not expressing them at a level lower than that normally expressed in nature, as well as not expressing them at all.
  • the number of molecules of the ligand per recombinant adeno-associated virus-like particle (VLP) in the host cell of the present disclosure (sometimes referred to as the "actual modification rate (number/particle)”) is a predetermined value, typically between 1 and 50.
  • This value (the "actual modification rate (number/particle)" is preferably 1 to 30 molecules of the ligand per recombinant adeno-associated virus vector particle, more preferably 1 to 22, 1 to 20, 1 to 16, 2 to 16, 2 to 13, etc. This is because, without wishing to be bound by theory, these values provide good values for both productivity and infection efficiency.
  • 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 serotype 9 adeno-associated virus, or amino acid residues at positions corresponding thereto, are absent and/or substituted with one or more other amino acid residues.
  • the mutated VPs may take any of the forms detailed elsewhere in this specification.
  • the ligand has specific affinity for another molecule.
  • the surface has two or more types of ligands.
  • the present disclosure provides a recombinant adeno-associated virus (rAAV) particle or vector having a VP3 modified with a ligand on its surface.
  • the rAAV particle or vector of the present disclosure is used to produce a VLP (rAAV particle or vector) 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.
  • 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 vector of the present disclosure is extremely useful in the field of gene therapy, for example, for selective delivery to specific diseased tissues and reduction of 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.
  • the present disclosure provides a composition comprising an rAAV particle or rAAV vector having VP3 modified with a ligand on its surface.
  • VP3 the main protein forming the capsid
  • 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 in this aspect of the composition.
  • Such improved targeting is particularly advantageous for pharmaceutical applications, as it maximizes therapeutic efficacy and reduces the risk of side effects.
  • the present disclosure provides a virus-like particle (VLP) having VP3 modified with a ligand on its surface.
  • VLP virus-like particle
  • An important aspect of this VLP is that it can be used as a pharmaceutical containing a protein for treatment or prevention as the desired protein.
  • 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 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.
  • the present disclosure provides a composition comprising a virus-like particle having VP3 modified with a ligand on its surface.
  • VP3 the main protein forming the capsid
  • a ligand capable of binding to specific cells or tissues. This allows the composition to have improved selectivity and specific infectivity for the intended target cells, and to 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.
  • the recombinant adeno-associated virus vector disclosed herein can be considered a blueprint that is introduced into host cells to provide recombinant AAV particles.
  • 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.
  • the rAAV particles or rAAV vectors disclosed herein are highly useful in the field of gene therapy, enabling selective delivery to specific diseased tissues and reducing side effects.
  • Ligand modification can be performed on the N-terminus, C-terminus, or internal region of the VP3 protein, but modification is particularly preferred in regions highly exposed to the capsid surface. This allows the ligand to interact with host cell receptors with high efficiency, achieving specific infection efficiency.
  • rAAV vectors containing the ligand-modified VP3 disclosed herein can maintain stable particle formation and efficient cell infection without impairing the function of the native AAV capsid protein.
  • target cells can be selected according to therapeutic or research purposes, such as neurons, hepatocytes, muscle cells, cancer cells, immune cells, and endothelial cells.
  • cancer cell-specific infection can be achieved by using a tumor-specific antibody fragment as the ligand.
  • a neuron-specific peptide ligand enables specific gene transfer to the central nervous system.
  • the rAAV vector disclosed herein can achieve stable, long-term expression of target genes, and therefore has the potential to provide technological therapeutic effects in the treatment of intractable diseases, rare diseases, and genetic diseases.
  • 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 upper limits including, but not limited to, 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 greater than a predetermined molecular weight.
  • the predetermined molecular weight may be, for example, a polypeptide or other substance having a size of 4.5 kDa or more, or 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.
  • the upper limit can include, but is not limited to, 100 kDa or less, 50 kDa or less, 30 kDa or less, etc.
  • the ligands used in the present disclosure may be, but are not limited to, VHHs, VNARs (shark heavy chain antibody variable regions), etc.
  • the ligand used in the present disclosure has specific affinity for a protein present on the surface of vascular endothelial cells.
  • the viral vector of the present disclosure does not have ligand-modified VP1 and/or ligand-modified VP2 on its surface.
  • “not having” includes not having them on the surface in amounts less than those normally expressed in nature, as well as not having them on the surface or not having them at all.
  • the number of molecules of the ligand per particle of the rAAV particles or rAAV vectors of the present disclosure is a predetermined value, typically between 1 and 50.
  • This value (the "actual modification rate (number/particle)" may preferably be between 1 and 30, more preferably between 1 and 20, or between 1 and 16, 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.
  • 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 detailed elsewhere in this specification.
  • 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 adeno-associated virus amino acid residues of other serotypes when aligned with serotype 9 adeno-associated virus amino acids.
  • serotype 9 adeno-associated virus amino acid residues e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03
  • the actual modification rate of the number of molecules of the ligand in the VLP1 particle produced is a predetermined percentage.
  • the actual modification rate is typically 1 to 50%.
  • the actual modification rate (%) is the ratio of VLPs having the modified VP in a vector comprising VLPs having VP3 modified with a ligand on their surface and VLPs having unmodified VP3.
  • the actual modification rate of the ligands disclosed herein may be 3-30%, more preferably 4-25%, or 4-10%. It has been found that these actual modification rates result in good clearance rates and infection efficiencies.
  • the number of molecules of the ligand per VLP particle is a predetermined value, typically 1 to 50.
  • This value is preferably 1 to 30, more preferably 1 to 22, 1 to 20, 1 to 16, 2 to 16, 2 to 13, etc., 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.
  • the ligand has specific affinity for another molecule.
  • the surface has two or more types of ligands.
  • 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, 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.
  • serotypes e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV
  • 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.
  • the VP includes a mutant in which one or more specific amino acid residues are deleted in the Loop-4 region of the VP.
  • 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.
  • 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.
  • loop regions are expected to improve properties such as host cell infection efficiency, tissue specificity, immunogenicity, or in vivo stability.
  • the Loop-4, Loop-5, and Loop-8 regions are highly exposed on the capsid surface and are directly involved in interactions with host cell surface receptors and immune recognition. Therefore, introducing mutations into these regions may alter the infection specificity of target cells and recognition by the immune system.
  • AAV vector particles produced using the mutated VPs of the present disclosure 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.
  • vector particles containing the mutated VPs of the present disclosure may have reduced in vivo immunogenicity compared to vector particles derived from conventional wild-type AAV9, allowing for repeated administration.
  • One specific example is an embodiment in which mutant vector particles with improved selective infection of nerve cells or muscle cells are obtained by substituting specific amino acids in Loop-4 or Loop-5.
  • Yet 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 mutated VP can be prepared, for example, by mutagenesis that identifies the position of a specific amino acid (site-directed mutagenesis).
  • mutated VPs provided in these various embodiments significantly expand the range of therapeutic and research applications of rAAV vectors, and contribute to improved therapeutic efficacy and safety for specific diseases, particularly in the field of gene therapy.
  • the present invention relates to rAAV particles that suppress infection of the liver when administered intravenously or otherwise.
  • rAAV particles have a mutation added to the Loop-8 region of the capsid protein VP.
  • the Loop-8 region in the case of serotype 9 AAV, corresponds to amino acids 582 to 604 from the N-terminus of VP1, and is a region that largely overlaps with the variable region.
  • Preferred mutations in the Loop-8 region include deletion of amino acids that make up the Loop-8 region. As long as infection in the liver is suppressed, there is no particular limit to the number of amino acids to be deleted, but preferably 1 to 8, for example 1 to 6, 1 to 4, 1 to 3, or 1 to 2.
  • a specific example of a suitable mutation is one in which the 591st alanine residue, the 592nd glutamine residue, the 593rd threonine residue, and the 594th glycine residue are deleted from the N-terminus of VP1.
  • deletion of the 591st alanine residue, the 592nd glutamine residue, and the 593rd threonine residue from the N-terminus of VP1 is more preferred, and deletion of the 593rd threonine residue is particularly preferred.
  • 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.
  • mutations similar to those described above can be added to 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.
  • a preferred mutation in the Loop-8 region is the addition of an amino acid to the Loop-8 region.
  • an amino acid to the Loop-8 region As long as liver infection is suppressed, there is no particular limit to the number of amino acids to be added, but it is preferably 1 to 8, for example, 1 to 6, 1 to 4, 2 to 6, or 2 to 4.
  • a specific example is a mutation in which the 591st alanine residue, the 592nd glutamine residue, the 593rd threonine residue, and the 594th glycine residue are deleted from the N-terminus of VP1.
  • deletion of the 591st alanine residue, the 592nd glutamine residue, and the 593rd threonine residue from the N-terminus of VP1 is more preferred, and deletion of the 593rd threonine residue is particularly preferred.
  • AAV of other serotypes similar mutations to those described above can be made in the region corresponding to the Loop-8 region of serotype 9 AAV.
  • rAAV particles with such mutations are administered by intravenous injection, infection of the liver is suppressed, thereby increasing the amount taken up by other organs.
  • the above-described mutation in the capsid protein that suppresses liver infection of rAAV particles is one embodiment that can be used in combination with other embodiments.
  • the following two examples are examples of applying this mutation to rAAV particles in which the CAP protein contains a fusion protein of the CAP protein and another protein.
  • the mutation is introduced into all CAP proteins, including fusion proteins of the CAP protein with other proteins.
  • the mutation is not introduced into a fusion protein of the CAP protein with another protein, but is introduced only into the other CAP protein.
  • the present disclosure provides a method for producing a recombinant adeno-associated virus vector, comprising the step of 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 has substituted with 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 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 the adeno-associated virus of the other serotype when aligned with the serotype 9 adeno-associated virus amino acid residues.
  • 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 the adeno-associated virus of the other serotype when aligned with the ser
  • 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 one, two, or all of VP1, VP2, and VP3 at the amino acid residues exemplified herein in Loop-4, Loop-5, and Loop-8 derived from VP1, or at positions corresponding to those residues in other serotypes.
  • a VP hereinafter referred to as a "mutated VP”
  • 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 amino acid residues in other serotypes of adeno-associated virus when aligned with serotype 9 adeno-associated virus amino acids.
  • serotypes e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03
  • 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.
  • 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).
  • rAAV particles After transfection, expression of the VP protein and assembly and packaging of rAAV particles are induced within 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.
  • a host cell in another aspect, has a group of genes necessary for producing a recombinant adeno-associated virus vector, and 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 one or more amino acid residues are substituted with other amino acid residues.
  • 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 other serotypes that correspond to these when aligned with the amino acid residues of serotype 9 adeno-associated virus.
  • serotype 9 adeno-associated virus e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03
  • 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 have substituted with one or more amino acid residues of Loop-4 and/or Loop-5 and/or Loop-8, respectively.
  • 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 amino acid residues of adeno-associated virus of other serotypes when aligned with serotype 9 adeno-associated virus amino acids.
  • serotypes e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03
  • the mutated VP, recombinant adeno-associated virus vector, its production method, 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).
  • serotype 9 e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAVrhlO, AAV11, AAV-DJ, AAV-LK03
  • the site of mutation preferably does not contain 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 corresponding to these positions, when aligned to serotype 9 adeno-associated virus VP1.
  • 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 this 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 combination thereof.
  • 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).
  • 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).
  • 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)
  • 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.
  • 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.
  • 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.
  • pR2C8 (VHH456-462) was constructed using the following method.
  • pR2C8 (VHH456-462): Using pR2C8 constructed 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 was ligated to the DNA fragment encoding the linker-attached VHH described above 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).
  • 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-added 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-added VHH (linker-added VHH). This vector was designated pR2C9 (VHH455-460).
  • the amino acid sequence of the N-terminal linker was the cIgG2a Hinge linker of SEQ ID NO: 81
  • the amino acid sequence of the C-terminal linker was also the cIgG2a Hinge linker
  • the amino acid sequence of the VHH was that of SEQ ID NO: 9.
  • 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.
  • 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).
  • PCR was performed with primers 11 and 14 (SEQ ID NOs: 127 and 131), which were designed on the C-terminal side 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-terminal side, and this amplification product was allowed to self-anneal.
  • the resulting vector was designated pR2C9 (VHH2-455). This vector encodes a fusion protein in which VHH is present at the C-terminal side of the 455th amino acid residue from the N-terminus of the amino acid sequence of VP1 encoded by the Cap region of AAV9.
  • Example 5 Investigation of a method for producing rAAV particles (rAAV8 particles) bearing VHHs on their surface.
  • FBS was added to Dulbecco's modified Eagle's medium (DMEM) to a final concentration of 10% to prepare a 10% FBS-containing DMEM medium.
  • DMEM Dulbecco's modified Eagle's medium
  • HEK293T cells a cell line derived from human embryonic kidney cells expressing the SV40 virus large T antigen gene, were seeded in 10% FBS-containing DMEM medium onto a 6-well plate at a density of 6-8E4/ cm2 , cultured at 37°C in the presence of 5% CO2 , and used for transfection the following day.
  • Solutions containing mixed vectors were prepared by mixing pR2C8 (VHH456-462) and pR2C8 prepared in Example 3 so that the molar ratio of pR2C8 (VHH456-462) was 10%, 30%, and 50% of the total.
  • pHelper (mod) plasmid (Takara Bio Inc.)
  • each mixed vector pAAV-CMV-GFP vector
  • polyethyleneimine PEI MAX-Transfection Grade Linear Polyethyleneimine Hydrochloride (MW 40,000)
  • transfection reagent 0.35 ⁇ g of vector was added per cm2 of culture area.
  • a transfection reagent containing only pR2C8 instead of the mixed vector was prepared in the same manner. After removing all of the medium from the culture vessel, 2% FBS-containing DMEM medium supplemented with valproic acid was added to the culture vessel, and the transfection reagent was then added to transduce the vector into the cells. After vector transfection, the cells were cultured at 37°C in the presence of 5% CO2 for 3 to 4 days, and the post-culture supernatant was recovered. The amount of rAAV particles contained in the recovered supernatant was quantified using the method described in Example 45. The results are shown in Figure 1.
  • Example 6-1 Investigation of a method for producing rAAV particles (AAV9) having VHH on their surface (1) As a result of examining the method for producing rAAV particles having VHH on their surface, as described in Example 5 above, it was shown that when the AAV is AAV8, highly infectious rAAV particles can be obtained by using a solution containing a mixed vector prepared by mixing pR2C8 (VHH456-462) and pR2C8 so that the ratio (molar ratio) of pR2C8 (VHH456-462) is 10% of the total.
  • VHH455 Hinge-Hinge a vector in which a gene encoding a 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 of pR2C9 prepared in Example 4.
  • pR2C9 (VHH455 Hinge-Hinge) is a vector in which a gene encoding a linkered VHH in which the N- and C-terminal linkers are cIgG2a Hinge with the amino acid sequence of SEQ ID NO: 81, and the VHH has the amino acid sequence of SEQ ID NO: 9, was inserted at a position corresponding to the C-terminal side of the 455th amino acid residue from the N-terminus of VP1.
  • This vector was prepared based on pR2C9 (VHH455) using an In-Fusion HD cloning kit (Clontech).
  • Solutions containing mixed vectors were prepared by mixing pR2C9 (VHH455 Hinge-Hinge) and pR2C9 so that the molar ratio of pR2C9 (VHH455 Hinge-Hinge) was 0%, 5%, 10%, 30%, or 50% of the total.
  • pR2C9 VHH455 Hinge-Hinge
  • pR2C9 VHH455 Hinge-Hinge
  • the amounts of rAAV particles obtained when transfected with mixed vectors prepared so that the ratio of pR2C9 (VHH455 Hinge-Hinge) to pR2C9 was 5%, 10%, 30%, and 50% of the total were 0.65, 0.5, 0.38, and less than 0.1, respectively. Furthermore, when pR2C9 (VHH455 Hinge-Hinge) was set to 100%, almost no rAAV particles were obtained.
  • Example 6-2 Investigation of the method for producing rAAV particles (rAAV9 particles) having VHH on their surface (2) Furthermore, the vector mixing ratio when producing rAAV particles was examined using pR2C9(VHH455), a vector in which a gene encoding 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 of pR2C9 prepared in Example 4. The examination was performed using a solution containing a mixed vector prepared by mixing pR2C9(VHH455) and pR2C9 so that the proportion (molar ratio) of pR2C9(VHH455) was 5% and 10% of the total.
  • rAAV particles were produced according to the method described in Example 11.
  • the obtained rAAV particles were administered to hTfRKI mice (14-week-old, male) at a dose of 1 x 10 13 vg/kg.
  • the brains were removed according to the method described in Example 30 and subjected to immunohistochemical staining according to the method described in Example 31 to observe GFP expression in the brain tissue.
  • the GFP contained in the brain tissue was quantified according to the method described in Example 32.
  • pR2C9 VHH2-455 prepared in Example 4, which is a vector into which a gene encoding a VHH having the amino acid sequence of SEQ ID NO: 11 was inserted at the C-terminal side of the 455th amino acid residue from the N-terminus of VP1 encoded in the Cap region of pR2C9.
  • FIG. 3 The results of immunohistochemical staining are shown in Figure 3.
  • (a) is an immunohistochemical staining image of an untreated mouse
  • (b) is an immunohistochemical staining image of a mouse administered with anti-TfR VHH(455)(CAG-GFP-WPRE) AAV9 (5%)
  • (c) is an immunohistochemical staining image of a mouse administered with anti-TfR VHH(455)(CAG-GFP-WPRE) AAV9 (10%).
  • Figures (b) and (c) show that the tissues are strongly stained.
  • the amount of GFP in the brain of mice administered with (CAG-GFP-WPRE)-AAV9 was approximately 0.275 ⁇ g/wet tissue weight
  • the amount of GFP in the brain of mice administered with anti-TfR VHH(455)(CAG-GFP-WPRE)AAV9 (5%) and anti-TfR VHH(455)(CAG-GFP-WPRE)AAV9 (10%) was approximately 12.27 and 10.17 ⁇ g/wet tissue weight, respectively, which was more than 35 times higher.
  • rAAV particles (anti-TfR VHH(455)(CAG-GFP-WPRE) AAV9(5%) and anti-TfR VHH(455)(CAG-GFP-WPRE) AAV9(10%) prepared using mixed solutions containing either 5% or 10% (molar ratio) of pR2C9(VHH455) can be suitably used as rAAV particles for expressing foreign genes in brain tissue.
  • pAAV-CAG-GFP-WPRE was constructed using the method described in Example 18. Similar results were obtained when pR2C9 (VHH2-455) was used (results not shown).
  • Example 7 Investigation of the insertion site of the VHH-encoding gene into the cap region (rAAV particle production and infectivity) FBS was added to Dulbecco's modified Eagle's medium (DMEM) to a final concentration of 10% to prepare a 10% FBS-containing DMEM medium.
  • DMEM Dulbecco's modified Eagle's medium
  • HEK293T cells a cell line derived from human embryonic kidney cells expressing the SV40 virus large T antigen gene, were seeded in 10% FBS-containing DMEM medium onto a 6-well plate at a density of 6-8E4/cm2, cultured at 37°C in the presence of 5% CO2 , and used for transfection the following day.
  • a solution containing a mixed vector was prepared by mixing one of the pR2C8 vectors incorporating VHHs produced in Example 3 and pR2C8, with the proportion (molar ratio) of pR2C8 incorporating VHHs being 10% of the total.
  • pHelper (mod) plasmid (Takara Bio Inc.)
  • the mixed vector pAAV-CMV-GFP vector
  • polyethyleneimine PEI MAX-Transfection Grade Linear Polyethyleneimine Hydrochloride (MW 40,000)
  • a control transfection reagent was also prepared in the same manner, using only pR2C8 instead of the mixed vector. After removing all of the medium from the culture vessel, 2% FBS-containing DMEM medium supplemented with valproic acid was added to the culture vessel, and then a transfection reagent was added to introduce the vector into the cells. After vector introduction, the cells were cultured at 37°C in the presence of 5% CO2 for 3 to 4 days, and the post-culture supernatant was collected.
  • the amount of rAAV particles contained in the recovered supernatant was quantified by the method described in Example 45, and the infectivity of the rAAV particles was measured by the method described in Example 29.
  • the quantification results for rAAV particles are shown in Figure 4.
  • the quantitative value of rAAV particles is taken as 1 for infection using only pR2C8, the quantitative value when pR2C8 (456-462) was used was roughly consistent with the value obtained in Example 5 (i.e., 0.38).
  • Some pR2C8s incorporating VHHs, such as pR2C8 (VHH359) can produce high yields of rAAV particles, but these generally tend to have significantly reduced infectivity. Those marked with an asterisk in Figure 4 have significantly reduced infectivity.
  • the quantitative values for each rAAV particle indicate that pR2C8 (VHH456-462), pR2C8 (VHH455), pR2C8 (VHH457), pR2C8 (VHH462), pR2C8 (VHH501), pR2C8 (VHH588), and pR2C8 (VHH599) are suitable vectors for producing rAAV particles with VHHs on their surface in terms of the yield and infectivity of the resulting rAAV particles.
  • Example 8 Examination of the amino acid sequence of the linker (1)
  • the vectors used to investigate the insertion site of the gene encoding VHH into the Cap region all contained a base sequence encoding an N-terminal linker (GGGGS x 1), a base sequence encoding a VHH having affinity for human TfR, including the amino acid sequence of SEQ ID NO: 9, and a base sequence encoding a C-terminal linker (GGGGS x 3).
  • the amino acid sequences of the linkers used here, 2xEAAAK and cIgG2a Hinge, are shown in SEQ ID NO: 82 and SEQ ID NO: 81, respectively.
  • 2xEAAAK consists of 12 amino acids
  • cIgG2a Hinge consists of 22 amino acids.
  • the amino acid sequence of cIgG2a is the amino acid sequence of the hinge region of camel cIgG2a.
  • FBS was added to Dulbecco's modified Eagle's medium (DMEM) to a final concentration of 10% to prepare a 10% FBS-containing DMEM medium.
  • DMEM Dulbecco's modified Eagle's medium
  • HEK293T cells a cell line derived from human embryonic kidney cells expressing the SV40 virus large T antigen gene, were seeded in 10% FBS-containing DMEM medium onto a 6-well plate at a density of 6-8E4/cm2, cultured at 37°C in the presence of 5% CO2 , and used for transfection the following day.
  • a solution containing a mixed vector was prepared by combining pR2C8 with one of the pR2C8 vectors incorporating a VHH having the amino acid sequence of each linker shown in Table 2, with pR2C8, so that the proportion (molar ratio) of pR2C8 incorporating the VHH accounted for 10% of the total.
  • pHelper (mod) plasmid (Takara Bio Inc.), the mixed vector, pAAV-CMV-GFP vector, and polyethyleneimine (PEI MAX-Transfection Grade Linear Polyethyleneimine Hydrochloride (MW 40,000)) was added to DMEM medium in a weight ratio of approximately 10:6:5:42, mixed, and allowed to stand at room temperature for 15 to 30 minutes to prepare a transfection reagent.
  • a control transfection reagent was also prepared in the same manner, using only pR2C8 instead of the mixed vector.
  • 2% FBS-containing DMEM medium supplemented with valproic acid was added to the culture vessel, and a transfection reagent was then added to transduce the vector into the cells.
  • the cells were cultured at 37°C in the presence of 5% CO2 for 3 to 4 days, and the post-culture supernatant was recovered.
  • the amount of rAAV particles contained in the recovered supernatant was quantified by the method described in Example 45, and the infectivity of the rAAV particles was measured by the method described in Example 29.
  • the C-terminal linker of the VHH of pR2C8(VHH456-462) consists of 15 amino acids.
  • pR2C8(VHH456-462)GS1-GS1 and pR2C8(VHH456-462)GS1-GA1 which have a C-terminal linker of five amino acids, both rAAV particle yields decreased and their infectivity was also weakened. Similar results were obtained when using pR2C8(VHH456-462)GS1-0, which has a deleted C-terminal linker.
  • the rAAV particle yield obtained using the 12-amino acid C-terminal linker 2xEAAAK was comparable to that of pR2C8(VHH456-462).
  • a vector containing the amino acid sequence of the linker shown in Table 8 was constructed, and the yield and infectivity of the resulting rAAV particles were further examined.
  • the vector shown in Table 8 was constructed using the In-Fusion HD cloning kit (Clontech) according to the method described in Example 3.
  • the amino acid sequences of the newly used linkers, P9 and PPR5, are shown in SEQ ID NO: 88 and SEQ ID NO: 84, respectively.
  • P9 consists of 9 amino acids
  • PPR5 and cIgG2a Hinge consist of 15 amino acids.
  • Example 9 Examination of the amino acid sequence of the linker (2) In Example 8, pR2C8 (VHH456-462) was used as a model vector. The results showed that, when a gene encoding a VHH is inserted into the Cap region, rAAV particles bearing a VHH on their surface and having high infectivity can be produced in good yields by using a VHH C-terminal linker consisting of 12 or more amino acids and by using an N-terminal or/and C-terminal linker with the amino acid sequence of a camel IgG2a hinge region.
  • GGGGSx1-cIgG2a Hinge The amino acid sequences of the newly used linkers, GGGGSx1-cIgG2a Hinge, CDC-GGGGGSx3, and GGGGAx3-CFC, are shown in SEQ ID NOs: 85, 83, and 87, respectively.
  • GGGGSx1-cIgG2a Hinge consists of 27 amino acids
  • CDC-GGGGGSx3 consists of 18 amino acids
  • GGGAx3-CFC consists of 18 amino acids.
  • Example 10 Examination of the amino acid sequence of the linker (3) Furthermore, vectors in which the linker was replaced with various other linkers were prepared, and their effects on rAAV particle yield and infectivity were examined.
  • the vectors examined here and the sequences of the linkers contained therein are shown in Table 10.
  • the vectors shown in Table 10 were constructed according to the method described in Example 3 using an In-Fusion HD cloning kit (Clontech).
  • the amino acid sequences of the newly used linkers, PA4, PQ4, IgA Hinge, and cIgG2c are shown in SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:86, and SEQ ID NO:91, respectively.
  • PA4 consists of 8 amino acids
  • PQ4 consists of 8 amino acids
  • gA Hinge consists of 11 amino acids
  • cIgG2c consists of 7 amino acids.
  • Example 11 Examination of the amino acid sequence of the linker (4)
  • pR2C8(VHH456-462)GS1-Hinge In order to further examine pR2C8(VHH456-462)GS1-Hinge, pR2C8(VHH501)GS1-Hinge, and pR2C8(VHH599)GS1-Hinge, which were shown to have high yields and infectivity in Example 8, purified rAAV particles were obtained using these vectors by the method described in detail below. The vectors examined here and the linker sequences contained therein are shown in Table 11.
  • FBS was added to Dulbecco's modified Eagle's medium (DMEM) to a final concentration of 10% to prepare a 10% FBS-containing DMEM medium.
  • DMEM Dulbecco's modified Eagle's medium
  • HEK293T cells a cell line derived from human embryonic kidney cells expressing the SV40 virus large T antigen gene, were seeded onto a T2 25 plate at 6-8E4/ cm2 in 10% FBS-containing DMEM medium, cultured at 37°C in the presence of 5% CO2 , and used for transfection the following day.
  • a solution containing a mixed vector was prepared by mixing one of the pR2C8 vectors incorporating VHH prepared in Example 5 and pR2C8, with the proportion (molar ratio) of pR2C8 incorporating VHH being 10% of the total.
  • pHelper (mod) plasmid (Takara Bio Inc.), mixed vector, pAAV-CMV-GFP vector, and polyethyleneimine (PEI MAX-Transfection Grade Linear Polyethyleneimine Hydrochloride (MW 40,000)) were added to DMEM medium in a weight ratio of approximately 10:6:5:42, mixed, and left to stand at room temperature for 15 to 30 minutes to prepare a transfection reagent.
  • a control transfection reagent containing only pR2C8 instead of the mixed vector was also prepared in the same manner.
  • 2% FBS-containing DMEM medium supplemented with valproic acid was added to the culture vessel, followed by the addition of the transfection reagent to introduce the vector into the cells. After vector introduction, the cells were cultured at 37°C in the presence of 5% CO2 for 3 to 4 days.
  • a 40% iodixanol solution was prepared by adding PBS-MK buffer (20 mmol/L sodium phosphate buffer (pH 7.4 ) containing 136.9 mmol/L sodium chloride, 28.2 mmol/L potassium chloride, and 1 mmol/L magnesium chloride) to 60% iodixanol (multipurpose density gradient centrifugation medium OptiPrepTM, Cosmo Bio Co., Ltd.).
  • a 25% iodixanol solution was prepared by adding PBS-MK buffer to 60% iodixanol.
  • a 15% iodixanol solution was prepared by adding PBS-MK buffer to 60% iodixanol.
  • PA Quick-Seal TM Round-Top Polypropylene Tube QSPP
  • the amount of rAAV particles contained in this purified product was quantified by the method described in Example 28, and the infectivity of the rAAV particles was measured by the method described in Example 29.
  • the yield of rAAV particles contained in the purified rAAV particle product is shown in Figure 9.
  • the yield of rAAV particles obtained was higher than when pR2C8(VHH456-462) was used.
  • infective rAAV particles were obtained.
  • rAAV particles obtained using pR2C8(VHH456-462)Hinge-Hinge and pR2C8(VHH599)GS1-Hinge showed high infectivity.
  • Example 12 Infectivity test of rAAV particles using mice (1)
  • pR2C8 VHH456-462 Hinge-Hinge enables the production of highly infectious rAAV particles in high yield. Therefore, an infectivity test was conducted using mice with rAAV particles obtained using this vector.
  • a similar test was also conducted on rAAV particles obtained using only pR2C8 as a control. The method for producing rAAV particles is described in detail below.
  • FBS was added to Dulbecco's modified Eagle's medium (DMEM) to a final concentration of 10% to prepare a 10% FBS-containing DMEM medium.
  • DMEM Dulbecco's modified Eagle's medium
  • HEK293T cells a cell line derived from human embryonic kidney cells expressing the SV40 virus large T antigen gene, were seeded onto T75 plates at 6-8E4/ cm2 in 10% FBS-containing DMEM medium, cultured at 37°C in the presence of 5% CO2 , and used for transfection the following day.
  • a solution containing a mixed vector of pR2C8 (VHH456-462) Hinge-Hinge and pR2C8 was prepared such that the proportion (molar ratio) of pR2C8 incorporating VHH was 10% of the total.
  • pHelper (mod) plasmid (Takara Bio Inc.), mixed vector, pAAV-CMV-GFP vector, and polyethyleneimine (PEI MAX-Transfection Grade Linear Polyethyleneimine Hydrochloride (MW 40,000)) were added to DMEM medium in a weight ratio of approximately 10:6:5:42, mixed, and left to stand at room temperature for 15 to 30 minutes to prepare a transfection reagent.
  • a control transfection reagent containing only pR2C8 instead of the mixed vector was also prepared in the same manner.
  • 2% FBS-containing DMEM medium supplemented with valproic acid was added to the culture vessel, followed by the addition of the transfection reagent to introduce the vector into the cells. After vector introduction, the cells were cultured at 37°C in the presence of 5% CO2 for 3 to 4 days.
  • cell lysis buffer 0.5 M HEPES, 20 mM MgCl2 , 5 w/v% polysorbate 20
  • 50 U/mL benzonase were added to the culture vessel.
  • the solution was recovered and filtered through a 0.22 ⁇ m filter to obtain an enzyme-treated solution containing AAV9 virions.
  • a column packed with POROS-AAVX resin (POROSTM CaptureSelect TM -AAVX Affinity Resin, Thermo Fisher Scientific) was filled with 5 column volumes of equilibration buffer (20 mmol/L trishydroxymethylaminomethane buffer (pH 7.5) containing 2 mmol/L MgCl 2 and 150 mmol/L NaCl), and then filtered.
  • the equilibrated enzyme-treated solution containing AAV9 virions was passed through the column, allowing the rAAV particles to adsorb onto the column.
  • the rAAV particles were eluted with 5 column volumes of elution buffer (10 mM citrate buffer (pH 3.5) containing 2 mM MgCl , 150 mmol/L NaCl, and 0.001% F-68).
  • the eluate was immediately neutralized by adding 1/19 volume of neutralization buffer (500 mM Tris, 2 mM MgCl pH 8.5).
  • the neutralized eluate was centrifuged using an Amicon Ultra 4 centrifugal filter unit, 100 kDa (Merck) to obtain a concentrate containing rAAV particles.
  • This concentrate was diluted with DPBS containing 0.001% F-68 and centrifuged again. This procedure was repeated to obtain a concentrated solution of rAAV particles in which the solvent was replaced with DPBS containing 0.001% F-68.
  • the obtained concentrated solution of rAAV particles was frozen and stored at -80°C.
  • the rAAV particles obtained using pR2C8 (VHH456-462) Hinge-Hinge were designated Anti-TfRAAV8, and the control rAAV particles were designated AAV8 (control).
  • the animals infected with each rAAV particle were IDS-KO/hTfR-KI mice (14 weeks old, male) for Anti-TfRAAV8 and C57BL6/N (6 weeks old, male) for AAV8 (control).
  • Each rAAV particle was administered via tail vein injection at a dose of 1 x 10 13 vg/kg to mice in the low-dose group and at a dose of 1 x 10 13 vg/kg to mice in the high-dose group.
  • the IDS-KO/hTfR-KI mice used here were generated by the method described in Example 36. Table 12 outlines the dosing regimen.
  • mice Two weeks after administration or two weeks after administration, the mice were euthanized, systemically perfused with saline, and tissues were excised.
  • the GFP concentration in each tissue was measured using the method described in Example 32.
  • the brains were divided in the sagittal plane, and one section was used to measure the GFP concentration in the tissue, while the other section was subjected to immunohistochemical staining as described in Example 31.
  • the amount of rAAV particles in each tissue was quantified using the method described in Example 45.
  • Figure 10 shows the results of measuring the GFP concentration in each tissue. In the brain, in the low-dose group, expression levels were low in those administered with Anti-TfRAAV8.
  • Example 13 Preparation of rAAV particles with antibodies bound to the surface via the Tag sequence
  • the results of Example 12 demonstrated that by administering to mice rAAV particles in which a portion of the capsid protein was made into a fusion protein with a VHH that has affinity for human TfR, the foreign gene encapsulated in the rAAV particles was expressed in the brain.
  • rAAV particles were produced that have a capsid protein with an ALFA Tag sequence attached to their surface, which has affinity for another desired protein.
  • An antibody with affinity for human TfR produced as a fusion protein with a single-chain antibody with affinity for the ALFA Tag sequence, was then bound to these rAAV particles.
  • rAAV particles with an antibody with affinity for human TfR bound to their surface via the Tag sequence were produced by the method described in Examples 14 to 16. An investigation was then conducted to determine whether rAAV particles produced by this method could exhibit the same functions as the rAAV particles investigated in Example 12.
  • Example 14 Preparation of rAAV particles with an ALFA Tag on their surface
  • Primer 5 SEQ ID NO: 92
  • Primer 6 SEQ ID NO: 92
  • a DNA fragment containing the nucleotide sequence of SEQ ID NO: 95 encoding the ALFA Tag having the amino acid sequence of SEQ ID NO: 94 and a DNA fragment consisting of its complementary sequence were mixed and heated to 98°C, followed by gradual cooling to allow annealing.
  • this annealed product and the one-round PCR product were ligated using an In-Fusion HD cloning kit (Clontech).
  • the resulting plasmid was designated pR2C9 (ALFATag 707).
  • a solution containing purified rAAV particles bearing an ALFA Tag on their surface was prepared by the method described in Example 11. However, in the method described in Example 11, the phrase "prepared so that the proportion (molar ratio) was 10% of the total” was changed to "prepared so that the proportion (molar ratio) was 5% of the total.”
  • the resulting rAAV particles were designated ALFATag-AAV9, and the solution containing this purified product was designated ALFATag-AAV9 solution.
  • Example 15 Production of rAAV particles with antibodies (VHH) bound to the surface via a tag sequence
  • VHH5-GS3-hIDS expression vector described in International Publication WO 2023/090409 was digested with BamHI and NotI to obtain a DNA fragment from which the nucleotide sequence encoding I2S had been removed.
  • the resulting vector was designated pCI-4R15-GS3-ALFANb. Furthermore, inverse PCR was performed using primer 7 (SEQ ID NO: 98) and primer 8 (SEQ ID NO: 99) and pCI-4R15-GS3-ALFANb as a template, and the gap was ligated using an In-Fusion HD cloning kit (Clontech). The resulting plasmid was designated pCI-4R15-GS1-ALFANb.
  • This plasmid encodes a protein (anti-TfR VHH-ALFANb) in which ALFANb is attached, via the amino acid sequence GGGGS, to the C-terminus of a VHH having affinity for human TfR, comprising the amino acid sequence of SEQ ID NO: 9.
  • ExpiCHO cells were transformed with pCI-4R15-GS1-ALFANb using an ExpiCHO Expression System (ThermoFisher Scientific). The transformed cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air at an agitation speed of approximately 120 rpm for one day. The Feed solution and Enhancer solution provided with the ExpiCHO Expression System were added, and the cells were cultured at 32°C in a humidified atmosphere of 5% CO2 and 95% air at an agitation speed of approximately 120 rpm for six days to express 4R15-ALFANb.
  • ThermoFisher Scientific ThermoFisher Scientific. The transformed cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air at an agitation speed of approximately 120 rpm for one day.
  • the Feed solution and Enhancer solution provided with the ExpiCHO Expression System were added, and the cells were cultured at 32°C in a humidified atmosphere of 5% CO
  • the culture medium was centrifuged (3000 g, 5 minutes) and the resulting supernatant was filtered through a 0.22 ⁇ m filter (Millipore) and collected as the culture supernatant.
  • This culture supernatant contains anti-TfR VHH-ALFANb.
  • the Mabselect Xtra resin was equilibrated by passing 10 column volumes of DPBS through the resin.
  • the culture supernatant was passed through the resin to adsorb 4R15-ALFANb, and the resin was washed with 10 column volumes of DPBS.
  • the resin was eluted with 3 column volumes of 100 mM glycine, 100 mM NaCl, pH 3.0.
  • the eluate was immediately neutralized by adding 1/17 volume of 1 M Tris-HCl, pH 8.0.
  • the neutralized eluate was loaded onto an Amicon Ultra centrifugal filter unit 10 kDa (Merck) and centrifuged.
  • DPBS containing 0.001% F-68 was added to the concentrated solution, and the filter was further centrifuged.
  • Example 16 Production of rAAV Particles Having an Antibody (Fab) Bound to the Surface via a Tag Sequence Primer 9 (SEQ ID NO: 100) and primer 10 (SEQ ID NO: 101), which were designed to remove 4R15-GS1 from pCI-4R15-GS1-ALFANb prepared in Example 15, were used to perform one-round PCR using pCI-4R15-GS1-ALFANb as a template to amplify a DNA fragment.
  • Fab Antibody
  • a DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 103 encoding the Fab heavy chain (SEQ ID NO: 102) of an antibody having affinity for human TfR was synthesized, and this DNA fragment and the previously amplified DNA fragment were ligated using an In-Fusion HD cloning kit (Clontech).
  • the resulting plasmid was designated pCI-hu459 Fab HC-ALFANb.
  • This plasmid encodes a protein (anti-TfR FabH-ALFANb) in which ALFANb is attached to the C-terminus of the Fab heavy chain having affinity for human TfR.
  • pCI-neo Mammalian Expression Vector (Promega) was digested with MLuI and NotI.
  • This DNA fragment was ligated to the vector previously digested with restriction enzymes using the In-Fusion HD cloning kit (Clontech).
  • the resulting plasmid was designated pCI-hu459 Fab LC. This plasmid encodes the Fab light chain with affinity for human TfR.
  • a mixture containing pCI-hu459 Fab HC-ALFANb and pCI-hu459 Fab LC at a weight ratio of 1:1 was prepared.
  • ExpiCHO cells were transformed using the mixture and an ExpiCHO Expression System (ThermoFisher Scientific). The transformed cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air at an agitation speed of approximately 120 rpm for one day. The Feed solution and Enhancer solution provided with the ExpiCHO Expression System were added, and the cells were cultured at 32°C in a humidified atmosphere of 5% CO2 and 95% air at an agitation speed of approximately 120 rpm for six days.
  • This culture supernatant contains a protein in which ALFANb is bound to the C-terminus of Fab having affinity for human TfR (anti-TfR Fab-ALFANb).
  • the CH1-XL resin was equilibrated by passing 10 column volumes of DPBS through it. The culture supernatant was then passed through the column, allowing anti-TfR Fab-ALFANb to adsorb to the resin. After washing the column with 10 column volumes of DPBS, anti-TfR Fab-ALFANb was eluted with 3 column volumes of 100 mM glycine buffer (pH 3.0) containing 100 mM NaCl. The eluate was immediately neutralized by adding 1/17 volume of 1 M Tris-HCl pH 8.0 to the eluate. The neutralized eluate was loaded onto an Amicon Ultra centrifugal filter unit 10 kDa (Merck) and centrifuged.
  • DPBS containing 0.001% F-68 was added to the concentrated solution, and the mixture was further centrifuged. This procedure was repeated several times to exchange the buffer and concentrate the sample. The concentrated solution was used as the anti-TfR Fab-ALFANb solution.
  • Anti-TfR Fab-ALFANb solution was added to the ALFATag-AAV9 solution prepared in Example 14 so that 400 ⁇ g of anti-TfR Fab-ALFANb was used per 1E12 ⁇ g of ALFATag-AAV9, and the mixture was left to stand at room temperature for 30 minutes or more to allow ALFATag and ALFANb to bind.
  • These rAAV particles were designated anti-TfR Fab-(ALFANb ALFATag)-AAV9.
  • Example 17 Infectivity test of rAAV particles using mice (2) Using pR2C9 prepared in Example 2, a solution containing purified rAAV particles having wild-type AAV9 capsids was prepared by the method described in Example 11. These rAAV particles were designated AAV9-WT, and the solution containing this purified product was designated an rAAV9-WT solution. Furthermore, using pR2C9 (VHH455-460) prepared in Example 4, a solution containing purified rAAV particles having VHH with affinity for human TfR in the form of a fusion protein with a capsid protein was prepared by the method described in Example 11.
  • anti-TfR VHH(455-460)-AAV9 contains a capsid in which 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 that shown in SEQ ID NO:9.
  • the IDS-KO/hTfR-KI mice used here were prepared by the method of Example 36.
  • mice Four weeks after administration, the mice were euthanized, perfused with saline, and tissues were removed.
  • the GFP concentration in each tissue was measured using the method described in Example 32.
  • rAAV particles were also quantified using the method described in Example 45.
  • the brains were divided in the sagittal plane, and one section was used to measure the GFP concentration in the tissue, and the other section was used for immunohistochemical staining as described in Example 31.
  • Figure 12 shows the quantitative values of rAAV particles and the measured GFP concentration in the brain.
  • the quantitative values of rAAV particles in the brain increased by approximately 50-fold compared to the control group.
  • the rAAV particles that had VHH as an antibody with affinity for human TfR on their surface had approximately four times the measured GFP values compared to rAAV particles that had Fab as the antibody.
  • rAAV particles with equivalent infectivity and other functions can also be produced by contacting a separately prepared VHH with the surface of rAAV particles modified with a Tag, thereby binding the VHH to the surface of the rAAV particles via the Tag.
  • This method is also considered to be particularly effective when rAAV particles are modified with proteins with complex structures, such as Fab, which consists of two peptide chains, an H chain and an L chain.
  • rAAV particles that have a VHH on their surface as an antibody with affinity for human TfR are expected to express the gene at a higher level than rAAV particles that have a Fab as the antibody. Therefore, to achieve this goal, VHH is preferable to Fab as an antibody with affinity for human TfR on its surface.
  • Example 18 Promoter study (1) In the experiments up to Example 17 above, the cytomegalovirus immediate-early promoter (CMV) was used as the promoter for expressing GFP. Here, various promoters were examined for their function as promoters for expressing genes in the brain. In addition to CMV, promoter examination was also performed on the CAG, CBh, and PGK promoters.
  • CMV cytomegalovirus immediate-early promoter
  • vectors in which the GFP gene was placed under the control of these promoters were constructed by the method described below.
  • pAAV-CMV-GFP-WPRE The pAAV-CMV-GFP vector having the base sequence shown in SEQ ID NO: 106 was digested with EcoRV. A DNA fragment having the base sequence shown in SEQ ID NO: 107, which contains the WPRE sequence, was synthesized. This DNA fragment was ligated to pAAV-CMV-GFP digested with EcoRV using the In-Fusion HD cloning kit (Clontech). The resulting plasmid was designated pAAV-CMV-GFP-WPRE.
  • pAAV-CAG-GFP-WPRE pAAV-CMV-GFP was digested with HindIII and BglII to remove the fragment containing CMV, GFP, and polyA, and a sequence (sequence number 155) containing a synthetic CAG promoter and bGH polyA sequence was ligated. The resulting vector was digested with MLuI and NotI, and a synthetic I2S sequence (sequence number 156) was ligated to create pAAV-CAG-I2S-WPRE. pAAV-CAG-I2S-WPRE was digested with MLuI and NotI to remove the I2S sequence.
  • the DNA fragment (sequence number 108) encoding GFP from pAAV-CMV-GFP was amplified by PCR. This amplification product was ligated to pAAV-CAG-I2S-WPRE digested with MLuI and NotI using the In-Fusion HD cloning kit (Clontech). The resulting plasmid was named pAAV-CAG-GFP-WPRE.
  • pAAV-CBh-GFP-WPRE A plasmid fragment was prepared by digesting pAAV-CAG-GFP-WPRE with EcoRI and KpnI to remove the CMV promoter sequence.
  • DNA fragment 1 SEQ ID NO: 109
  • DNA fragment 2 with the base sequence shown in SEQ ID NO: 110, was also synthesized.
  • DNA fragment 1, DNA fragment 2, and pAAV-CAG-GFP-WPRE digested with EcoRI and KpnI were ligated using the In-Fusion HD cloning kit (Clontech). The resulting plasmid was designated pAAV-CBh-GFP-WPRE.
  • pAAVPGK-GFP-WPRE pAAV-CAG-I2S-WPRE was digested with ClaI and MLuI to remove the CAG promoter.
  • a DNA fragment containing the PGK promoter and having the base sequence of SEQ ID NO: 111 was synthesized. This DNA fragment was ligated to pAAV-CAG-I2S-WPRE digested with ClaI and MLuI using the In-Fusion HD cloning kit (Clontech).
  • the resulting plasmid was named pAAVPGK-I2S-WPRE.
  • pAAVPGK-I2S-WPRE was digested with MLuI and NotI to remove the portion encoding I2S.
  • the GFP-containing sequence of pAAV-CMV-GFP (SEQ ID NO: 112) was amplified by PCR. This amplification product was ligated to pAAVPGK-I2S-WPRE digested with MLuI and NotI using the In-Fusion HD cloning kit (Clontech). The resulting plasmid was named pAAVPGK-GFP-WPRE.
  • rAAV particles were produced by the method described in Example 11. However, in the method described in Example 11, "prepared so that the proportion (molar ratio) was 10% of the total” was changed to "prepared so that the proportion (molar ratio) was 5% of the total.”
  • the pR2C9 (VHH455-460) used was the one produced in Example 4.
  • mice were infected with the rAAV particles shown in Table 14, and the levels of GFP expression in the brain were compared.
  • Each rAAV particle was administered at a dose of 1 x 10 13 vg/kg to IDS-KO/hTfR-KI mice (14-week-old, male) via tail vein injection.
  • AAV9-WT was also administered at a dose of 1 x 10 13 vg/kg to IDS-KO/hTfR-KI mice (14-week-old, male) via tail vein injection as a control group.
  • mice Four weeks after administration, the mice were euthanized, perfused whole body with saline, and the brains were removed. The brains were sectioned in the sagittal plane and subjected to immunohistochemical staining as described in detail in Example 31. The results are shown in Figures 13 to 15.
  • Figure 13 shows the staining results for the whole brain, Figure 14 for the cerebrum, and Figure 15 for the cerebellum. It was found that the highest level of GFP expression in the brain was achieved when CAG was used as the promoter.
  • CBh was also shown to have high function as a promoter for protein expression in the brain.
  • CMV had lower function as a promoter for protein expression in the brain than CAG and CBh, and that the PGK promoter had the lowest function as a promoter for protein expression in the brain among the promoters examined.
  • Example 19 Promoter study (2) Example 18 demonstrated that CAG functions highly as a promoter for protein expression in the brain.
  • the data in Example 18 was obtained using rAAV particles having a VHH with affinity for human TfR in the form of a fusion protein with a capsid protein.
  • rAAV particles prepared with a capsid protein containing an ALFA Tag sequence were prepared as a fusion protein with a single-chain antibody with affinity for the ALFA Tag sequence.
  • the rAAV particles used in this study were prepared by the method described in Example 11 using pR2C9(ALFATag 707) prepared in Example 14 and pAAV-CAG-GFP-WPRE prepared in Example 18.
  • the rAAV particles thus obtained were designated anti-TfR Fab-[(ALFANb ALFATag)(CAG-GFP-WPRE)]-AAV9.
  • Example 20 Pharmacological test of rAAV particles using MPS-II model mice Based on the experimental results up to Example 19 above, rAAV particles designed to express I2S in the brain as shown in Table 16 were prepared, and MPS-II model mice were infected with these particles to verify their pharmacological effects.
  • the method for preparing the plasmids used to prepare the rAAV particles is shown below.
  • the rAAV particles were prepared using the combination of plasmids shown in Table 16 by the method shown in Example 11. However, in the method described in Example 11, the phrase "prepared so that the proportion (molar ratio) is 10% of the total” was changed to "prepared so that the proportion (molar ratio) is 5% of the total.” Furthermore, infection of mice was performed by the method described in Example 12.
  • pR2C9 Prepared by the method described in Example 2.
  • pR2C9 (VHH455-460): Prepared by the method described in Example 4.
  • pR2C9 (ALFATag 707): Prepared by the method described in Example 14.
  • pAAV-CAG-I2S-WPRE pAAV-CMV-GFP was digested with HindIII and BglII to remove the fragment containing CMV, GFP, and polyA, and a sequence (sequence number 151) containing a synthetic CAG promoter and bGH polyA sequence was ligated. The resulting vector was digested with MLuI and NotI, and a synthetic I2S sequence (sequence number 152) was ligated to create pAAV-CAG-I2S-WPRE.
  • the rAAV particles shown in Table 16 were administered at a dose of 1 x 10 13 vg/kg to IDS-KO/hTfR-KI mice (14-week-old, male) via tail vein injection.
  • AAV9-WT was also administered at a dose of 1 x 10 13 vg/kg to IDS-KO/hTfR-KI mice (14-week-old, male) via tail vein injection.
  • mice Four weeks after administration, the mice were euthanized, perfused with saline, and tissues were removed.
  • the GFP concentration in each tissue was measured using the method described in Example 32.
  • rAAV particles were also quantified using the method described in Example 28.
  • the brains were divided in the sagittal plane, and one section was used to measure the GFP concentration in the tissue, and the other section was used for immunohistochemical staining as described in Example 31.
  • Figure 17 shows the concentration of heparan sulfate, a substrate for I2S, in the brain. It can be seen that HS accumulated abnormally in the untreated mice compared to wild-type mice. In the group administered with (CAG-I2S-WPRE)-AAV9, which has a wild-type capsid, the concentration of heparan sulfate in the brain was reduced by approximately 64% compared to the untreated group.
  • mice administered with anti-TfR VHH(CAG-I2S-WPRE)-AAV9 mice administered with anti-TfR VHH-[(ALFANb ALFATag)(CAG-I2S-WPRE)]-AAV9
  • concentration of heparan sulfate in the brain was significantly reduced compared to the untreated group, indicating that in both cases, most (more than 96%) of the heparan sulfate accumulated in the brain was degraded and removed from the brain.
  • anti-TfR VHH(CAG-I2S-WPRE)-AAV9 and anti-TfR VHH-[(ALFANb ALFATag)(CAG-I2S-WPRE)]-AAV9 can be used as drugs to degrade and remove I2S substrates, including heparan sulfate, that abnormally accumulate in the brains of MPS-II patients.
  • the measured values of I2S concentration in the brain correlated with the measured values of heparan sulfate, with a tendency for lower measured values of heparan sulfate to correspond to higher I2S concentrations in the brain.
  • Example 21 Pharmacological test of rAAV particles using GM-1 gangliosidosis model mice rAAV particles designed to be able to express GLB1 in the brain as shown in Table 17 were prepared and used to infect GM-1 gangliosidosis model mice described in Example 37 to examine their pharmacological effects.
  • the method for preparing the plasmids used to prepare the rAAV particles is described below.
  • rAAV particles were produced using the combination of plasmids shown in Table 17 by the method described in Example 11. However, in the method described in Example 11, the phrase "prepared so that the proportion (molar ratio) was 10% of the total” was changed to "prepared so that the proportion (molar ratio) was 5% of the total.” Furthermore, infection of mice was performed by the method described in Example 12.
  • pR2C9 Prepared by the method described in Example 2.
  • pR2C9 (VHH455-460): Prepared by the method described in Example 4.
  • pAAV-CAG-GLB1-WPRE pAAV-CAG-I2S-WPRE prepared in Example 20 was digested with MLuI and NotI to remove the I2S sequence.
  • the synthesized GLB1 sequence (SEQ ID NO: 118) was ligated with the vector using the In-Fusion HD cloning kit (Clontech) to create pAAV-CAG-GLB1-WPRE.
  • pAAV-CBh-VHH-GLB1-WPRE Construction of pAAV-CBh-VHH-GLB1-WPRE: pAAV-CBh-GFP-WPRE prepared in Example 18 was digested with KpnI and BsrGI to produce a fragment from which GFP had been removed. This fragment was then ligated to a synthesized GLB1 fragment (SEQ ID NO: 119) and a synthesized VHH-GS3 fragment (SEQ ID NO: 120) using an In-Fusion HD cloning kit (Clontech) to produce pAAV-CBh-VHH-GLB1-WPRE.
  • the rAAV particles shown in Table 17 were administered at a dose of 1 x 10 vg/kg via tail vein injection to hTfRKIxGLB1 KO mice (14-week-old, male), a pathological mouse model of GM-1 gangliosidosis.
  • AAV9-WT was also administered at a dose of 1 x 10 vg/kg via tail vein injection to hTfRKIxGLB1 KO mice (14-week-old, male) as a control group.
  • mice Four weeks after administration, the mice were euthanized, peripheral blood was collected, and the mice were perfused with physiological saline. The brains and livers were then removed. Plasma was prepared from the peripheral blood using standard methods. The GLB1 concentrations in the plasma and each tissue were measured using the method described in Example 35. The Lyso-GM1 concentration in the brain was also quantified using the method described in Example 33. Figure 19 shows the quantified values for Lyso-GM1 in the brain. Compared to wild-type mice, the non-administered pathological model mice showed abnormal accumulation of Lyso-GM1 in the brain.
  • mice administered with (CAG-GLB1-WPRE)-AAV9 which has a wild-type capsid and contains a gene encoding wild-type GLB1
  • brain Lyso-GM1 was reduced by only about 7% compared to the control group.
  • mice administered with (CBh-VHH-GLB1-WPRE)-AAV9 which has a wild-type capsid but contains a gene encoding a fusion protein of VHH and GLB1
  • brain Lyso-GM1 was reduced by approximately 80% compared to the control group.
  • mice administered with anti-TfR VHH(CAG-GLB1-WPRE)-AAV9 brain Lyso-GM1 substrate was reduced by approximately 85% compared to the control group.
  • Figure 20 also shows the quantitative values of GLB1 in the brain.
  • the quantitative value of GLB1 in mice administered with (CBh-VHH-GLB1-WPRE)-AAV9 was approximately 1.45 ⁇ g/g wet tissue weight, which was approximately 4.5 times the quantitative value of GLB1 in mice administered with (CAG-GLB1-WPRE)-AAV9 (approximately 0.33 ⁇ g/g wet tissue weight).
  • This result indicates that in mice administered with (CBh-VHH-GLB1-WPRE)-AAV9, the fusion protein of VHH and GLB1 expressed in tissues other than the brain infected with rAAV particles crossed the BBB and degraded substrates accumulated in the brain.
  • mice administered anti-TfR VHH(CAG-GLB1-WPRE)-AAV9 was 16 ⁇ g/g wet tissue weight, approximately 11 times higher than that of (CAG-GLB1-WPRE)-AAV9. This result indicates that by using rAAV particles containing VHH with affinity for TfR on the capsid surface, it is possible to achieve high-level expression of the GLB1 gene encoded by the rAAV genome encapsulated within these particles.
  • Example 22 Pharmacological Test of rAAV Particles Using Demyelinating Disease Model Mice rAAV particles designed to express ASPA in the brain as shown in Table 18 were prepared, and demyelinating disease model mice were infected with these particles to verify their pharmacological effects.
  • the method for preparing the plasmids used to prepare the rAAV particles is shown below.
  • the rAAV particles were prepared using the combination of plasmids shown in Table 18 by the method shown in Example 11. However, in the method described in Example 11, the phrase "prepared so that the proportion (molar ratio) is 10% of the total” was changed to "prepared so that the proportion (molar ratio) is 5% of the total” here. Infection of mice, etc. was performed by the method described in Example 12.
  • pR2C9 Prepared by the method described in Example 2.
  • pR2C9 (VHH455-460) Prepared by the method described in Example 4.
  • pscAAV-CBh-ASPA The pAAV-CMV-GFP-WPRE vector prepared in Example 18 was digested with MLuI to remove the 5' ITR sequence and CMV promoter, and then a fragment containing an ITR sequence lacking a TRS sequence (sequence number 113) amplified by PCR from the pAAV-CMV-GFP-WPRE vector was ligated to the vector using an In-Fusion HD cloning kit (Clontech) to create the pscAAV-GFP-WPRE vector.
  • the GFP fragment was removed with MLuI and PmLI, and then the I2S fragment (SEQ ID NO: 114) amplified by PCR from the pAAV-CAG-I2S-WPRE vector and the synthesized synthetic polyA fragment (SEQ ID NO: 115) were ligated to the vector using an In-Fusion HD cloning kit (Clontech).
  • the vector was digested with MLuI, and CMV promoter fragment 1 (SEQ ID NO: 116) and fragment 2 (SEQ ID NO: 117) synthesized by PCR from pAAV-CMV-GFP-WPRE were ligated to the vector using an In-Fusion HD cloning kit (Clontech) to create pscAAV-shortCMV-I2S.
  • psc-AAV-shortCMV-I2S was digested with MLuI to remove the CMV sequence.
  • the CBh promoter (SEQ ID NO: 121) was ligated to the vector using the In-Fusion HD cloning kit (Clontech) to create pscAAV-CBh-I2S.
  • pscAAV-CBh-I2S was digested with MLuI and BglII, and the synthesized ASPA fragment (SEQ ID NO: 122) was ligated to the vector using the In-Fusion HD cloning kit (Clontech) to create pscAAV-CBh-ASPA.
  • pAAV-CAG-ASPA-WPRE pAAV-CAG-I2S-WPRE prepared in Example 20 was digested with MLuI and NotI to remove the I2S sequence.
  • the synthesized ASPA fragment (SEQ ID NO: 123) was ligated with the vector using the In-Fusion HD cloning kit (Clontech) to create pAAV-CAG-ASPA-WPRE.
  • the rAAV particles shown in Table 18 were each administered at a dose of 1 x 10 vg/kg to demyelinating disease model mice (13-week-old, male and female) via tail vein injection.
  • -AAV9-WT was also administered at a dose of 1 x 10 vg/kg to demyelinating disease model mice (13-week-old, male and female) via tail vein injection.
  • mice Four and eight weeks after administration, grip strength tests and rotarod tests were performed to examine the motor function of the mice.
  • the grip strength test was performed using the method described in Example 41, and the rotarod test was performed using the method described in Example 42.
  • Eight weeks after administration the mice were euthanized, perfused whole body with saline, and their brains were removed. The removed brains were subjected to immunohistochemical staining of myelin using the method described in Example 43.
  • mice grip strength test The results of the mouse grip strength test are shown in Figure 22(a). Eight weeks after administration, the mouse grip strength test scores improved with both anti-TfR VHH(CBh-ASPA)-scAAV9 and anti-TfR VHH(CAG-ASPA-WPRE)-AAV9. The results of the rotorad test are shown in Figure 22(b). Eight weeks after administration, the mouse grip strength test scores improved with both anti-TfR VHH(CBh-ASPA)-scAAV9 and anti-TfR VHH(CAG-ASPA-WPRE)-AAV9.
  • rAAV particles that have VHH on their surface in the form of a capsid fusion protein and that encapsulate a DNA fragment incorporating the ASPA gene can be used as a drug to prevent demyelination and/or promote remyelination in demyelinating diseases, and as a drug to suppress decline in motor function or improve motor function in demyelinating diseases.
  • they show that they can be used as a therapeutic agent for Canavan disease, which develops due to genetic abnormalities in ASPA.
  • Example 23 Examination of rAAV particle productivity Two types of rAAV particles, AAV9-WT and anti-TfR VHH(455-460)-AAV9, were produced, and the productivity of these two types was examined. rAAV particle production was performed by the production method using the CellStack culture chamber described in detail in Example 26. Production was performed twice for each type.
  • the capsid contains an N-terminal linker with the amino acid sequence cIgG2a Hinge linker of SEQ ID NO:81, a C-terminal linker with the amino acid sequence cIgG2a Hinge linker, and a VHH with the amino acid sequence shown in SEQ ID NO:9.
  • the amount of rAAV particles contained in the purified rAAV particle product was measured by rAAV genome quantification as described in Example 28. The results are shown in Figure 23.
  • the production amount of anti-TfR VHH(455-460)-AAV9 was approximately 65% of that of AAV9-WT, demonstrating that anti-TfR VHH(455-460)-AAV9 can be produced with higher production efficiency than AAV9-WT.
  • Example 24 Investigation of the stability of rAAV particles Solutions were prepared by diluting purified AAV9-WT and anti-TfR VHH(455-460)-AAV9 to 2.4E+07 to 1.0E+11, respectively. These were allowed to stand at 2 to 8°C for 14 days. The cell infectivity of the solutions immediately after dilution and after 14 days of standing was measured using the method described in Example 29.
  • Example 25 Examination of the quality of rAAV particles The proportion of virus packaging the viral genome among the rAAV particles contained in the purified AAV9-WT and anti-TfR VHH(455-460)-AAV9 products was measured by the method described in Example 39. The results showed that approximately 83% of AAV9-WT and approximately 85% of anti-TfR VHH(455-460)-AAV9 particles were intact rAAV particles packaging the viral genome.
  • the proportion of VHH fusion protein (proportion by molecular number) among the capsid proteins was measured using the CE-SDS method described in Example 40. The results are shown in Figure 25.
  • Lot 1 was produced by the method using a T225 flask described in Example 11, and Lots 2 and 3 were produced by the method using a CellStack culture chamber described in Example 26.
  • the proportion of fusion protein in VP was calculated as follows for VP2: [amount of VP2 and VHH fusion protein / (amount of VP2 and VHH fusion protein + amount of VP2)] x 100(%), and for VP3: [amount of VP3 and VHH fusion protein / (amount of VP3 and VHH fusion protein + amount of VP2)] x 100(%).
  • Example 26 Method for producing rAAV particles using a CellStack culture chamber.
  • FBS was added to Dulbecco's modified Eagle's medium (DMEM) to a final concentration of 10% to prepare a 10% FBS-containing DMEM medium.
  • DMEM Dulbecco's modified Eagle's medium
  • HEK293T cells a cell line derived from human embryonic kidney cells expressing the SV40 virus large T antigen gene, were seeded in a CellStack culture chamber (cell culture surface treatment, 10 chambers, Corning) at a density of 6-8E4/cm2 in the 10% FBS-containing DMEM medium. The cells were cultured at 37°C in the presence of 5% CO2 and used for transfection the following day.
  • anti-TfR VHH(455-460)-AAV9 a solution containing a mixed vector was prepared by mixing anti-TfR VHH(455-460)-AAV9 and pR2C9, with anti-TfR VHH(455-460)-AAV9 accounting for 5% of the total. This was the solution containing the mixed vector.
  • AAV9-WT a solution containing only pR2C9 was used instead of the solution containing the mixed vector.
  • pHelper (mod) plasmid (Takara Bio Inc.), mixed vector, pAAV-CMV-GFP, and polyethyleneimine (PEI MAX-Transfection Grade Linear Polyethyleneimine Hydrochloride (MW 40,000)) were added to DMEM medium in a weight ratio of approximately 10:6:5:42, mixed, and left to stand at room temperature for 15 to 30 minutes to form the transfection reagent.
  • the medium was completely removed from the culture vessel, and 2% FBS-containing DMEM medium supplemented with valproic acid was added, followed by the addition of a transfection reagent (0.35 ⁇ g of vector per 1 cm2 of culture area).
  • the cells were cultured at 37°C in the presence of 5% CO2 for 3 to 4 days.
  • the AAV produced in the CellStack culture chamber was purified as follows. After adding one-ninth the volume of cell lysis buffer to the cell culture medium, benzonase (Merck) was added to a concentration of 10 U/mL and mixed to uniformly disperse the solution in the cell stack. The mixture was then left to stand at room temperature for 30 minutes. The solution was then transferred from the cell stack to a bottle and stirred for 2 hours at room temperature using a stirrer and a stirrer. Then, one-ninth the volume of the liquid was added with 10% (w/v) sucrose and 5M NaCl solution and stirred to obtain a cell lysis solution.
  • benzonase benzonase
  • the resulting cell lysate was divided equally into 1000 mL flat-bottom centrifuge vessels and centrifuged at 3000 g, 4°C, and 4°C for 20 minutes in a Sorvall LYNX 6000 large-capacity high-speed refrigerated centrifuge (Thermo Fisher Scientific). The supernatant was filtered using a 0.2 ⁇ m pore size Nalgene Rapid-Flow PES filter unit (Thermo Fisher Scientific) to obtain a cell lysate containing rAAV particles.
  • the column was then washed with 20 mmol/L trishydroxymethylaminomethane buffer (pH 7.5) containing 2 mmol/L MgCl2 at a volume equal to or greater than five column volumes, followed by 20 mmol/L trishydroxymethylaminomethane buffer (pH 7.5) containing 2 mmol/L MgCl2 and 150 mmol/L NaCl at a volume equal to or greater than three column volumes.
  • the rAAV particles adsorbed to the resin were then eluted by passing an elution buffer at a volume equal to or greater than three column volumes through the column, thereby obtaining a fraction containing rAAV particles.
  • Neutralization buffer 500 mM Tris, 2 mM MgCl 2 , pH 8.5
  • the affinity column elution fraction was concentrated to approximately 8 mL by circulating it through Spectrum MicroKros Hollow Fiber Modules 100 kDa, 75 cm (Repligen) equilibrated with 2 mmol/L MgCl2, 150 mmol/L NaCl - containing 20 mmol/L trishydroxymethylaminomethane buffer (pH 7.5) at a flux of 7 L/min/m2 or less, and collected.
  • the ultrafiltration membrane was then washed twice with 2 mL of 2 mmol/L MgCl2 and 150 mmol/L NaCl-containing 20 mmol/L trishydroxymethylaminomethane buffer (pH 7.5), and this washing solution was combined with the previously collected solution to obtain a total of approximately 12 mL of concentrated solution.
  • a 40% iodixanol solution was prepared by adding PBS-MK buffer (20 mmol/L sodium phosphate buffer (pH 7.4 ) containing 136.9 mmol/L sodium chloride, 28.2 mmol/L potassium chloride, and 1 mmol/L magnesium chloride) to 60% iodixanol (multipurpose density gradient centrifugation medium OptiPrepTM, Cosmobio Co., Ltd.).
  • a 25% iodixanol solution was prepared by adding PBS-MK buffer to 60% iodixanol.
  • a 15% iodixanol solution was prepared by adding PBS-MK buffer to 60% iodixanol.
  • PA Quick-Seal TM Round-Top Polypropylene Tube QSPP
  • the concentrate containing rAAV particles was diluted 50-fold with 350 mmol/L NaCl/PBS buffer (20 mmol/L sodium phosphate buffer (pH 7.4) containing 486.9 mmol/L sodium chloride and 2.7 mmol/L potassium chloride, 0.001% F-68) and concentrated to approximately 15 mL by circulating at a flux of 7 L/min/m or less through a Spectrum MicroKros Hollow Fiber Modules 100 kDa, 75 cm2 (Repligen) equilibrated with 350 mmol/L NaCl/PBS buffer.
  • a 10 DV buffer exchange was performed with 350 mmol/L NaCl/PBS buffer.
  • the concentrate was further concentrated to approximately 8 mL by circulating at a flux of 7 L/min/m2 or less.
  • the ultrafiltration membrane was then washed twice with 2 mL of 350 mmol/L NaCl/PBS buffer, and this washing solution was combined with the previously collected solution to obtain a total of approximately 12 mL of concentrated solution.
  • This concentrated solution was further centrifuged using an Amicon Ultra 15 centrifugal filter unit 50 kDa (Merck) at 3200 rpm (2100 ⁇ g) or less for 20 minutes or less, yielding a concentrated solution of rAAV particles in which the solvent had been replaced with 350 mmol/L NaCl/PBS buffer. This was used as a purified rAAV particle product.
  • anti-TfR VHH(455-460)-AAV9 as a model AAV, various variants were created by adding mutations to the Loop-8 region of the capsid protein that constitutes it, and the yield and infectivity of these variants when infected into mice were examined by measuring the expression levels of GFP in the brains and livers of infected mice.
  • Mutations were introduced into the Loop-8 region by performing one-round PCR using primers designed to introduce the desired mutations, using pR2C9 prepared in Example 4 as a template.
  • the mutation sites into the Loop-8 region are shown in Table 19.
  • the name of the plasmid containing these mutations is also shown in Table 19 as Plasmid 2.
  • the positions where point mutations were introduced are indicated by the amino acid numbering from the N-terminus of VP1 of wild-type AAV9.
  • pAAV-CAG-GFP-WPRE prepared in Example 18 was digested with NheI, and a 10-bp DNA fragment (barcode sequence, SEQ ID NOs: 134-143) was inserted into pAAV-CAG-GFP-WPRE.
  • rAAV particles were obtained using the combinations shown in Table 19 of pR2C9 with a mutation introduced into the Loop-8 region and pAAV-CAG-GFP-WPRE with a barcode sequence inserted, as well as pR2C9 (VHH455-460). rAAV particles were produced by the method described in Example 12.
  • Each of the rAAV particles with mutations introduced into the Loop-8 region shown in Table 19 was mixed with anti-TfR VHH(455-460)-AAV9[1] without a mutation in the Loop-8 region at a concentration of 1E13 vg/mL, and administered via the tail vein of one hTfR KI mouse.
  • the amount of rAAV particles was measured using a CFX96 Touch real-time PCR analysis system (BioRad). Forward primers (sequence numbers 144-153) corresponding to each barcode and a common reverse primer (sequence number 154) were used, and experiments were performed in duplicate wells. Powers were calculated using the formula 2 ⁇ (-Ct), and relative values were graphed.
  • Figure 26 shows the yield results. Looking at the yield for each, rAAV particles with mutations introduced into Loop-8 showed increased yields compared to the wild-type type, with the exception of anti-TfR VHH(455-460) ⁇ 594G-AAV9[9]. There was also no significant decrease in yield with anti-TfR VHH(455-460) ⁇ 594G-AAV9[9]. Next, looking at the level of GFP expression in the brain, GFP expression increased with the exception of anti-TfR VHH(455-460) ⁇ 593T-AAV9[19] and anti-TfR VHH(455-460) ⁇ 598N-AAV9[13].
  • anti-TfR VHH(455-460) ⁇ 598N-AAV9[13] did not show a significant decrease in GFP expression (Figure 27).
  • GFP expression in the liver was significantly decreased in anti-TfR VHH(455-460) ⁇ 591A-AAV9[13], anti-TfR VHH(455-460) ⁇ 592Q-AAV9[14], anti-TfR VHH(455-460) ⁇ 593T-AAV9[19], and anti-TfR VHH(455-460) ⁇ 594G-AAV9[9] ( Figure 28).
  • the GFP expression levels in anti-TfR VHH(455-460) ⁇ 591A-AAV9[13] and anti-TfR VHH(455-460) ⁇ 592Q-AAV9[14] were less than 5% of those in the wild type, and the GFP expression level in anti-TfR VHH(455-460) ⁇ 593T-AAV9[19] was less than 1%.
  • anti-TfR VHH(455-460) ⁇ 592Q-AAV9[14] are preferably used.
  • Example 28 Quantification of AAV genome by ddPCR
  • a solution containing rAAV particles was appropriately diluted with TE buffer containing 0.05% F-68 to prepare a sample solution for droplet digital PCR.
  • a HEX-labeled 20x primer/probe mix was prepared containing 1.0 ⁇ M forward primer (primer SI-3, SEQ ID NO: 124), 1.0 ⁇ M reverse primer (primer SI-4, SEQ ID NO: 125), and 0.25 ⁇ M probe (probe SI-2, SEQ ID NO: 126, modified at the 5' end with HEX as a reporter dye and at the 3' end with BHQ1 as a quencher dye).
  • a PCR reaction mixture was prepared by adding 12 ⁇ L of ddPCR Supermix for probes (no dUTP) (BioRad), 1.2 ⁇ L of HEX-labeled 20x primer/probe mix, and 6.8 ⁇ L of water to 4 ⁇ L of droplet digital PCR sample solution.
  • a droplet (droplet) was prepared by suspending 20 ⁇ L of PCR reaction mixture and 70 ⁇ L of Droplet Generator Oil for Probes (BioRad) using a droplet generator (BioRad). This droplet was then used in a QX200 Droplet Digital PCR System (BioRad).
  • PCR conditions were a denaturation reaction (95°C, 10 minutes), 40 cycles of three-step PCR (95°C, 30 seconds ⁇ 60°C, 60 seconds ⁇ 72°C, 15 seconds), and PCR enzyme inactivation treatment (98°C, 10 minutes).
  • HEX-positive droplets were defined as rAAV-positive droplets, and the rAAV genome amount (vg: viral genome) was determined using QuantaSoft Version 1.7 (BioRad).
  • the DNA region amplified in this PCR was the internal region of the ITR.
  • Example 29 Test of cell infectivity of rAAV particles HEK293T cells were seeded onto a 6-well plate at 6-8E4/ cm2 in 10% FBS-containing DMEM medium. The next day, a solution containing rAAV particles diluted with DMEM to an appropriate concentration was added to each well to infect the cells with the rAAV particles. After 3 days, the cells were photographed using a fluorescence microscope to observe intracellular GFP expression. In addition, TrypLE (a porcine trypsin substitute, Thermo Fisher Scientific) was added to the wells to detach the cells, which were then collected and the proportion of cells expressing GFP (AAV positive rate) was measured using a flow cytometer.
  • TrypLE a porcine trypsin substitute, Thermo Fisher Scientific
  • Example 30 Test of infectivity of rAAV particles in mice Purified rAAV particles were intravenously administered to hTfR-KI mice at a dose of 2.5E12 vg/kg or 1E13 vg/kg. Two or four weeks after administration, the mice were euthanized, perfused whole body with saline, and tissues were collected. The excised brains were divided in the sagittal plane, and one aliquot was used to measure the concentration of the transgene product in the tissue, while the other aliquot was used for immunohistochemical staining as described in Example 31.
  • Example 31 Immunohistochemical staining of GFP Immunohistochemical staining of GFP in brain tissue was performed generally according to the following procedure. Brain tissue collected for immunohistochemical staining was immersed in OCT compound (Sakura Finetech Japan Co., Ltd.) and rapidly frozen to -80°C using Histotech Pino (Sakura Finetech Japan Co., Ltd.) to prepare frozen tissue blocks. These frozen blocks were sectioned at 7 ⁇ m and attached to MAS-coated glass slides (Matsunami Glass Co., Ltd.). The slides were immersed in 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd.) at 4°C for 5 minutes to fix the specimens.
  • OCT compound Sekura Finetech Japan Co., Ltd.
  • Histotech Pino Sekura Finetech Japan Co., Ltd.
  • the slides were then immersed in SuperBlock Blocking Buffer (ThermoFisher) for blocking.
  • An appropriately diluted primary antibody was then added dropwise to the tissue slices and allowed to react for 1 hour.
  • Histofine Simple Stain Mouse MAX-PO® was then added dropwise and allowed to react for 1 hour.
  • the tissue slices were then reacted with DAB substrate (3,3'-diaminobenzidine, Vector Laboratories) to develop color. They were then counterstained with Mayer's hematoxylin (Merck), dehydrated, cleared, mounted, and observed under a light microscope.
  • Example 32 Quantification of GFP in Each Tissue Measurement of GFP concentrations in brain tissue, liver, and plasma was generally carried out by the following method. Collected brain tissue and liver were homogenized in RIPA Buffer (Wako Pure Chemical Industries, Ltd.) containing 0.025% PROTEASE Inhibitor Cocktail (Sigma-Aldrich), centrifuged, and the supernatant was collected. Each of these was used as a sample solution for measurement. Rabbit Anti-GFP pAb (MBL) was added to PBS (Sigma-Aldrich) and mixed to prepare a solid-phase antibody solution.
  • the blocking solution was removed from each well, and after washing with 0.05% Tween 20-containing PBS (PBST, Sigma-Aldrich), 25 ⁇ L of the standard sample solution and the specimen were added to each well and shaken for at least 1 hour.
  • Mouse-Anti-GFP mAb (Novus) was added to Superblock Blocking Buffer in PBS (Thermo Fisher Scientific) and mixed to prepare antibody reaction solution 1.
  • the standard sample solution and specimen were then removed, washed with PBST, and antibody reaction solution 1 was added and shaken for at least 1 hour.
  • SULFO-anti-mouse IgG (Mesoscale) was added to Superblock Blocking Buffer in PBS (Thermo Fisher Scientific) and mixed to prepare antibody reaction solution 2.
  • antibody reaction solution 1 was removed, and after washing with PBST, antibody reaction solution 2 was added and allowed to react for at least 1 hour.
  • antibody reaction solution 2 was removed, and after washing with PBST, 150 ⁇ L of 4 ⁇ Read Buffer (Mesoscale diagnostics) diluted with an equal volume of water for injection (Otsuka Pharmaceutical Factory) was added, and the amount of luminescence from each well was measured using a Sector TM Imager 6000 (Mesoscale diagnostics).
  • a calibration curve was created from the measured values of the standard samples for each expressed protein calibration curve, and the amount of expressed protein contained in each sample solution was calculated by interpolating the measured values of each sample solution.Furthermore, the amount of each expressed protein contained per gram of brain tissue and liver, or per mL of plasma, was calculated from the amount of each expressed protein contained in each sample solution.
  • Lyso-GM1 is a substrate for ⁇ -galactosidase.
  • the solutions (a) to (i) used in the test were prepared according to the following procedure.
  • (a) Mobile phase A Mobile phase A was prepared by mixing 998 mL of ultrapure water and 2.0 mL of formic acid (Fujifilm Wako Pure Chemical Industries, Ltd.).
  • Mobile phase B was a mixture of 998 mL of acetonitrile (Fujifilm Wako Pure Chemical Industries, Ltd.) and 2.0 mL of formic acid (Fujifilm Wako Pure Chemical Industries, Ltd.).
  • Standard stock solution 0.5 mg of Lyso-Monosialoganglioside GM1 (ammonium salt) (Matreya LLC) was dissolved in 0.5 mL of methanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a solution containing 1.0 mg/mL of Lyso-GM1.
  • Equal volumes (50 ⁇ L) of the solutions containing Lyso-GM1 at concentrations of 0.06, 0.02, 0.6, 2.0, and 6.0 ng/mL were mixed with IS-1 to prepare solutions containing 0.03, 0.1, 0.3, 1.0, 3.0, and 10 ng/mL of Lyso-GM1 and 5 ng/mL of N-glycinated lyso-ceramide trihexoside, respectively. 20 ⁇ L of this solution was measured and filled into an LC vial. This solution was used as a calibration curve sample (STD-IS).
  • a solution containing 16 ng/mL of Lyso-GM1 was prepared by measuring out 920 ⁇ L of methanol and adding 80 ⁇ L of a solution containing 200 ng/mL of Lyso-GM1. Equal volumes (50 ⁇ L) of solutions containing 0.06, 0.2, 2.0, and 16.0 ng/mL of Lyso-GM1 were mixed with IS-1 to prepare solutions containing 0.03, 0.1, 1.0, and 8.0 ng/mL of Lyso-GM1 and 5 ng/mL of N-glycinated lyso-ceramide trihexoside. 20 ⁇ L of this solution was measured and filled into an LC vial. This solution was used as the QC standard sample (QC-IS).
  • the extracted brain tissue was freeze-dried and the dry weight was measured.
  • the freeze-dried tissue was crushed in water for injection (Otsuka Pharmaceutical). 20 ⁇ L of this was measured and mixed with 390 ⁇ L of water for injection, diluting the crushed tissue 40-fold. This was used as the sample solution.
  • LC/MS/MS analysis was performed using a combination of hydrophilic interaction ultra-high performance liquid chromatography and a tandem quadrupole mass spectrometer.
  • the mass spectrometer (MS/MS device) used was a QTRAP5500 (AB Sciex), equipped with a Nexera X2 (Shimadzu) HPLC device.
  • the LC column used was a Cadenza CW-C18 [3.0 ⁇ m (2.0 x 150 mm, Intact Co., Ltd.)], and the guard cartridge was a CW-C18 (5 mm x 2 mm, Intact Co., Ltd.).
  • Mobile phases A and B were used.
  • the column temperature was set to 40°C.
  • the area of the Lyso-GM1 detection peak in each calibration curve sample relative to the detection peak area derived from the internal standard solution was plotted on the vertical axis, and the Lyso-GM1 concentration of each calibration curve sample was plotted on the horizontal axis.
  • a regression equation was obtained using quadratic discriminant analysis. LC/MS/MS analysis was performed on the brain tissue sample solution, and the Lyso-GM1 contained in the sample solution was quantified by interpolating it into the regression equation.
  • the area of the Lyso-GM1 detection peak in each calibration curve sample relative to the detection peak area derived from the internal standard solution was plotted on the vertical axis, and the Lyso-GM1 concentration of each calibration curve sample was plotted on the horizontal axis.
  • a regression equation was obtained using quadratic discriminant analysis. LC/MS/MS analysis was performed on the brain tissue sample solution, and the Lyso-GM1 contained in the sample solution was quantified by interpolating it into the regression equation.
  • Example 34 Quantification of Human Iduronate-2-Sulfatase (I2S) in Each Tissue Measurement of the hI2S protein concentration in brain tissue, liver, and plasma was generally carried out by the following method: Collected brain tissue and liver were homogenized in RIPA Buffer (Wako Pure Chemical Industries, Ltd.) containing 0.025% Protease Inhibitor Cocktail (Sigma-Aldrich), centrifuged, and the supernatants were collected and used as sample solutions for measurement. 150 ⁇ L of Superblock Blocking Buffer in PBS (Thermo Fisher Scientific) was added to each well of a Streptavidin Gold plate (Mesoscale diagnostics), and the plate was blocked by shaking for at least 1 hour.
  • hI2S standard sample a standard sample containing a known concentration of hI2S (hI2S standard sample) was added to the mixed solution of biotinylated anti-hI2S monoclonal antibody and sulfo-anti-hI2S monoclonal antibody, and the mixture was shaken for at least 1 hour to prepare an antibody reaction solution.
  • the anti-hI2S monoclonal antibody was obtained by culturing an anti-hI2S-producing hybridoma prepared by a standard method using splenocytes obtained from a mouse immunized with human iduronate-2-sulfatase.
  • One of the obtained monoclonal antibodies was modified using Biotin Labeling Kit-NH 2 (Dojindo Laboratories) according to the attached protocol to obtain a biotinylated anti-hI2S monoclonal antibody.
  • Another of the obtained monoclonal antibodies was modified using MSD GOLD SULFO-TAG NHS-Ester (Mesoscale Diagnostics) according to the attached protocol to obtain a sulfo-anti-hI2S monoclonal antibody.
  • the blocking solution was removed from each well, and the wells were washed with 0.05% Tween 20-containing PBS (PBST, Sigma-Aldrich). 25 ⁇ L of antibody reaction solution was then added to each well, and the wells were shaken for at least 1 hour.
  • the antibody reaction solution was then removed, and the wells were washed with PBST. Then, 150 ⁇ L of 4x Read Buffer (Meso Scale Diagnostics) diluted with an equal volume of water for injection (Otsuka Pharmaceutical Factory) was added, and the luminescence intensity from each well was measured using a Sector TM Imager 6000 (Meso Scale Diagnostics).
  • a calibration curve was created from the measured values of the standard samples for each expressed protein calibration curve, and the amount of expressed protein contained in each sample solution was calculated by interpolating the measured values of each sample solution onto this curve. Furthermore, the amount of each expressed protein contained in 1 g of brain tissue and liver, or 1 mL of plasma was calculated from the amount of each expressed protein contained in each sample solution. Note that hI2S contained in the standard sample for the calibration curve was obtained by expressing it in CHO cells using a conventional method.
  • Example 35 Quantification of ⁇ -galactosidase (GLB1) in Each Tissue GLB1 was determined generally by the following method: Collected brain tissue and liver were homogenized in RIPA Buffer (Wako Pure Chemical Industries, Ltd.) containing 0.025% Protease Inhibitor Cocktail (Sigma-Aldrich), centrifuged, and the supernatant was collected. These were then used as sample solutions for measurement. 150 ⁇ L of Superblock Blocking Buffer in PBS (Thermo Fisher Scientific) was added to each well of a Streptavidin Gold plate (Mesoscale diagnostics), and the plate was blocked by shaking for at least 1 hour.
  • biotinylated anti-GLB1 monoclonal antibody was added and immobilized for at least 1 hour.
  • a standard sample for a calibration curve containing known concentrations of hI2S standard sample for hI2S calibration curve
  • Rabbit anti-GLB1 polyclonal antibody was added and allowed to react for at least 1 hour.
  • SULFO-TAG Anti-Rabbit Antibody was added and allowed to react for at least 1 hour.
  • the antibody reaction solution was then removed, washed with PBST, and 150 ⁇ L of 4x Read Buffer (Meso Scale Diagnostics) diluted with an equal volume of water for injection (Otsuka Pharmaceutical Factory) was added.
  • the luminescence intensity from each well was measured using a Sector TM Imager 6000 (Meso Scale Diagnostics).
  • a calibration curve was created from the measured values of the standard samples for each expressed protein calibration curve, and the amount of expressed protein contained in each sample solution was calculated by interpolating the measured values of each sample solution. Furthermore, the amount of expressed protein contained per gram of brain tissue and liver, or per mL of plasma, was calculated from the amount of each expressed protein contained in each sample solution.
  • the GLB1 contained in the standard sample for the calibration curve was obtained by expressing it in CHO cells using a conventional method.
  • IDS-KO/hTfR-KI mice are mice that are hemi-deficient in the iduronate-2-sulfatase (IDS) gene and heterozygous for the chimeric TfR gene.
  • IDS-KO/hTfR-KI mice were generated generally by the following method. A DNA fragment was chemically synthesized containing a neomycin resistance gene flanked by loxP sequences at the 3' end of a cDNA encoding a chimeric TfR whose intracellular domain is the amino acid sequence of mouse TfR and whose extracellular domain is the amino acid sequence of human TfR.
  • This DNA fragment was incorporated into a targeting vector having 5' and 3' arm sequences by standard methods, and then introduced into mouse ES cells by electroporation. After gene introduction, the mouse ES cells were selectively cultured in the presence of neomycin, and mouse ES cells in which the targeting vector had been integrated into the chromosome by homologous recombination were selected. The resulting genetically modified mouse ES cells were injected into 8-cell embryos (host embryos) of ICR mice and transplanted into pseudopregnant mice (recipient mice) obtained by mating with vasoligated mice.
  • mice The resulting offspring (chimeric mice) were assessed for coat color, and individuals in which the ES cells contributed highly efficiently to the formation of the organism, i.e., individuals with a high proportion of white hair relative to their total hair, were selected. These chimeric mice were crossed with ICR mice to obtain F1 mice. White F1 mice were selected, and DNA extracted from their tail tissue was analyzed. Mice in which the mouse hTfR gene had been replaced on the chromosome with the chimeric TfR were designated hTfR-KI mice. Based on these mice, mice with a hemi-deficient IDS gene and a heterozygous chimeric TfR gene (IDS-KO/hTfR-KI mice) were generated. The IIDS-KO/hTfR-KI mice were produced in accordance with the method described in patent document (WO2016/208695).
  • Example 37 Preparation of GM-1 gangliosidosis model mice GM-1 gangliosidosis model mice (GLB1-KO/hTfR-KI) were prepared according to the method for preparing IDS-KO/hTfR-KI mice described in Example 36.
  • Example 38 Generation of a demyelinating disease mouse model Cas9 gRNA was designed upstream of Exon 2 and downstream of Exon 4 of the mouse ASPA locus. Cas9 and gRNA were injected into fertilized eggs of C57BL/6J mice. DNA was extracted from the tail tissue of the resulting offspring, and DNA fragments were amplified by PCR using primers designed outside the predicted deletion region. The DNA was confirmed by band size and sequencing via electrophoresis to obtain heterozygous knockout mice. ASPA homozygous knockout mice were obtained by mating the heterozygous knockout mice.
  • Example 39 Measurement of the proportion of intact AAV virus
  • a 1 x 10 to 1 x 10 vg/mL rAAV particle solution was prepared in 180 mmol/L NaCl/PBS buffer (180 mmol/L sodium chloride and 2.7 mmol/L potassium chloride, 8.1 mmol /L disodium hydrogen phosphate, 1.5 mmol/L potassium dihydrogen phosphate (pH 7.4) containing 0.001% F- 68 ).
  • 2 ⁇ L of the sample was loaded into duplicate wells of a Stunner plate.
  • 2 ⁇ L of 180 mmol/L NaCl/PBS buffer was loaded into a Stunner (Unchained Labs) plate as a blank.
  • UV/Vis and DLS were measured.
  • DLS was acquired four times at 5-second intervals. The average values of the measurement results (average particle size, particle number, target substance content, and aggregate content) from the two wells were calculated.
  • Example 40 CE-SDS Method: 10 ⁇ L of rAAV particle solution was transferred, followed by the addition of 10 ⁇ L of SDS sample buffer and 2 ⁇ L of 1 M DTT solution, and incubation at 70°C for 10 minutes. 2 ⁇ L of Chromeo P503 solution was then added, and incubation at 60°C for 20 minutes. 26 ⁇ L of ultrapure water was added, and the mixture was transferred to a nanovial to serve as the electrophoresis sample. According to the buffer tray configuration, the appropriate number of vials containing SDS-MW Gel Buffer, 0.1 M NaOH, and 0.1 M HCl were placed in the tray.
  • Inlet buffer tray A-1 contained 1.5 mL of ultrapure water
  • B-1 contained 1.2 mL of SDS-MW Gel Buffer
  • C-1 contained 1.1 mL of SDS-MW Gel Buffer
  • D-1 contained 1.5 mL of 0.1 M NaOH
  • E-1 contained 1.5 mL of 0.1 M HCl
  • F-1 contained 1.5 mL of ultrapure water
  • A-4, B-4, and C-4 contained 1.5 mL of ultrapure water.
  • Outlet buffer tray A-1 contained 1.5 mL of ultrapure water
  • B-1 contained 1.0 mL of ultrapure water
  • C-1 contained 1.1 mL of SDS-MW Gel Buffer
  • D-1, E-1, and F-1 contained 1.0 mL of ultrapure water
  • B-4 contained 1.5 mL of ultrapure water.
  • the Dynamic Range of Electrogram Channel 1 was set to 100 RFU, Filter Settings to Normal, Signal to Direct, Laser / filter description - information only Excitation wavelength: 488 nm, Emission wavelength: 600 nm, Data rate Both channels: 4 Hz.
  • the conditioning method and separation method were executed. After analysis of all samples was completed, the shutdown method was executed. Tables 23 to 25 show the conditions for each method.
  • Example 41 Measurement of mouse grip strength Measurements were performed using a smart grip strength measurement device for rats and mice (Muromachi Kikai MK-380CM/F). Mice were made to grasp the grip and their tails were pulled with their bodies held horizontally to measure grip strength. Measurements were taken three times for each individual at 12 weeks (before AAV administration), 17 weeks (4 weeks after AAV administration), and 21 weeks (8 weeks after AAV administration).
  • Example 42 Rotarod Test in Mice This test was performed using a mouse rotarod (Muromachi Kikai MK-610A). On the first day, mice were allowed to acclimate to the machine. For the acclimatization method, mice were placed on the rotating rod at 4 RPM for 2 minutes, with a 10-minute rest period between each. Mice that jumped off during the measurement were placed on the rod again after the timer was stopped. Mice that were unable to learn and jumped off the rod during the second acclimatization were excluded from the measurement. On the day of measurement, mice were allowed to acclimate for 1 minute, and then the speed was accelerated from 4 RPM to 40 RPM over 5 minutes.
  • mice were allowed to acclimate for 1 minute, and then the speed was accelerated from 4 RPM to 40 RPM over 5 minutes.
  • mice Even if no mice dropped off, the 5-minute point was considered the end time. Three trials were performed with a 15-minute rest period between each trial. This test was performed on two consecutive days, and the average of six trials was obtained. Each mouse was measured three times at 12 weeks (before AAV administration), 17 weeks (4 weeks after AAV administration), and 21 weeks (8 weeks after AAV administration).
  • Example 43 Immunohistochemical staining of myelin The procedure was generally as follows. Brain tissue collected for immunohistochemical staining was immersed in a 30% sucrose solution and rapidly frozen to -80°C using a Histotech Pino (Sakura Finetech Japan Co., Ltd.) to prepare frozen tissue blocks. These frozen blocks were sectioned at 7 ⁇ m and attached to MAS-coated glass slides (Matsunami Glass Co., Ltd.). The slides were immersed in 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd.) at 4°C for 5 minutes to fix the specimens. After washing with PBST, the slides were immersed in SuperBlock Blocking Buffer (ThermoFisher) for blocking.
  • SuperBlock Blocking Buffer ThermoFisher
  • a primary antibody (NeuN: 1:200; Millipore; #MAB377) diluted in Can Get Signal Immunostain Solution A (Toyobo) was added dropwise to the tissue slices and incubated for 1 hour.
  • a secondary antibody (1:500; Invitrogen; Alexa Fluor TM 488 #ab150113) diluted in Can Get Signal Immunostain Solution A was added dropwise and incubated for 1 hour.
  • a solution of FluoroMyelin TM Red (1:300 Thermo Fisher Scientific; #F34652) diluted with SuperBlock Blocking Buffer was added dropwise and allowed to react for 30 minutes.
  • Example 44 Evaluation of the binding activity of VHHs with affinity for human TfR to human TfR.
  • the binding activity of VHHs with affinity for human TfR to human TfR can be measured using the OctetRED96 (ForteBio, a division of Pall Corporation), a biomolecular interaction analysis system that uses biolayer interferometry (BLI).
  • BBI biolayer interferometry
  • the thickness of the layer at the tip of the sensor increases, resulting in a wavelength shift in the interference wave.
  • the number of molecules bound to the biomolecules immobilized on the sensor chip surface can be quantified and analyzed kinetically in real time. Measurements are generally performed according to the operating manual provided with the OctetRED 96.
  • the human TfR used is a recombinant hTfR (rhTfR, Sino Biological Co., Ltd.) with an N-terminal histidine tag and the amino acid sequence of the extracellular domain of hTfR, spanning from the 89th cysteine residue from the N-terminus to the phenylalanine residue at the C-terminus, as shown in SEQ ID NO: 4.
  • Purified VHHs with affinity for human TfR are diluted with HBS-P+ (10 mM HEPES containing 150 mM NaCl, 1% BSA, 50 ⁇ M EDTA, and 0.05% Surfactant P20) to prepare solutions with three concentrations ranging from 10 to 40 nM. These solutions are used as sample solutions.
  • rhTfR is diluted with HBS-P+ to prepare a 15 ⁇ g/mL solution, which is used as the hTfR-ECD (HisTag) solution.
  • the above sample solution was dispensed into a 96-well black plate (Greiner Bio-One) at 200 ⁇ L/well.
  • the prepared hTfR-ECD (HisTag) solution was dispensed into the designated wells at 200 ⁇ L/well.
  • HBS-P+ was dispensed into the baseline, dissociation, and washing wells at 200 ⁇ L/well.
  • 10 mM Glycine-HCl (pH 1.7) was dispensed into the regeneration wells at 200 ⁇ L/well.
  • 0.5 mM NiCl2 solution containing 1% BSA was dispensed into the activation wells at 200 ⁇ L/well.
  • This plate and a biosensor Biosensor/Ni-NTA: ForteBio, a division of Pall Corporation
  • the analysis software included with OctetRED96 is used to fit the binding reaction curve to a 1:1 or 2:1 binding model, measure the association rate constant (k on ) and dissociation rate constant (k off ) for each hTfR affinity peptide with hTfR, monkey TfR, and mouse TfR, and calculate the dissociation constant (K D ). Measurements are performed at a temperature of 25-30°C.
  • Example 45 Quantification of AAV Genome by Real-Time PCR
  • a solution containing rAAV particles was appropriately diluted with TE buffer containing 0.05% F-68 to prepare a sample solution.
  • a HEX-labeled 20x primer/probe mix was prepared containing 1.0 ⁇ M forward primer (primer SI-3, SEQ ID NO: 124), 1.0 ⁇ M reverse primer (primer SI-4, SEQ ID NO: 125), and 0.25 ⁇ M probe (probe SI-2, SEQ ID NO: 126 modified at the 5' end with HEX as a reporter dye and at the 3' end with BHQ1 as a quencher dye).
  • the amount of rAAV particles was measured using a CFX96 Touch real-time PCR analysis system (BioRad). Reverse primers (sequences A16 to A18 (SEQ ID NOs: 184 to 186)) corresponding to no insertion at 455-460, VHH455 Hinge-Hinge insertion, and FMDV peptide insertion, respectively, and a common forward primer (sequence A19 (SEQ ID NO: 187)) were used, and assays were performed in duplicate wells for each.
  • a standard curve was prepared by simultaneously measuring serial dilutions of pR2C9, pR2C9 (VHH455 Hinge-Hinge), and pR2C9 (FMDV peptide 455-460) plasmids, and the relative values were graphed.
  • Example 47 Cell infectivity test of rAAV particles with VHH or peptide added only to VP3
  • the VP1 start codon ATG of pR2C9 (VHH455-460) was changed to CTG, and the VP2 start codon ACG to GCG to prevent the expression of VP1 and VP2.
  • the construct is shown in Figure 30.
  • pHelper (mod) plasmid (Takara Bio Inc.), mixed vector, pAAV-CMV-GFP vector, and polyethyleneimine (PEI MAX-Transfection Grade Linear Polyethyleneimine Hydrochloride (MW 40,000)) were added to DMEM medium in a weight ratio of approximately 10:6:5:42, mixed, and allowed to stand at room temperature for 15 to 30 minutes to prepare a transfection reagent. After removing all of the medium from the culture vessel, 2% FBS-containing DMEM medium supplemented with valproic acid was added to the culture vessel, and the transfection reagent was then added to introduce the vector into the cells. The cells after vector introduction were cultured at 37°C in the presence of 5% CO2 for 3 days. Thereafter, a solution containing rAAV particles was obtained using a method similar to the purification method described in Example 11.
  • HEK293T cells were seeded onto a 96-well plate at a density of 6-8E4/ cm2 in 10% FBS-containing DMEM medium. The next day, a solution containing rAAV particles, diluted with DMEM to an appropriate concentration, was added to each well to infect the cells with the rAAV particles. Three days later, TrypLE (a porcine trypsin substitute, Thermo Fisher Scientific) was added to the wells to detach the cells, which were then collected and the proportion of cells expressing GFP (AAV-positive rate) was measured using a flow cytometer.
  • TrypLE a porcine trypsin substitute, Thermo Fisher Scientific
  • rAAV particles were produced in which VHH was added only to VP3, and the yield was measured. The yield decreased as the ratio of VHH-added plasmid increased. The results are shown in Figure 31.
  • Cultured HEK293T cells were infected with each virus at concentrations of 2.5 x 10 vg/mL, 1.0 x 11 vg/mL, and 4.0 x 11 vg/mL, and the positive rate was measured using a flow cytometer. The positive rate increased up to 10%, but plateaued thereafter and decreased at 50%. The results are shown in Figure 33.
  • Example 48 Mutation Introduction into Loop4 or Loop8 Using anti-TfR VHH(455-460)-AAV9 as a model AAV, various AAVs were prepared by introducing mutations into the Loop-8 region of the capsid protein that constitutes the model AAV, and the yield and infectivity of these AAVs when infected into mice were examined by measuring the expression levels of GFP in the brains and livers of infected mice. The examination was performed in accordance with the method described in Example 27.
  • deletion mutations at 591, 592, 590-591, and 591-592 significantly reduced liver infection. Deletion of 591-592 slightly reduced brain infection, but the other mutations increased brain infection. The results are shown in Figure 41.
  • Example 49 Introduction of Mutations into the Loop Region of AAV5 or AAV8
  • Various AAV5 and AAV8 variants were prepared by introducing mutations into the Loop-4 or Loop-8 region of the capsid protein that constitutes these AAVs, and the yield and infectivity of these variants when infected into mice were examined by measuring the expression levels of GFP in the brains and livers of infected mice.
  • A1 (Construction of the vector used)
  • the pR2C5 used to prepare AAV5 was purchased from Vigene and had the following sequence A1 (SEQ ID NO: 174).
  • pR2C5 (Construction of pR2C5(VHH446) vector)
  • pR2C5 (VHH446) was prepared by the following method.
  • the resulting vector encodes a fusion protein lacking amino acid sequence 446 of the VP1 amino acid sequence (sequence A5 (SEQ ID NO: 178)) encoded in the Cap region, and instead containing the amino acid sequence of a VHH to which a linker has been added (linker-added VHH).
  • This vector was designated pR2C5 (VHH446). Using these vectors, solutions containing rAAV particles were obtained according to the method described in Example 11.
  • Example 50 Mouse Testing of Mutated AAV5 and AAV8 Using pR2C5(VHH446) prepared in Example 48, the efficiency of infection of mutated particles in the brain and liver was examined. The study was performed using a solution containing a mixed vector prepared by mixing pR2C5(VHH446) with pR2C5 or pR2C5 with mutations introduced into the Loop-4 or Loop-8 regions, with the proportion (molar ratio) of pR2C5(VHH446) at 5% of the total. The combinations of plasmids used to prepare rAAV particles are shown in Table 25. rAAV particles were prepared according to the method described in Example 11.
  • the obtained rAAV particles were administered to hTfRKI mice (31-week-old, male) at a dose of 1 x 10 13 vg/kg. Two weeks after administration, the mice were whole-body perfused with saline, and the brain and liver were isolated. GFP in the brain tissue was quantified by the method described in Example 32. The amount of rAAV particles in each tissue was quantified by the method described in Example 45. GFP mRNA was quantified by the same method as in Example 27. Similar studies were also performed using pR2C8 (VHH456-462) and pR2C8 or pR2C8 with mutations introduced into the Loop-4 or Loop-8 regions. The results confirmed that AAV5 with the ⁇ P580 or ⁇ A581 mutation, and AAV8 with the ⁇ N498, ⁇ P593, or ⁇ Q594 mutation introduced suppressed liver infection and increased brain infection.
  • Example 51 Liver-Avoiding Effect of ⁇ 498, ⁇ 497, 499, 504, ⁇ 496, 502 in Loop-5 Following the procedures of Examples 48 and 49, a similar experiment was carried out for Loop-5.
  • deletion mutations of 498 and 503 belonging to Loop-5 were found to significantly reduce infection in the liver. Of these, deletion mutations of 498 did not reduce infection in the brain.
  • deletion mutations of 500, 501, 505, and 506 belonging to Loop-5 significantly reduced infection in the liver.
  • mutations 500 and 505 reduced infection in the brain relatively little.
  • deletion mutations of 591, 592, 590-591, and 591-592 which are different types of two-residue deletions in Loop-8, significantly reduced liver infection. Deletion of 591-592 slightly reduced brain infection, while the other deletions increased brain infection. The results are shown in Figure 41.
  • Example 53 Example of function with FMDV (construction of pR2C9 (FMDV peptide 455-460) vector) pR2C9 (FMDV peptide455-460):
  • a DNA fragment was obtained by PCR using pR2C9 prepared in Example 2, primer 11 (sequence number 127), and primer 12 (sequence number 128). This DNA fragment was ligated to a DNA fragment (sequence number A6) encoding the synthesized FMDV peptide using an In-Fusion HD cloning kit (Clontech). The resulting vector encodes a fusion protein lacking amino acid sequences 455-460 of the VP1 sequence encoded in the Cap region, and instead containing the amino acid sequence of the FMDV peptide. This vector was designated pR2C9 (FMDV peptide 455-460). The amino acid sequence of the FMDV peptide is shown in sequence number A7.
  • Virus production Using the vector thus obtained, a solution containing rAAV particles was obtained by a method similar to that of Example 11, except that the ratio of pR2C9 (FMDV peptide 455-460) was set to 10%.
  • mice were administered a peptide sequence known to bind to Integrin ⁇ V ⁇ 5 (excluding the IGF2 sequence) inserted at position 455, and infection of the heart and quadriceps was evaluated.
  • MyoAAV2a is a modified serotype reported in the Broad Institute literature. Improved infection in muscle tissue was observed with the FMDV peptide and MyoAAV2a. The results are shown in Figure 42-1.
  • Example 54 An example of the muscle transfer and liver escape effects of the combination of FMDV and ⁇ 592 The experimental procedure was almost the same as in Example 53, but with mutations introduced.
  • pR2C9 (FMDV peptide 455-460) vector)
  • pR2C9 (FMDV peptide455-460): PCR was performed using pR2C9 prepared in Example 2 with primer 11 (SEQ ID NO: 127) and primer 12 (SEQ ID NO: 128) to obtain a DNA fragment. This DNA fragment was ligated with a DNA fragment (sequence A6 (SEQ ID NO: 179)) encoding the synthesized FMDV peptide 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 by the Cap region, and instead containing the amino acid sequence of the FMDV peptide.
  • This vector was designated pR2C9 (FMDV peptide 455-460).
  • the amino acid sequence of the FMDV peptide is represented by sequence A7 (SEQ ID NO: 180).
  • one-round PCR was performed according to the method described in Example 14 to obtain each vector.
  • a solution containing rAAV particles was obtained by a method similar to that of Example 11, except that the ratio of pR2C9 (FMDV peptide 455-460) was set to 10%.
  • ⁇ 591 and ⁇ 592 were found to be preferable (FIGS. 43-44).
  • Example 55 Example of function with another ligand, POD (construction of pR2C9 (POD 455-460)- ⁇ VP1/VP2 vector) PCR was performed using pR2C9 (VHH455 Hinge-Hinge)- ⁇ VP1/VP2 and primers (sequence A13 (SEQ ID NO: 181)) and (sequence A14 (SEQ ID NO: 182)) to obtain a DNA fragment.
  • This DNA fragment was ligated to a sequence containing the synthesized POD sequence (sequence A15 (SEQ ID NO: 183)) using an In-Fusion HD cloning kit (Clontech).
  • the viral vector was a solution containing rAAV particles, prepared in a manner similar to that described in Example 11, using the plasmid obtained in Example 11.
  • Example 56 4R15VHH-AAV9-CAG monkey test pAAV9-CAG-mCherry-WPRE: pAAV-CAG-GFP-WPRE was digested with Mlu1 and Not1 to remove the GFP-containing fragment, and a synthesized fragment containing the mCherry sequence was ligated using an In-Fusion HD cloning kit (Clontech). The resulting plasmid was named pAAV-CAG-mCherry-WPRE.
  • rAAV particles were produced using the same method as in Example 11. However, pR2C9 (VHH455-460) was added at a ratio of 5%, and the AAV genome plasmids used were pAAV-CAG-GFP-WPRE and pAAV-CAG-mCherry-WPRE.
  • Cynomolgus monkeys were intravenously administered a mixture of Anti-TfR VHH(CAG-GFP-WPRE)-AAV9 and (CAG-mCherry-WPRE)-AAV9 at 5x10 VG/mL each to AN1, and a mixture of (CAG-GFP-WPRE)-AAV9 and Anti-TfR VHH(CAG-mCherry-WPRE)-AAV9 at 5x10 VG/mL each to AN2. Each individual was administered 2mL/kg (1x10 13 vg/kg). After 4 weeks, the monkeys were euthanized, and tissues were collected and cryopreserved. The experimental design is shown in Figure 45.
  • RNA and DNA were extracted from each tissue using AllPrep DNA/RNA Kits (QIAGEN). RNA was reverse transcribed into cDNA using ReverTra Ace TM qPCR RT Master Mix with gDNA Remover (Toyobo). The reverse-transcribed solution was diluted 5-fold with water for injection.
  • 1.0 ⁇ M forward primer (GFP GACAAGCAGAAGAACGGCATC (SEQ ID NO: 189), mCherry CAAGGGCGAGGAGGATAACA (SEQ ID NO: 190)), 1.0 ⁇ M reverse primer (GFP TGGGTGCTCAGGTAGTGGTT (SEQ ID NO: 191), mCherry ACCTTCAGCTTGGCGGTCT (SEQ ID NO: 192)), and 0.25 ⁇ M probe (GFP AACTTCAAGATCCGCCACAACAT (SEQ ID NO: 193), mCherry A HEX-labeled 20x primer/probe mix containing ATCATCAAGGAGTTCATGCGCTT (SEQ ID NO: 194), modified at the end with HEX as a reporter dye and at the 3' end with BHQ1 as a quencher dye, was prepared.
  • PCR conditions were a denaturation reaction (95°C, 10 minutes), 40 cycles of three-step PCR (95°C, 30 seconds ⁇ 60°C, 60 seconds ⁇ 72°C, 15 seconds), and PCR enzyme inactivation treatment (98°C, 10 minutes).
  • HEX-positive droplets were defined as rAAV-positive droplets, and the rAAV genome amount (vg: viral genome) was determined using QuantaSoft Version 1.7 (BioRad).
  • the DNA region amplified in this PCR was the internal region of the ITR.
  • rAAV particles with high infectivity whose capsid contains a fusion protein of a capsid protein and another protein (A)
  • can be produced in good yield making it possible to steadily provide such rAAV particles to the market.
  • SEQ ID NO: 1 Amino acid sequence of VP1 of AAV of serotype 6, wild type SEQ ID NO: 2: Amino acid sequence of VP1 of AAV of serotype 8, wild type SEQ ID NO: 3: Amino acid sequence of VP1 of AAV of serotype 9, wild type SEQ ID NO: 4: Amino acid sequence of human transferrin receptor SEQ ID NO: 5: Amino acid sequence of VHH1 SEQ ID NO: 6: Amino acid sequence of VHH2 SEQ ID NO: 7: Amino acid sequence of VHH3 SEQ ID NO: 8: Amino acid sequence of VHH4 SEQ ID NO: 9: Amino acid sequence of VHH5 SEQ ID NO: 10: Amino acid sequence of VHH6 SEQ ID NO: 11: Amino acid sequence of VHH7 SEQ ID NO: 12: Amino acid sequence of VHH8 SEQ ID NO: 13: Amino acid sequence of VHH9 SEQ ID NO: 14: Amino acid sequence of CDR
  • Sequence number 175 Sequence A2 (base sequence).
  • Sequence number 176 Sequence A3 (base sequence).
  • Sequence number 177 Sequence A4 (base sequence).
  • Sequence number 178 Sequence A5 (amino acid sequence).
  • Sequence number 179 Sequence A6 (base sequence).
  • SEQ ID NO: 180 Sequence A7 (amino acid sequence).
  • SEQ ID NO: 181 Primer 1 of pR2C9 (VHH455 Hinge-Hinge)- ⁇ VP1/VP2
  • SEQ ID NO: 182 Primer 2 of pR2C9 (VHH455 Hinge-Hinge)- ⁇ VP1/VP2
  • SEQ ID NO: 183 Sequence containing the synthesized POD sequence
  • SEQ ID NO: 185 Reverse primer corresponding to VHH455 Hinge-Hinge insertion
  • SEQ ID NO: 191 GFP reverse primer (1.0 ⁇ M)

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Abstract

La présente invention concerne un procédé de production de particules virales adéno-associées recombinées présentant un ligand à leur surface, de particules pseudo-virales, de cellules hôtes et de virions (VP) à faible hépatotoxicité, ledit procédé comportant les étapes suivantes : (A) introduction, dans des cellules hôtes par transfert génique, d'une molécule d'acide nucléique contenant une séquence nucléotidique codant pour une protéine souhaitée, et d'un ou plusieurs types de molécules d'acide nucléique contenant une séquence nucléotidique permettant l'expression de VP1, VP2, VP3 et VP3 modifiées avec le ligand lors du transfert génique ; et (B) soumission des cellules hôtes à des conditions permettant la génération des particules virales adéno-associées recombinées.
PCT/JP2025/016439 2024-05-01 2025-04-30 Particules d'aav modifiées, procédé de production, médicament et production associés Pending WO2025230000A1 (fr)

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