Fusion protein of Anti-transferrin receptor antibody and bioactive protein for safe gene therapy

Abstract

The purpose of the present invention is to provide a nucleic acid molecule into which a gene for encoding a fusion protein of an anti-transferrin receptor (TfR) antibody and a bioactive protein has been incorporated, with which it is possible to reduce side effects such as anemia occurring due to the anti-TfR antibody binding to a transferrin receptor when the gene for encoding the fusion protein of the anti-TfR antibody and the bioactive protein is used in gene therapy, for example, and in which the TfR-binding activity is regulated. Another purpose of the present invention is to provide a virus virion including said nucleic acid molecule.

Description

Fusion proteins of anti-transferrin receptor antibodies and biologically active proteins for safe gene therapy

[0001] The present invention relates to a gene encoding an anti-transferrin receptor antibody, and more specifically, to a gene encoding an anti-transferrin receptor antibody that is used, for example, in an AAV vector system, when creating a vector into which a nucleic acid molecule encoding a fusion protein of an anti-transferrin receptor antibody and a protein having physiological activity has been incorporated.

[0002] Many recombinant proteins used as medicines are administered to patients by parenteral means such as subcutaneous injection, intramuscular injection, intravenous injection, etc. When a patient has a chronic disease, repeated administration of medicines over a long period of time is necessary, and parenteral administration imposes a heavy burden on the patient.

[0003] Some recombinant proteins are fusion proteins of antibodies and biologically active proteins, such as fusion proteins of antibodies and lysosomal enzymes (Patent Documents 1 to 5, Non-Patent Document 1).

[0004] Lysosomal diseases are genetic disorders caused by a decrease or absence of lysosomal enzyme activity due to abnormalities in the genes encoding lysosomal enzymes that should be present in lysosomes. Patients with lysosomal diseases undergo enzyme replacement therapy, in which recombinant lysosomal enzymes are administered intravenously to replenish the decreased or missing lysosomal enzymes. Patients with lysosomal diseases, which are genetic disorders, must undergo this enzyme replacement therapy for the rest of their lives.

[0005] Some lysosomal diseases affect the brain. To treat brain damage, it is necessary to replenish lysosomal enzymes in the brain. However, lysosomal enzymes administered intravenously rarely cross the blood-brain barrier (BBB), making it impossible to replenish lysosomal enzymes in the brain.

[0006] One method reported for replenishing lysosomal enzymes in the brain is to bind lysosomal enzymes to antibodies that recognize molecules present on the surface of vascular endothelial cells as antigens, creating fusion proteins that are then administered intravenously (Non-Patent Document 2). Such fusion proteins bind to the surface of vascular endothelial cells via the antibody moiety and can then pass through the BBB to reach the brain. Therefore, such fusion proteins can replenish lysosomal enzymes in the brain. Even when lysosomal enzymes are converted into such fusion proteins, patients with lysosomal diseases, which are genetic disorders, must undergo lifelong enzyme replacement therapy using these fusion proteins.

[0007] Human transferrin receptor (hTfR) is a molecule present on the surface of vascular endothelial cells. In recent years, Pavinafusp alfa, a fusion protein of hTfR and human iduronate-2-sulfatase, a lysosomal enzyme, has been marketed as a therapeutic agent effective for central nervous system disorders associated with Hunter syndrome. Patients must receive Pavinafusp alfa via intravenous infusion once a week (Non-Patent Document 3). It has also been reported that administration of anti-TfR antibodies to animals reduces the number of reticulocytes by acting on reticulocytes that express high levels of TfR (Non-Patent Document 4).

[0008] WO2016 / 208695WO2018 / 124121US20070082380US20090053219US20110110935

[0009] Sonoda H. et al., Mol Ther. 26. 1366-74 (2018) Okuyama T. et al., Mol Ther. 26. 27. 456-64 (2019) Izucargo TM Intravenous Infusion 10mg Package Insert. 1st Edition. Created March 2021 Couch JA. Sci Transl Med. 5.183ra57(2013)

[0010] An object of the present invention is to provide a nucleic acid molecule incorporating a gene encoding a fusion protein of an anti-transferrin receptor antibody (anti-TfR antibody) and a physiologically active protein, which has binding activity to a specific TfR, and which can reduce side effects such as anemia caused by the binding of the anti-TfR antibody to the transferrin receptor (TfR), for example, when the gene encoding the fusion protein of the anti-TfR antibody and a physiologically active protein is used in gene therapy.

[0011] In research aimed at the above-mentioned object, the present inventors conducted extensive research and found that highly safe viral virions that can be used in gene therapy can be produced by using a nucleic acid molecule containing a gene encoding a fusion protein of an anti-TfR antibody and a physiologically active protein, which has a certain level of binding activity (affinity) with TfR, and thus completed the present invention. That is, the present invention includes the following: 1. A nucleic acid molecule containing any of the nucleotide sequences (1) to (6) below, wherein the fusion protein of an anti-transferrin receptor antibody and a physiologically active protein encoded by the nucleic acid molecule has a binding activity (EC ５０) (1) a nucleotide sequence containing a first inverted terminal repeat (ITR) or a functional equivalent thereof, downstream of which is a nucleotide sequence encoding a fusion protein of an anti-transferrin receptor antibody and a protein having physiological activity, and further downstream of which is a nucleotide sequence containing a second inverted terminal repeat (ITR) or a functional equivalent thereof; (2) a nucleotide sequence containing a first inverted terminal repeat (ITR) or a functional equivalent thereof, downstream of which is a nucleotide sequence containing a gene expression control site, further downstream of which is a nucleotide sequence encoding a fusion protein of an anti-transferrin receptor antibody and a protein having physiological activity, and further downstream of which is a nucleotide sequence containing a second inverted terminal repeat (ITR) or a functional equivalent thereof; (3) a nucleotide sequence containing a first long terminal repeat (LTR) or a functional equivalent thereof, downstream of which is a nucleotide sequence encoding a fusion protein of an anti-transferrin receptor antibody and a protein having physiological activity, and further downstream of which is a nucleotide sequence containing a second long terminal repeat (LTR) or a functional equivalent thereof; (4) A base sequence containing a first long terminal repeat (LTR) or a functional equivalent thereof, a base sequence containing a gene expression control site downstream thereof, a base sequence further downstream encoding a fusion protein of an anti-transferrin receptor antibody and a protein having physiological activity, and a base sequence further downstream containing a second long terminal repeat (LTR) or a functional equivalent thereof; (5) A base sequence containing a leader or a functional equivalent thereof, a base sequence further downstream encoding a fusion protein of an anti-transferrin receptor antibody and a protein having physiological activity, and a base sequence further downstream containing a trailer or a functional equivalent thereof; or (6) A base sequence containing a leader or a functional equivalent thereof, a base sequence further downstream containing a gene expression control site downstream thereof, a base sequence further downstream encoding a fusion protein of an anti-transferrin receptor antibody and a protein having physiological activity, and a base sequence further downstream containing a trailer or a functional equivalent thereof.2. The nucleic acid molecule according to 1 above, which is selected from the following (1) to (4): (1) a nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein, which comprises a nucleotide sequence encoding a conjugate in which the physiologically active protein is bound to the C-terminus of the heavy chain (which may be the variable region) of the anti-transferrin receptor antibody, and a nucleotide sequence encoding the light chain (which may be the variable region) of the anti-transferrin receptor antibody; (2) a nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein, which comprises a nucleotide sequence encoding a conjugate in which the physiologically active protein is bound to the N-terminus of the heavy chain (which may be the variable region) of the anti-transferrin receptor antibody, and a nucleotide sequence encoding the light chain (which may be the variable region) of the anti-transferrin receptor antibody; (3) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a nucleotide sequence encoding a conjugate in which a physiologically active protein is bound to the C-terminus of the light chain (which may be the variable region) of the anti-transferrin receptor antibody, and a nucleotide sequence encoding the heavy chain (which may be the variable region) of the anti-transferrin receptor antibody; or (4) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a nucleotide sequence encoding a conjugate in which a physiologically active protein is bound to the N-terminus of the light chain (which may be the variable region) of the anti-transferrin receptor antibody, and a nucleotide sequence encoding the heavy chain (which may be the variable region) of the anti-transferrin receptor antibody.3. The nucleic acid molecule according to 1 above, which is selected from the following (1) to (4): (1) a nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein, which comprises a nucleotide sequence encoding a conjugate in which the physiologically active protein is linked via a linker to the C-terminus of the heavy chain (which may be the variable region) of the anti-transferrin receptor antibody, and a nucleotide sequence encoding the light chain (which may be the variable region) of the anti-transferrin receptor antibody; (2) a nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein, which comprises a nucleotide sequence encoding a conjugate in which the physiologically active protein is linked via a linker to the N-terminus of the heavy chain (which may be the variable region) of the anti-transferrin receptor antibody, and a nucleotide sequence encoding the light chain (which may be the variable region) of the anti-transferrin receptor antibody; (3) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein comprises a nucleotide sequence encoding a conjugate in which a physiologically active protein is linked via a linker to the C-terminus of the light chain of the anti-transferrin receptor antibody (which may be its variable region), and a nucleotide sequence encoding the heavy chain of the anti-transferrin receptor antibody (which may be its variable region); or (4) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein comprises a nucleotide sequence encoding a conjugate in which a physiologically active protein is linked via a linker to the N-terminus of the light chain of the anti-transferrin receptor antibody (which may be its variable region), and a nucleotide sequence encoding the heavy chain of the anti-transferrin receptor antibody (which may be its variable region). 4. The nucleic acid molecule according to 3 above, wherein the linker is a peptide consisting of 1 to 50 amino acid residues. 5. 5. The nucleic acid molecule according to 4 above, wherein the linker is a peptide comprising an amino acid sequence selected from the group consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, and an amino acid sequence consisting of 2 to 10 consecutive amino acids of these amino acid sequences.6. The nucleic acid molecule according to 1 above, which is selected from the following (1) to (4): (1) a nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein, the nucleotide sequence comprising a nucleotide sequence encoding a conjugate in which the heavy chain (which may be the variable region) of the anti-transferrin receptor antibody is linked via a second linker to the C-terminus of the light chain (which may be the variable region) of the anti-transferrin receptor antibody, and the physiologically active protein is further linked to the C-terminus, directly or via a linker; (2) a nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein, the nucleotide sequence comprising a nucleotide sequence encoding a conjugate in which the light chain (which may be the variable region) of the anti-transferrin receptor antibody is linked via a second linker to the C-terminus of the heavy chain (which may be the variable region) of the anti-transferrin receptor antibody, and the physiologically active protein is further linked to the C-terminus, directly or via a linker; (3) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a nucleotide sequence encoding a conjugate in which the heavy chain of the anti-transferrin receptor antibody (which may be its variable region) is bound to the C-terminus of the physiologically active protein, either directly or via a linker, and the light chain of the anti-transferrin receptor antibody (which may be its variable region) is further bound to the C-terminus via a second linker; or (4) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a nucleotide sequence encoding a conjugate in which the light chain of the anti-transferrin receptor antibody (which may be its variable region) is bound to the C-terminus of the physiologically active protein, either directly or via a linker, and the heavy chain of the anti-transferrin receptor antibody (which may be its variable region) is further bound to the C-terminus via a second linker. 7. The nucleic acid molecule according to 6 above, wherein the linker is a peptide consisting of 1 to 50 amino acid residues.8. The nucleic acid molecule according to claim 7, wherein the linker is a peptide comprising an amino acid sequence selected from the group consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, and an amino acid sequence consisting of 2 to 10 consecutive amino acids of these amino acid sequences. 9. The nucleic acid molecule according to claim 8, wherein the second linker consists of 8 to 50 amino acid residues. 10. The nucleic acid molecule according to claim 9, wherein the second linker is selected from the group consisting of the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, each of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, an amino acid sequence consisting of three consecutive amino acids of the amino acid sequence of SEQ ID NO: 1, and an amino acid sequence consisting of 2 to 10 consecutive amino acids of these amino acid sequences.11. The nucleic acid molecule according to 2 above, which is selected from the following (1) to (4): (1) a nucleotide sequence encoding a fusion protein of an anti-transferrin receptor antibody and a physiologically active protein, which comprises a nucleotide sequence encoding a conjugate in which the physiologically active protein is bound to the C-terminus of the heavy chain (which may be the variable region) of the anti-transferrin receptor antibody, a nucleotide sequence encoding an internal ribosome binding site downstream thereof, and a nucleotide sequence encoding the light chain (which may be the variable region) of the anti-transferrin receptor antibody further downstream thereof; (2) a nucleotide sequence encoding a fusion protein of an anti-transferrin receptor antibody and a physiologically active protein, which comprises a nucleotide sequence encoding a conjugate in which the physiologically active protein is bound to the N-terminus of the heavy chain (which may be the variable region) of the anti-transferrin receptor antibody, a nucleotide sequence encoding an internal ribosome binding site downstream thereof, and a nucleotide sequence encoding the light chain (which may be the variable region) of the anti-transferrin receptor antibody further downstream thereof; (3) The base sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a base sequence encoding a conjugate in which the physiologically active protein is bound to the C-terminus of the light chain (which may be the variable region) of the anti-transferrin receptor antibody, a base sequence encoding an internal ribosome binding site downstream thereof, and a base sequence encoding the heavy chain of the anti-transferrin receptor antibody (which may be the variable region) further downstream thereof; or (4) The base sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a base sequence encoding a conjugate in which the physiologically active protein is bound to the N-terminus of the light chain (which may be the variable region) of the anti-transferrin receptor antibody, a base sequence encoding an internal ribosome binding site downstream thereof, and a base sequence encoding the heavy chain of the anti-transferrin receptor antibody (which may be the variable region) further downstream thereof.12. The nucleic acid molecule according to any one of items 3 to 5 above, which is selected from the following (1) to (4): (1) a nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein, which comprises a nucleotide sequence encoding a conjugate in which the physiologically active protein is linked via a linker to the C-terminus of the heavy chain (which may be the variable region) of the anti-transferrin receptor antibody, a nucleotide sequence encoding an internal ribosome binding site downstream thereof, and a nucleotide sequence encoding the light chain of the anti-transferrin receptor antibody (which may be the variable region) further downstream thereof; (2) a nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein, which comprises a nucleotide sequence encoding a conjugate in which the physiologically active protein is linked via a linker to the N-terminus of the heavy chain (which may be the variable region) of the anti-transferrin receptor antibody, a nucleotide sequence encoding an internal ribosome binding site downstream thereof, and a nucleotide sequence encoding the light chain of the anti-transferrin receptor antibody (which may be the variable region) further downstream thereof; (3) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a nucleotide sequence encoding a conjugate in which the physiologically active protein is linked via a linker to the C-terminus of the light chain (which may be the variable region) of the anti-transferrin receptor antibody, a nucleotide sequence encoding an internal ribosome binding site downstream thereof, and a nucleotide sequence encoding the heavy chain (which may be the variable region) of the anti-transferrin receptor antibody further downstream thereof; or (4) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a nucleotide sequence encoding a conjugate in which the physiologically active protein is linked via a linker to the N-terminus of the light chain (which may be the variable region) of the anti-transferrin receptor antibody, a nucleotide sequence encoding an internal ribosome binding site downstream thereof, and a nucleotide sequence encoding the heavy chain (which may be the variable region) of the anti-transferrin receptor antibody further downstream thereof.13. The nucleic acid molecule according to claim 11 or 12, wherein the internal ribosome binding site is derived from the 5' untranslated region of a virus or gene selected from the group consisting of a Picornaviridae virus, foot-and-mouth disease virus, hepatitis A virus, hepatitis C virus, coronavirus, bovine enterovirus, Theiler's murine encephalomyelitis virus, Coxsackie B virus, human immunoglobulin heavy chain binding protein gene, Drosophila antennapedia gene, and Drosophila ultravithorax gene. 14. The nucleic acid molecule according to claim 11 or 12, wherein the internal ribosome binding site is derived from the 5' untranslated region of a Picornaviridae virus. 15. 15. The nucleic acid molecule according to any one of 1 to 14 above, wherein the gene expression regulatory site is selected from the group consisting of a cytomegalovirus-derived promoter, an SV40 early promoter, a human elongation factor-1α (EF-1α) promoter, a human ubiquitin C promoter, a retroviral Rous sarcoma virus LTR promoter, a dihydrofolate reductase promoter, a β-actin promoter, a phosphoglycerate kinase (PGK) promoter, a mouse albumin promoter, a human albumin promoter, a human α-1 antitrypsin promoter, and a mouse α-fetoprotein enhancer / mouse albumin promoter. 16. The nucleic acid molecule according to any one of 1 to 15 above, wherein the anti-transferrin receptor antibody is an antigen-binding fragment. 17. The nucleic acid molecule according to any one of 1 to 15 above, wherein the anti-transferrin receptor antibody is a Fab.18. The physiologically active protein is selected from the group consisting of growth hormone, lysosomal enzyme, somatomedin, insulin, glucagon, cytokine, lymphokine, blood coagulation factor, anti-transferrin receptor antibody, fusion protein of anti-transferrin receptor antibody and other 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 (FSH), gonadotropin-releasing hormone (GnRH), gonadotropin, nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor, 18. The nucleic acid molecule according to any one of 1 to 17 above, wherein the nucleic acid molecule is selected from the group consisting of: fibroblast growth factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor α receptor (TNF-α receptor), an enzyme having an activity of degrading beta-amyloid, etanercept, pegvisomant, metreleptin, abatacept, asfotase, GLP-1 receptor agonist, and antibody pharmaceutical.19. The physiologically active protein is α-L-iduronidase, iduronate-2-sulfatase, acid α-glucosidase, glucocerebrosidase, β-galactosidase, GM2-activating protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sulfatase, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sulfatase, α-L-fucosidase, α-L-aspartate-2-sulfatase ... 19. The nucleic acid molecule according to any one of items 1 to 17, wherein the first inverted terminal repeat and the second inverted terminal repeat are selected from the group consisting of digomyelinase, α-galactosidase A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2. 20. The nucleic acid molecule according to any one of items 1 to 19, wherein the first inverted terminal repeat and the second inverted terminal repeat are derived from an adeno-associated virus, an adenovirus, or a mutant thereof. 21. 20. The nucleic acid molecule according to any one of 1 to 19 above, wherein the first inverted terminal repeat comprises the base sequence shown in SEQ ID NO: 5 and the second inverted terminal repeat comprises the base sequence shown in SEQ ID NO: 6.22. The nucleic acid molecule according to any one of 1 to 19 above, wherein the first inverted terminal repeat is selected from the following (1) to (3), and the second inverted terminal repeat is selected from the following (4) to (6): (1) comprising a nucleotide sequence that is 80% or more identical to the nucleotide sequence shown in SEQ ID NO:5, (2) comprising a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO:5, or (3) the nucleotide sequence shown in SEQ ID NO:5 modified by 1 to 20 substitutions, deletions, or additions; and (4) comprising a nucleotide sequence that is 80% or more identical to the nucleotide sequence shown in SEQ ID NO:6, (5) comprising a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO:6, or (6) the nucleotide sequence shown in SEQ ID NO:6 modified by 1 to 20 substitutions, deletions, or additions. 23. The nucleic acid molecule according to any one of 1 to 19 above, wherein the functional equivalent of the first inverted terminal repeat comprises the nucleotide sequence shown in SEQ ID NO: 7, and the functional equivalent of the second inverted terminal repeat comprises the nucleotide sequence shown in SEQ ID NO: 8. 24. The nucleic acid molecule according to any one of 1 to 19 above, wherein the functional equivalent of the first inverted terminal repeat is selected from the following (1) to (3), and the functional equivalent of the second inverted terminal repeat is selected from the following (4) to (6): (1) comprising a nucleotide sequence that is 80% or more identical to the nucleotide sequence shown in SEQ ID NO:7, (2) comprising a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO:7, or (3) the nucleotide sequence shown in SEQ ID NO:7 modified by 1 to 20 substitutions, deletions, or additions; and (4) comprising a nucleotide sequence that is 80% or more identical to the nucleotide sequence shown in SEQ ID NO:8, (5) comprising a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO:8, or (6) the nucleotide sequence shown in SEQ ID NO:8 modified by 1 to 20 substitutions, deletions, or additions. 20. The nucleic acid molecule according to any one of 1 to 19 above, wherein the first long terminal repeat and the second long terminal repeat are derived from a lentivirus or retrovirus or a mutant thereof.26. The nucleic acid molecule according to any one of 1 to 19 above, wherein the first long terminal repeat comprises the nucleotide sequence shown in SEQ ID NO: 52, and the second long terminal repeat comprises the nucleotide sequence shown in SEQ ID NO: 53. 27. The nucleic acid molecule according to any one of 1 to 19 above, wherein the first long terminal repeat is selected from the following (1) to (3), and the second long terminal repeat is selected from the following (4) to (6): (1) a nucleic acid molecule comprising a nucleotide sequence that is 80% or more identical to the nucleotide sequence shown in SEQ ID NO:52, (2) a nucleic acid molecule comprising a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO:52, or (3) a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:52, which has been modified by 1 to 20 substitutions, deletions, or additions; and (4) a nucleic acid molecule comprising a nucleotide sequence that is 80% or more identical to the nucleotide sequence shown in SEQ ID NO:53, (5) a nucleic acid molecule comprising a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO:53, or (6) a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:53, which has been modified by 1 to 20 substitutions, deletions, or additions. 29. The nucleic acid molecule according to any one of 1 to 19 above, wherein the leader and the trailer are derived from Sendai virus or a mutant thereof. 29. The nucleic acid molecule according to any one of 1 to 19 above, wherein the leader comprises the nucleotide sequence shown in SEQ ID NO: 54, and the trailer comprises the nucleotide sequence shown in SEQ ID NO: 55. 30. 30. The nucleic acid molecule according to any one of 1 to 19 above, wherein the leader is selected from the following (1) to (3), and the trailer is selected from the following (4) to (6): (1) a nucleic acid molecule comprising a nucleotide sequence showing 80% or more identity to the nucleotide sequence shown in SEQ ID NO: 54, (2) a nucleic acid molecule comprising a nucleotide sequence showing 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 54, or (3) a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO: 54 modified by 1 to 20 substitutions, deletions, or additions; and (4) a nucleic acid molecule comprising a nucleotide sequence showing 80% or more identity to the nucleotide sequence shown in SEQ ID NO: 55, (5) a nucleic acid molecule comprising a nucleotide sequence showing 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 55, or (6) a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO: 55 modified by 1 to 20 substitutions, deletions, or additions. ５０31. The nucleic acid molecule according to any one of claims 1 to 30, wherein the binding activity (EC ５０ 32. The nucleic acid molecule according to any one of claims 1 to 30, wherein the nucleic acid molecule (A) is 3 to 500 nM, 3 to 100 nM, 4 nM to 1 μM, 4 to 500 nM, 4 to 100 nM, 5 nM to 1 μM, 5 to 500 nM, or 5 to 100 nM. 33. A cell, tissue, or animal into which the nucleic acid molecule according to any one of claims 1 to 32 has been introduced. 34. A stem cell into which the nucleic acid molecule according to any one of claims 1 to 32 has been introduced. 35. The stem cell according to claim 34, which is selected from the group consisting of mesenchymal stem cells, dental pulp-derived stem cells, hematopoietic stem cells, embryonic stem cells, endothelial stem cells, mammary stem cells, intestinal stem cells, hepatic stem cells, pancreatic stem cells, neural stem cells, and iPS cells. 36. A plasmid comprising the nucleic acid molecule according to any one of claims 1 to 32. 37. A viral virion comprising the nucleic acid molecule according to any one of claims 1 to 32. 38. A pharmaceutical composition comprising the viral virion according to claim 37 and a carrier. 39. A viral virion comprising the nucleic acid molecule according to claim 31. 40. A pharmaceutical composition comprising the viral virion according to claim 39. 41. A single dose of 1.0 x 10 10 ~7.5×10 12 41. The pharmaceutical composition according to claim 40, wherein the saturation is 0.015 mg / kg.

[0012] According to the present invention, it is possible to provide a fusion protein of an anti-TfR antibody with a physiologically active protein, which has a reduced possibility of inducing anemia symptoms, or a safe nucleic acid molecule containing a gene encoding such a fusion protein, which can be used to express the fusion protein in cells, tissues, or the living body.

[0013] Schematic diagram showing the structure of the pAAV-mMAP-mscFv-GS3-hI2S vector (plasmid). Schematic diagram showing the structure of the pAAV-mMAP-mscFv2-GS3-hI2S vector (plasmid). Schematic diagram showing the structure of the pAAV-mMAP-hI2S-mscFv2 vector (plasmid). Schematic diagram showing the structure of the pAAV-mMAP-hI2S vector (plasmid). Schematic diagram showing the structure of the pR2(mod)C8 vector. Figure 1 shows the results of measuring plasma rAAV-derived protein concentrations (Example 17). The vertical axis of the bar graph shows the logarithmic representation of the plasma rAAV-derived protein concentration (μg / mL). Black bars show the measured values ​​of plasma rAAV-derived protein concentrations 1 week after administration of each rAAV virion, and white bars show the measured values ​​of plasma rAAV-derived protein concentrations 6 weeks after administration of each rAAV virion. (1) is a normal control group, (2) is a pathological control group, and (3) is rAAV-mscFv-GS3-hI2S 1.0 × 10 13 vg / kg administration group, (4) rAAV-mscFv-GS3-hI2S 1.0 × 10 12 vg / kg administration group, (5) rAAV-mscFv-GS3-hI2S 1.0 × 10 11 vg / kg administration group, (6) rAAV-mscFv2-GS3-hI2S 1.0 × 10 13 vg / kg administration group, (7) rAAV-mscFv2-GS3-hI2S 1.0 × 10 12 vg / kg administration group, (8) rAAV-mscFv2-GS3-hI2S 1.0 × 10 11 vg / kg administration group, (9) rAAV-hI2S-mscFv2 1.0 × 10 13 vg / kg administration group, (10) is rAAV-hI2S-mscFv2 1.0 × 10 12 vg / kg administration group, (11) rAAV-hI2S-mscFv2 1.0 × 10 11 vg / kg administration group, (12) rAAV-hI2S 1.0 × 10 13 vg / kg administration group, (13) rAAV-hI2S 1.0 × 10 12 vg / kg administration group, and (14) rAAV-hI2S 1.0 × 10 11The results for the 1.0×10 rAAV-derived protein administration group and the 1.0×10 rAAV-mscFv-GS3-hI2S ... 13 vg / kg administration group, (4) rAAV-mscFv-GS3-hI2S 1.0 × 10 12 vg / kg administration group, (5) rAAV-mscFv-GS3-hI2S 1.0 × 10 11 vg / kg administration group, (6) rAAV-mscFv2-GS3-hI2S 1.0 × 10 13 vg / kg administration group, (7) rAAV-mscFv2-GS3-hI2S 1.0 × 10 12 vg / kg administration group, (8) rAAV-mscFv2-GS3-hI2S 1.0 × 10 11 vg / kg administration group, (9) rAAV-hI2S-mscFv2 1.0 × 10 13 vg / kg administration group, (10) is rAAV-hI2S-mscFv2 1.0 × 10 12 vg / kg administration group, (11) rAAV-hI2S-mscFv2 1.0 × 10 11 vg / kg administration group, (12) rAAV-hI2S 1.0 × 10 13 vg / kg administration group, (13) rAAV-hI2S 1.0 × 10 12 vg / kg administration group, and (14) rAAV-hI2S 1.0 × 10 11The results for the 1.0×10 rAAV virion administration group and the 1.0×10 rAAV mscFv-GS3-hI2S ... 13 vg / kg administration group, (4) rAAV-mscFv-GS3-hI2S 1.0 × 10 12 vg / kg administration group, (5) rAAV-mscFv-GS3-hI2S 1.0 × 10 11 vg / kg administration group, (6) rAAV-mscFv2-GS3-hI2S 1.0 × 10 13 vg / kg administration group, (7) rAAV-mscFv2-GS3-hI2S 1.0 × 10 12 vg / kg administration group, (8) rAAV-mscFv2-GS3-hI2S 1.0 × 10 11 vg / kg administration group, (9) rAAV-hI2S-mscFv2 1.0 × 10 13 vg / kg administration group, (10) is rAAV-hI2S-mscFv2 1.0 × 10 12 vg / kg administration group, (11) rAAV-hI2S-mscFv2 1.0 × 10 11 vg / kg administration group, (12) rAAV-hI2S 1.0 × 10 13 vg / kg administration group, (13) rAAV-hI2S 1.0 × 10 12 vg / kg administration group, and (14) rAAV-hI2S 1.0 × 10 11The results for the 1.0×10 rAAV-mscFv-GS3-hI2S ... 13 vg / kg administration group, (4) rAAV-mscFv-GS3-hI2S 1.0 × 10 12 vg / kg administration group, (5) rAAV-mscFv-GS3-hI2S 1.0 × 10 11 vg / kg administration group, (6) rAAV-mscFv2-GS3-hI2S 1.0 × 10 13 vg / kg administration group, (7) rAAV-mscFv2-GS3-hI2S 1.0 × 10 12 vg / kg administration group, (8) rAAV-mscFv2-GS3-hI2S 1.0 × 10 11 vg / kg administration group, (9) rAAV-hI2S-mscFv2 1.0 × 10 13 vg / kg administration group, (10) is rAAV-hI2S-mscFv2 1.0 × 10 12 vg / kg administration group, (11) rAAV-hI2S-mscFv2 1.0 × 10 11 vg / kg administration group, (12) rAAV-hI2S 1.0 × 10 13 vg / kg administration group, (13) rAAV-hI2S 1.0 × 10 12 vg / kg administration group, and (14) rAAV-hI2S 1.0 × 10 11 The results for the rAAV-mscFv3-GS3-hI2S administration group are shown in Table 1. The values ​​are mean values, and the error bars indicate standard deviation (n=3). Figure 2 shows the results of measuring the concentration of mscFv3-GS3-hI2S in plasma (Example 26). The vertical axis of the bar graph indicates the concentration (μg / mL) of mscFv3-GS3-hI2S in plasma. The vertical bars indicate the concentration of mscFv3-GS3-hI2S in plasma collected 6 weeks after administration of rAAV-mscFv3-GS3-hI2S. (1) is 1.0 × 10 11vg / kg administration group, (2) 1.0 × 10 12 vg / kg administration group, (3) 1.0 × 10 13 The results for the rAAV-mscFv3-GS3-hI2S administration group are shown in Table 2. The values ​​are average values, and the error bars indicate standard deviations. Figure 2 shows the results of measuring the concentration of mscFv3-GS3-hI2S in the brain (Example 26). The vertical axis of the bar graph indicates the concentration of mscFv3-GS3-hI2S in the brain (μg / g wet weight). The vertical bars indicate the concentration of rAAV-derived proteins in the brain collected 6 weeks after administration of rAAV-mscFv3-GS3-hI2S. (1) is 1.0 × 10 11 vg / kg administration group, (2) 1.0 × 10 12 vg / kg administration group, (3) 1.0 × 10 13 The results for the rAAV-mscFv3-GS3-hI2S administration group and the rAAV-mscFv3-GS3-hI2S administration group are shown in Table 1. The values ​​are averages, and the error bars indicate standard deviations. Figure 2 shows the results of measuring heparan sulfate concentrations in brain tissue (Example 27). The vertical axis of the bar graph indicates heparan sulfate concentration (μg / mg wet weight). The vertical bars indicate heparan sulfate concentrations in brain samples taken 6 weeks after administration of rAAV-mscFv3-GS3-hI2S. (1) is the normal control group, (2) is the pathological control group, and (3) is the 1.0 × 10 11 vg / kg administration group, (4) 1.0 × 10 12 vg / kg administration group, (5) 1.0 × 10 13 The results for the rAAV-mscFv3-GS3-hI2S administration group and the rAAV-mscFv3-GS3-hI2S administration group are shown in Table 2. The values ​​are the mean values, and the error bars indicate the standard deviation. Figure 2 shows the results of measuring heparan sulfate concentration in CSF (Example 27). The vertical axis of the bar graph indicates the heparan sulfate concentration (μg / mg). The vertical bars indicate the heparan sulfate concentration in CSF collected 6 weeks after administration of rAAV-mscFv3-GS3-hI2S. (1) is the normal control group, (2) is the pathological control group, and (3) is the 1.0 × 10 11 vg / kg administration group, (4) 1.0 × 10 12 vg / kg administration group, (5) 1.0 × 10 13The results for the rAAV-mscFv3-GS3-hI2S administration group and the rAAV-mscFv3-GS3-hI2S administration group are shown in Table 2. The values ​​are mean values, and the error bars indicate standard deviations. Figure 2 shows the results of measuring hemoglobin concentration in the blood (Example 28). The vertical axis of the bar graph indicates hemoglobin concentration (g / dL). The vertical bars indicate hemoglobin concentration in blood collected 6 weeks after administration of rAAV-mscFv3-GS3-hI2S. (1) is the normal control group, (2) is the pathological control group, and (3) is the 1.0 × 10 11 vg / kg administration group, (4) 1.0 × 10 12 vg / kg administration group, (5) 1.0 × 10 13 The results for the 1.0×10 rAAV-hGAA(Pro)-GS-mscFv2 ... 13 vg / kg administration group, (4) rAAV-mscFv-GS3-hGAA 1.0 × 10 13 vg / kg administration group, (5) rAAV-hGAA 1.0 × 10 13 The results for the 1.0×10 rAAV-hGAA(Pro)-GS-mscFv2 ... 13 vg / kg administration group, (4) rAAV-mscFv-GS3-hGAA 1.0 × 10 13 vg / kg administration group, (5) rAAV-hGAA 1.0 × 10 13The results for the 1.0 × 10 rAAV-hGAA(Pro)-GS-mscFv2 ... 13 vg / kg administration group, (4) rAAV-mscFv-GS3-hGAA 1.0 × 10 13 vg / kg administration group, (5) rAAV-hGAA 1.0 × 10 13 The results for the 2000 mg / kg group are shown. The values ​​are the mean values, and the error bars indicate the standard deviation.

[0014] In the present invention, the term "nucleic acid molecule" refers primarily to either DNA, which is a polymer of deoxyribonucleotides formed by phosphodiester bonds, or RNA, which is a polymer of ribonucleotides formed by phosphodiester bonds.

[0015] When the "nucleic acid molecule" is DNA, the DNA may be single-stranded (single-stranded) or double-stranded with a complementary strand. When the DNA is single-stranded, the DNA may be either a (+) strand or a (-) strand. The individual deoxyribonucleotides constituting the DNA may be naturally occurring or modified from the natural type, as long as the gene encoding the protein contained in the DNA can be translated into mRNA in mammalian (particularly human) cells. In one embodiment of the present invention, the individual deoxyribonucleotides constituting the DNA may be naturally occurring or modified from the natural type, as long as the gene encoding the protein contained in the DNA can be translated into mRNA and all or part of the DNA can be replicated in mammalian (particularly human) cells.

[0016] Furthermore, when the "nucleic acid molecule" is RNA, the RNA may be single-stranded (single-stranded) or double-stranded with a complementary strand. When the RNA is single-stranded, the RNA may be either a (+) strand or a (-) strand. In one embodiment of the present invention, the individual ribonucleotides constituting the RNA may be naturally occurring or modified, as long as the gene encoding the protein contained in the RNA can be reverse transcribed into DNA in mammalian (particularly human) cells. In one embodiment of the present invention, the individual ribonucleotides constituting the RNA may be naturally occurring or modified, as long as the gene encoding the protein contained in the RNA can be translated into protein in mammalian (particularly human) cells. Ribonucleotide modifications are performed, for example, to suppress degradation of RNA by RNase and increase the stability of RNA in cells.

[0017] In one embodiment of the present invention, the term "inverted terminal repeat (ITR)" refers to a base sequence present at the end of a viral genome, in which the same sequence is repeated. ITRs derived from adeno-associated viruses and adenoviruses can be suitably used. For example, the ITR of an adeno-associated virus is a region approximately 145 bases long and functions as a replication origin, etc. In one embodiment of the present invention, a nucleic acid molecule contains two inverted terminal repeats (ITRs), referred to as the first inverted terminal repeat (ITR) and the second inverted terminal repeat (ITR). When a gene encoding a fusion protein is placed between two ITRs, the ITR located on the 5' side is referred to as the first inverted terminal repeat (ITR), and the ITR located on the 3' side is referred to as the second inverted terminal repeat (ITR). In the present invention, the inverted terminal repeats (ITRs) may be derived from any virus as long as they have at least one of the functions of an original ITR, such as functioning as a replication origin or inserting a gene into a host cell, and the ITRs of an adeno-associated virus are one of the preferred ones.

[0018] When the ITR is an adeno-associated virus, the serotype of AAV is not particularly limited and may be any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. For example, the inverted terminal repeat (ITR) of serotype 2 AAV has a first ITR containing the nucleotide sequence shown in SEQ ID NO:5 and a second ITR containing the nucleotide sequence shown in SEQ ID NO:6.

[0019] Furthermore, the ITR is not limited to wild-type ITRs, and may be modified by substitution, deletion, addition, or other alterations to the wild-type ITR nucleotide sequence. When nucleotides in the wild-type ITR nucleotide sequence are substituted with other nucleotides, the number of substituted nucleotides is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When nucleotides in the wild-type ITR nucleotide sequence are deleted, the number of deleted nucleotides is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Mutations combining these base substitutions and deletions can also be added. When nucleotides are added to the wild-type ITR, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 nucleotides are added within the nucleotide sequence or to the 5' or 3' end of the ITR. Mutations combining these base additions, substitutions, and deletions can also be added. The nucleotide sequence of the mutated ITR preferably exhibits 80% or more identity to the nucleotide sequence of the wild-type ITR, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.

[0020] A functional equivalent of an ITR is one that can be used functionally in place of an ITR. An artificially constructed ITR based on an ITR is also a functional equivalent of an ITR as long as it can replace the ITR.

[0021] A functional equivalent of an AAV ITR is one that can be used functionally in place of the AAV ITR. In addition, an artificially constructed ITR based on the AAV ITR is also a functional equivalent of the AAV ITR as long as it can replace the AAV ITR.

[0022] An example of a functional equivalent of the artificially constructed first AAV inverted terminal repeat (first AAV-ITR) is one having the nucleotide sequence set forth in SEQ ID NO:7 (functional equivalent of the first AAV-ITR). Substitutions, deletions, or mutations in the nucleotide sequence set forth in SEQ ID NO:7 are also included in the functional equivalent of the first AAV-ITR, as long as they can be used functionally in place of the first AAV ITR. When bases in the nucleotide sequence set forth in SEQ ID NO:7 are substituted with other bases, the number of substituted bases is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3. When bases in the nucleotide sequence set forth in SEQ ID NO:7 are deleted, the number of deleted bases is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, ITRs mutated by a combination of these base substitutions and deletions are also functional equivalents of the first AAV-ITR. When bases are added to the base sequence shown in SEQ ID NO:7, preferably 1 to 20 bases, more preferably 1 to 10 bases, even more preferably 1 to 5 bases, and even more preferably 1 to 3 bases are added within the base sequence or to the 5'-end or 3'-end. ITRs with mutations that combine these base additions, substitutions, and deletions are also included in the functional equivalents of the first AAV-ITR. The base sequence of the mutated ITR preferably exhibits 80% or more identity, more preferably 85% or more identity, even 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 shown in SEQ ID NO:7.

[0023] Furthermore, functional equivalents of the artificially constructed second inverted terminal repeat (second AAV-ITR) include those having the nucleotide sequence set forth in SEQ ID NO: 8 (functional equivalents of the second AAV-ITR). Substitutions, deletions, or mutations in the nucleotide sequence set forth in SEQ ID NO: 8 are also included in the functional equivalents of the second AAV ITR, as long as they can be used functionally in place of the second AAV ITR. When bases in the nucleotide sequence set forth in SEQ ID NO: 8 are substituted with other bases, the number of substituted bases is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3. When bases in the nucleotide sequence set forth in SEQ ID NO: 8 are deleted, the number of deleted bases is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, ITRs mutated by a combination of these base substitutions and deletions are also functional equivalents of the second AAV ITR. When bases are added to the base sequence shown in SEQ ID NO:8, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3 bases are added within the base sequence or to the 5'-end or 3'-end. ITRs mutated by a combination of these base additions, substitutions, and deletions are also included in the functional equivalents of the ITRs of the second AAV. The mutated ITR base sequence preferably exhibits 85% or more identity, more preferably 85% or more identity, even 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 shown in SEQ ID NO:8.

[0024] In one embodiment of the present invention, the term "long terminal repeat (LTR)" refers to a base sequence present at the end of, for example, a eukaryotic retrotransposon, or a retroviral genome or lentiviral genome, in which the same sequence is repeated hundreds to thousands of times. In one embodiment of the present invention, a nucleic acid molecule contains two long terminal repeats (LTRs), referred to as the first long terminal repeat (LTR) and the second long terminal repeat (LTR). When a gene encoding a fusion protein is placed between the two LTRs, the LTR located on the 5' side is referred to as the first long terminal repeat (LTR), and the LTR located on the 3' side is referred to as the second long terminal repeat (LTR). In the present invention, the long terminal repeat (LTR) may be derived from any virus, as long as it has at least one of the functions of an original LTR, such as a function as a replication origin or gene insertion into a host cell. Suitable examples include retroviral genomes and lentiviruses. Furthermore, the LTR is not limited to wild-type LTRs, and may be modified by substitution, deletion, addition, or other modifications to the wild-type LTR base sequence.

[0025] When bases in the wild-type LTR base sequence are substituted with other bases, the number of substituted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When the wild-type LTR base sequence is deleted, the number of deleted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Mutations combining these base substitutions and deletions can also be added. When bases are added to the wild-type LTR, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added within the LTR base sequence or to the 5'-end or 3'-end. Mutations combining these base additions, substitutions, and deletions can also be added. The mutated LTR base sequence preferably exhibits 80% or more identity to the wild-type LTR base sequence, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.

[0026] A functional equivalent of an LTR is one that can be used functionally in place of an LTR. An artificially constructed LTR based on an LTR is also a functional equivalent of an LTR as long as it can replace the LTR.

[0027] A functional equivalent of a lentiviral LTR is one that can be used in place of the lentiviral LTR. In addition, an artificially constructed LTR based on a lentiviral LTR is also a functional equivalent of a lentiviral LTR as long as it can replace the lentiviral LTR.

[0028] A functional equivalent of a retroviral LTR is one that can be used in place of a retroviral LTR. In addition, an artificially constructed LTR based on a retroviral LTR is also a functional equivalent of a retroviral LTR as long as it can replace the retroviral LTR.

[0029] An example of a functional equivalent of the first LTR is one having the nucleotide sequence shown in SEQ ID NO: 52. Substitutions, deletions, or mutations in the nucleotide sequence shown in SEQ ID NO: 52 are also included in the functional equivalent of the first LTR, as long as they can be functionally used as the first LTR. When bases in the nucleotide sequence shown in SEQ ID NO: 52 are substituted with other bases, the number of substituted bases is preferably 1 to 20, more preferably 1 to 20, even more preferably 1 to 5, and even more preferably 1 to 3. When bases in the nucleotide sequence shown in SEQ ID NO: 52 are deleted, the number of deleted bases is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, LTRs with mutations that combine these base substitutions and deletions are also functional equivalents of the first LTR. When bases are added to the base sequence shown in SEQ ID NO:52, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3 bases are added within the base sequence or to the 5'-end or 3'-end. LTRs with mutations that combine these base additions, substitutions, and deletions are also included in the functional equivalents of the first LTR. The base sequence of the mutated LTR preferably exhibits 80% or more identity, more preferably 85% or more identity, even 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 shown in SEQ ID NO:52.

[0030] Furthermore, functional equivalents of the second LTR include those having the nucleotide sequence shown in SEQ ID NO: 53. Substitutions, deletions, or mutations in the nucleotide sequence shown in SEQ ID NO: 53 are also included in the functional equivalents of the second LTR, as long as they can be functionally used as the second LTR. When nucleotides in the nucleotide sequence shown in SEQ ID NO: 53 are substituted with other nucleotides, the number of substituted nucleotides is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3. When nucleotides in the nucleotide sequence shown in SEQ ID NO: 53 are deleted, the number of deleted nucleotides is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, LTRs mutated by a combination of these nucleotide substitutions and deletions are also functional equivalents of the second LTR. When bases are added to the base sequence shown in SEQ ID NO: 53, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3 bases are added within the base sequence or to the 5'-end or 3'-end. LTRs with mutations that combine these base additions, substitutions, and deletions are also included in the functional equivalents of the second LTR. The base sequence of the mutated LTR preferably exhibits 80% or more identity, more preferably 85% or more identity, even 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 shown in SEQ ID NO: 53.

[0031] In one embodiment of the present invention, the terms leader and trailer refer to partially complementary base sequences present at the termini of the viral genome. Leaders and trailers derived from Sendai virus can be suitably used. The leader and trailer of Sendai virus are both regions of approximately 50 bases in length. Typically, the leader is located on the 5' side and the trailer is located on the 3' side.

[0032] Examples of a leader that can be suitably used in one embodiment of the present invention include a leader derived from Sendai virus having the base sequence shown in SEQ ID NO: 54 and a trailer derived from Sendai virus having the base sequence shown in SEQ ID NO: 55.

[0033] Mutations in the leader and trailer derived from wild-type Sendai virus can also be suitably used in the present invention. When bases in the base sequence are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When the base sequence of the wild-type leader and / or trailer is deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Mutations that combine these base substitutions and deletions can also be added. When 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 of the leader and / or trailer or to the 5' end or 3' end. Mutations that combine these base additions, substitutions, and deletions can also be added. The mutated base sequence preferably exhibits 80% or more identity to the wild-type base sequence, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.

[0034] In one embodiment of the present invention, the base sequence that can be used as the base sequence containing the gene expression control site for controlling the expression of the fusion protein gene is not particularly limited, as long as it allows the fusion protein to be expressed in the cells, tissues, or living organisms of a mammal (especially a human) into which the gene encoding the fusion protein is introduced. However, preferred base sequences include a cytomegalovirus-derived promoter (optionally including an enhancer), SV40 early promoter, human elongation factor-1α (EF-1α) promoter, human ubiquitin C promoter, Rous sarcoma virus long terminal repeat (LTR) promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerate kinase (PGK) promoter, mouse albumin promoter, human albumin promoter, and human α-1 antitrypsin promoter. For example, a synthetic promoter having the base sequence shown in SEQ ID NO: 9, which contains the mouse albumin promoter downstream of the mouse α-fetoprotein enhancer (mouse α-fetoprotein enhancer / mouse albumin promoter), can be suitably used as the gene expression control site. A chicken β-actin / MVM chimeric intron having the nucleotide sequence shown in SEQ ID NO: 10 may be placed downstream of the mouse α-fetoprotein enhancer / mouse albumin promoter. Placing such an intron can increase the expression level of the protein controlled by the gene expression control site. In the nucleotide sequence shown in SEQ ID NO: 9, the nucleotide sequence from 1 to 219 is the mouse α-fetoprotein enhancer, and the nucleotide sequence from 241 to 549 is the mouse albumin promoter.

[0035] The gene regulatory site may be a promoter of a gene that is expressed in an organ-specific or cell type-specific manner. By using an organ-specific expression promoter or a cell type-specific expression promoter, the gene encoding the fusion protein incorporated into the nucleic acid molecule can be expressed specifically in a desired organ or cell.

[0036] In one embodiment of the present invention, an "internal ribosome binding site" refers to a region (structure) present within an mRNA strand to which a ribosome can directly bind and initiate translation independent of a cap structure, or a region (structure) of a DNA strand that generates such a region upon transcription. Furthermore, in the present invention, a "gene encoding an internal ribosome binding site" refers to a region (structure) of a DNA strand that generates such a region upon transcription. Internal ribosome binding sites are generally referred to as IRES (internal ribosome entry sites) and have been found in the 5' untranslated regions of viruses such as Picornaviridae viruses (poliovirus, rhinovirus, murine encephalomyocarditis virus, etc.), foot-and-mouth disease virus, hepatitis A virus, hepatitis C virus, coronavirus, bovine enterovirus, Theiler's murine encephalomyelitis virus, and Coxsackie B virus, as well as in the 5' untranslated regions of genes for human immunoglobulin heavy chain binding protein, Drosophila antennapedia, and Drosophila ultravithorax. In the case of picornaviruses, the IRES is a region of approximately 450 bp located in the 5' untranslated region of the mRNA. Here, the "5' untranslated region of a virus" refers to the 5' untranslated region of the viral mRNA or the region (structure) of the DNA strand that generates the region upon transcription.

[0037] In one embodiment, the internal ribosome binding site is not particularly limited as long as it functions as an internal ribosome binding site in mammalian (particularly human) cells, tissues, or living organisms, and any internal ribosome binding site can be used. Among these, an internal ribosome binding site derived from the 5' untranslated region of a virus is preferred, an internal ribosome binding site derived from the 5' untranslated region of a virus of the Picornaviridae family is more preferred, and an internal ribosome binding site derived from the 5' untranslated region of a murine encephalomyocarditis virus is even more preferred. One embodiment of an internal ribosome binding site derived from the 5' untranslated region of a murine encephalomyocarditis virus is one having the nucleotide sequence shown in SEQ ID NO: 11.

[0038] In one embodiment, an internal ribosome binding site having a wild-type nucleotide sequence can be used as is. Also, mutant internal ribosome binding sites obtained by adding one or more mutations (e.g., substitutions, deletions, and / or insertions) to the nucleotide sequence of these wild-type internal ribosome binding sites can be used as long as they function as internal ribosome binding sites in mammalian (e.g., human) cells, tissues, or living organisms. Chimeric internal ribosome binding sites, in which two or more internal ribosome binding sites are fused, can also be used.

[0039] In one embodiment of the present invention, the physiologically active protein that binds to the anti-transferrin receptor antibody to form the fusion protein is not particularly limited, as long as it exhibits physiological activity in vivo. Suitable physiologically active proteins include pharmaceuticals that are administered to patients over a long period of time. Examples of such pharmaceuticals include growth hormone, lysosomal enzymes, neurotrophic factors, somatomedins, insulin, glucagon, 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, DNasel, thyroid-stimulating hormone (TSH), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and ciliary neurotrophic factor (CLF). Neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor α receptor (TNF-α receptor), enzymes with beta-amyloid degrading activity, etanercept, pegvisomant, metreleptin, abatacept, asfotase, and GLP-1 receptor agonists, or any one of the antibody drugs.

[0040] Suitable examples of the physiologically active protein being a lysosomal enzyme include α-L-iduronidase, iduronate-2-sulfatase, acid α-glucosidase, glucocerebrosidase, β-galactosidase, GM2-activating protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, and aspartylglucosaminidase. , α-N-acetylgalactosaminidase, acid sphingomyelinase, α-galactosidase A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2.

[0041] In one embodiment of the present invention, the physiologically active protein is a human protein. Here, the protein may be a wild-type protein, or may be a mutated protein as long as it retains its inherent physiological activity. Here, a protein having inherent physiological activity means that the protein has 10% or more of the physiological activity of the wild-type protein. The physiological activity of the wild-type protein is preferably 20% or more, more preferably 40% or more, even more preferably 50% or more, even more preferably 80% or more, and even more preferably 90% or more.

[0042] When bases in the base sequence of a wild-type protein having physiological activity are substituted with other bases, the number of substituted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence of the wild-type protein are deleted, the number of deleted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Mutations combining these base substitutions and deletions can also be added. When bases are added to the wild-type protein, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence or to the 5'-end or 3'-end of the protein. Mutations combining these base additions, substitutions, and deletions can also be added. The base sequence of the mutated protein preferably exhibits 80% or more identity, preferably 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 protein.

[0043] In the present invention, the position and type (deletion, substitution, addition) of each mutation compared to the wild-type protein can be easily confirmed by aligning the amino acid sequences of the wild-type and mutant proteins. In the present invention, the identity between the amino acid sequence of a wild-type protein and that of a mutant protein can be easily calculated using well-known homology calculation algorithms. Examples of such algorithms include BLAST (Altschul SF. 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)).

[0044] Substitution of an amino acid in the amino acid sequence of the above protein with another amino acid occurs within a family of amino acids that are related, for example, by their side chains and chemical properties, and is predicted not to significantly alter the function of the protein (i.e., it is a conservative amino acid substitution). Examples of such amino acid families include: (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) the hydroxyl amino acids serine and threonine, (5) the hydrophobic amino acids methionine, alanine, valine, leucine, and isoleucine, (6) the neutral hydrophilic amino acids cysteine, serine, threonine, asparagine, and glutamine, (7) the amino acids that influence the orientation of the peptide chain glycine and proline, (8) the amide amino acids (polar amino acids) asparagine and glutamine, (9) the aliphatic amino acids alanine, leucine, isoleucine, and valine, and (10) the amino acids with small side chains alanine, glycine, serine, and threonine. (11) Alanine and glycine are amino acids with particularly small side chains.

[0045] The anti-transferrin receptor antibody constituting the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein in the present invention will be described in detail below.

[0046] Anti-transferrin receptor antibodies (anti-TfR antibodies) are not particularly limited in terms of animal species, as long as they have the property of specifically binding to the antigen, transferrin receptor (TfR), but are particularly suitable for human or humanized anti-TfR antibodies. For example, anti-TfR antibodies may be anti-TfR antibodies of mammals other than humans, or chimeric anti-TfR antibodies composed of a human anti-TfR antibody and an anti-TfR antibody of a mammal other than humans.

[0047] In one embodiment of the present invention, the term "anti-TfR antibody" refers to an antibody that specifically binds to the transferrin receptor (TfR) of any animal species. Therefore, the antigen-binding fragments described below are also included in the anti-TfR antibody as long as they have the property of specifically binding to the transferrin receptor (TfR).

[0048] A human anti-transferrin receptor antibody (human anti-TfR antibody) is an anti-TfR antibody that is entirely encoded by a gene of human origin. However, an anti-TfR antibody encoded by a gene that has been mutated to increase gene expression efficiency, etc., is also a human anti-TfR antibody. An anti-TfR antibody that combines two or more genes encoding human anti-TfR antibodies and replaces a portion of one human anti-TfR antibody with a portion of another human anti-TfR antibody is also a human anti-TfR antibody. Human anti-TfR antibodies typically have three complementarity-determining regions (CDRs) in the immunoglobulin light chain and three complementarity-determining regions (CDRs) in the immunoglobulin heavy chain. The three CDRs in the immunoglobulin light chain are called CDR1, CDR2, and CDR3, respectively, from the N-terminus. The three CDRs in the immunoglobulin heavy chain are called CDR1, CDR2, and CDR3, respectively, from the N-terminus. An anti-TfR antibody in which the antigen specificity, affinity, etc. of a human anti-TfR antibody is modified by replacing the CDR of one human anti-TfR antibody with the CDR of another human anti-TfR antibody is also a human anti-TfR antibody.

[0049] In the present invention, antibodies obtained by modifying the gene of the original human anti-TfR antibody to add mutations such as substitutions, deletions, and additions to the amino acid sequence of the original human anti-TfR antibody are also referred to as human anti-TfR antibodies. When amino acids in the amino acid sequence of the original human anti-TfR antibody are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of the original human anti-TfR antibody are deleted, the number of amino acids to be deleted is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, antibodies that have been mutated by combining these amino acid substitutions and deletions are also human anti-TfR antibodies. When amino acids are added, preferably 1 to 20, more preferably 1 to 10, even 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 original human anti-TfR antibody. Antibodies with mutations that combine these amino acid additions, substitutions, and deletions are also human anti-TfR antibodies. The amino acid sequence of the mutated human anti-TfR antibody preferably exhibits 80% or more identity, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity to the amino acid sequence of the original human anti-TfR antibody. In other words, the term "human-derived gene" as used herein includes not only the original human gene, but also genes obtained by modifying the original human gene.

[0050] In the present invention, the term "humanized anti-TfR antibody" refers to an anti-TfR antibody in which the amino acid sequence of a portion of the variable region (e.g., all or part of the CDRs) is derived from a mammal other than human, while the remaining regions are derived from humans. For example, a humanized anti-TfR antibody may be an anti-TfR antibody prepared by replacing three complementarity-determining regions (CDRs) of the immunoglobulin light chain and three complementarity-determining regions (CDRs) of the immunoglobulin heavy chain constituting a human anti-TfR antibody with CDRs from another mammal. The species of other mammal from which the CDRs are grafted to appropriate positions in a human anti-TfR antibody are derived is not particularly limited as long as it is a mammal other than human, but is preferably a mouse, rat, rabbit, horse, or non-human primate, more preferably a mouse or rat, such as a mouse.

[0051] In the present invention, when the anti-TfR antibody is a human anti-TfR antibody or a humanized anti-TfR antibody, the following is a detailed description. The light chains of human anti-TfR antibodies and humanized anti-TfR antibodies include λ chains and κ chains. The light chains constituting anti-TfR antibodies may be either λ chains or κ chains. Furthermore, the heavy chains of human anti-TfR antibodies and humanized anti-TfR antibodies include γ chains, μ chains, α chains, σ chains, and ε chains, which correspond to IgG, IgM, IgA, IgD, and IgE, respectively. The heavy chains constituting anti-TfR antibodies may be γ chains, μ chains, α chains, σ chains, and ε chains, but are preferably γ chains. Furthermore, the γ chains of the heavy chains of anti-TfR antibodies include γ1 chains, γ2 chains, γ3 chains, and γ4 chains, which correspond to IgG1, IgG2, IgG3, and IgG4, respectively. When the heavy chain constituting the anti-TfR antibody is a γ chain, the γ chain may be any of the γ1 chain, γ2 chain, γ3 chain, and γ4 chain, but is preferably the γ1 chain or the γ4 chain. When the anti-TfR antibody is a humanized anti-TfR antibody or a human anti-TfR antibody and is an IgG, the light chain of the anti-TfR antibody may be either a λ chain or a κ chain, and the heavy chain of the anti-TfR antibody may be any of the γ1 chain, γ2 chain, γ3 chain, and γ4 chain, but is preferably the γ1 chain or the γ4 chain. For example, one preferred embodiment of the anti-TfR antibody is one in which the light chain is a λ chain and the heavy chain is a γ1 chain.

[0052] In the present invention, the term "chimeric anti-TfR antibody" refers to an anti-TfR antibody formed by linking fragments of two or more different anti-TfR antibodies derived from two or more different species.

[0053] A chimeric anti-TfR antibody composed of a human anti-TfR antibody and an anti-TfR antibody from another mammal is an anti-TfR antibody in which a portion of the human anti-TfR antibody is replaced with a portion of the anti-TfR antibody from a mammal other than human. The anti-TfR antibody consists of an Fc region, Fab region, and hinge region, as described below. Specific examples of such chimeric anti-TfR antibodies include chimeric anti-TfR antibodies in which the Fc region is derived from a human anti-TfR antibody and the Fab region is derived from an anti-TfR antibody from another mammal. The hinge region is derived from either a human anti-TfR antibody or an anti-TfR antibody from another mammal. Conversely, examples include chimeric anti-TfR antibodies in which the Fc region is derived from another mammal and the Fab region is derived from a human anti-TfR antibody. The hinge region may be derived from either a human anti-TfR antibody or an anti-TfR antibody from another mammal. The same applies to humanized anti-TfR antibodies.

[0054] Alternatively, an anti-TfR antibody can be said to consist of a variable region and a constant region. Other specific examples of chimeric anti-TfR antibodies include those in which the heavy chain constant region (CH) and light chain constant region (CL) are derived from a human anti-TfR antibody, while the heavy chain variable region (VH) and light chain variable region (VL) are derived from an anti-TfR antibody of another mammal. Conversely, those in which the heavy chain constant region (CH) and light chain constant region (CL) are derived from an anti-TfR antibody of another mammal, while the heavy chain variable region (VH) and light chain variable region (VL) are derived from a human anti-TfR antibody. The other mammalian species is not particularly limited as long as it is a mammal other than human, but is preferably mouse, rat, rabbit, horse, or non-human primate, and more preferably mouse. The same applies to humanized anti-TfR antibodies. In one embodiment of the present invention, when simply referring to a heavy chain, this can be appropriately replaced with a portion containing the variable region of the heavy chain, and when simply referring to a light chain, this can be appropriately replaced with a portion containing the variable region of the light chain.

[0055] Chimeric anti-TfR antibodies composed of a human anti-TfR antibody and a mouse anti-TfR antibody are specifically referred to as "human / mouse chimeric anti-TfR antibodies." Examples of human / mouse chimeric anti-TfR antibodies include those in which the Fc region is derived from a human anti-TfR antibody and the Fab region is derived from a mouse anti-TfR antibody, and conversely, those in which the Fc region is derived from a mouse anti-TfR antibody and the Fab region is derived from a human anti-TfR antibody. The hinge region is derived from either a human anti-TfR antibody or a mouse anti-TfR antibody. Other examples of human / mouse chimeric anti-TfR antibodies include those in which the heavy chain constant region (CH) and light chain constant region (CL) are derived from a human anti-TfR antibody, while the heavy chain variable region (VH) and light chain variable region (VL) are derived from a mouse anti-TfR antibody, and conversely, those in which the heavy chain constant region (CH) and light chain constant region (CL) are derived from a mouse anti-TfR antibody, while the heavy chain variable region (VH) and light chain variable region (VL) are derived from a human anti-TfR antibody. The same is true for humanized anti-TfR antibodies.

[0056] Anti-TfR antibodies originally have a basic structure consisting of four polypeptide chains: two immunoglobulin light chains and two immunoglobulin heavy chains. However, in the present invention, the term "anti-TfR antibody" includes, in addition to those having this basic structure, the following: (1) an antibody consisting of two polypeptide chains, one immunoglobulin light chain and one immunoglobulin heavy chain; and, as described in detail below, (2) a single-chain antibody formed by bonding a linker to the C-terminus of an immunoglobulin light chain and then bonding an immunoglobulin heavy chain to the C-terminus thereof; (3) a single-chain antibody formed by bonding a linker to the C-terminus of an immunoglobulin heavy chain and then bonding an immunoglobulin light chain to the C-terminus thereof; (4) a single-chain antibody (scFv) formed by bonding a linker to the C-terminus of the variable region of an immunoglobulin heavy chain and then bonding the variable region of an immunoglobulin light chain to the C-terminus thereof; and (5) a single-chain antibody (scFv) formed by bonding a linker to the C-terminus of the variable region of an immunoglobulin light chain and then bonding the variable region of an immunoglobulin heavy chain to the C-terminus thereof. The term "antibody" as used herein also includes (6) antibodies consisting of an Fab region, which is an Fc region deleted from the basic structure of an antibody in the true sense, and antibodies consisting of an Fab region and all or part of a hinge region (including Fab, F(ab') and F(ab')2), and (7) single-domain antibodies. Furthermore, the term "antibody" as used herein also includes scFv, a single-chain antibody formed by linking the light chain variable region and the heavy chain variable region via a linker. Furthermore, the term "antibody" as used herein also includes antibodies comprising all or part of an Fc region whose amino acid sequence has been modified to have affinity for TfR.

[0057] In the present invention, the term "linker" refers to, for example, a peptide chain in which multiple amino acids are linked by peptide bonds. A linker consisting of such a peptide chain can also be called a "peptide linker." In the context of this specification, "linker" can also be referred to as a "linker sequence." The N-terminus of this linker is linked to the C-terminus of a certain protein by a peptide bond, and the N-terminus of another protein is further linked to the C-terminus of the linker, thereby forming a conjugate between the two proteins via the linker.

[0058] Antibodies have a basic structure consisting of four polypeptide chains: two light chains and two heavy chains. L ) contains three complementarity-determining regions (CDRs) and a heavy chain variable region (V H ) have three complementarity-determining regions (CDRs). The three CDRs of the light chain are called CDR1, CDR2, and CDR3, starting from the N-terminus. The three CDRs of the heavy chain are also called CDR1, CDR2, and CDR3, starting from the N-terminus. However, even if some or all of these CDRs are incomplete or absent, they are included in the antibody as long as they have the property of specifically binding to a specific antigen. The variable regions (V L and V H The regions other than the CDRs of a protein are called framework regions (FRs). The FRs are called FR1, FR2, FR3, and FR4, starting from the N-terminus. Usually, the CDRs and FRs are present in the following order from the N-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0059] In one embodiment of the present invention, Fab is a fragment of a variable region and a C L One light chain containing the variable region and the C H Fab is a molecule in which one heavy chain containing one region (the constant region of the heavy chain) is bound by disulfide bonds between the cysteine ​​residues present in each heavy chain. In Fab, the heavy chain consists of a variable region and a C H In addition to the heavy chain constant region (C1), the Fab may also contain part of the hinge region, but in this case the hinge region lacks the cysteine ​​residues that are present in the hinge region and that bind the heavy chains of an antibody. In Fab, the light chain and heavy chain are connected by the light chain constant region (C2). L The cysteine ​​residues in the heavy chain constant region (C H 1 region) or hinge region. The heavy chains that form Fab are called Fab heavy chains. Fab lacks the cysteine ​​residues that bind the heavy chains of antibodies in the hinge region, so it consists of one light chain and one heavy chain. The light chain that makes up Fab consists of the variable region and C LThe heavy chain that makes up Fab contains a variable region and a C Ｈ It may consist of one domain, a variable domain, C H It may also contain a portion of the hinge region in addition to the variable region. In this case, however, the hinge region is selected so as not to contain cysteine ​​residues that link the heavy chains, so that disulfide bonds are not formed between the two heavy chains at the hinge region. In F(ab'), the heavy chain consists of a variable region and a C H In addition to the first region, the heavy chain includes all or part of the hinge region containing the cysteine ​​residues that link the heavy chains. F(ab')2 refers to a molecule in which two F(ab')s are bonded by disulfide bonds between cysteine ​​residues present in their hinge regions. The heavy chain that forms F(ab') or F(ab')2 is called a Fab' heavy chain. Furthermore, polymers such as dimers and trimers formed by multiple antibodies linked directly or via linkers are also antibodies. Furthermore, the term "antibody" as used herein includes any entity that contains a portion of an antibody molecule and has the property of specifically binding to an antigen. In other words, the term "light chain" as used herein includes those derived from a light chain and having all or part of the amino acid sequence of its variable region. Furthermore, the term "heavy chain" includes those derived from a heavy chain and having all or part of the amino acid sequence of its variable region. Therefore, as long as it has all or part of the amino acid sequence of the variable region, even those lacking the Fc region, for example, are heavy chains.

[0060] Here, Fc or Fc region refers to the C region in an antibody molecule. H 2 region (part 2 of the heavy chain constant region), and C H This refers to a region containing a fragment consisting of three regions (part 3 of the heavy chain constant region).

[0061] Furthermore, antibodies in one embodiment of the present invention also include (8) scFab, scF(ab'), and scF(ab')2, which are single-chain antibodies formed by linking the light chain and heavy chain constituting the Fab, F(ab'), or F(ab')2 shown in (6) above via a linker sequence. Here, scFab, scF(ab'), and scF(ab')2 may be formed by linking a linker sequence at the C-terminus of the light chain to which a heavy chain is further linked, or by linking a linker at the C-terminus of the heavy chain to which a light chain is further linked. Furthermore, antibodies in the present invention also include scFv, which are single-chain antibodies formed by linking the variable region of the light chain and the variable region of the heavy chain via a linker. In the case of scFv, it may be formed by attaching a linker sequence to the C-terminus of the light chain variable region and further attaching the heavy chain variable region to the C-terminus of that, or it may be formed by attaching a linker sequence to the C-terminus of the heavy chain variable region and further attaching the light chain variable region to the C-terminus of that.

[0062] Furthermore, the term "antibody" as used herein includes full-length antibodies, those shown in (1) to (8) above, and also any form of full-length antibodies in which a portion of the full-length antibody is deleted, which is a broader concept including (1) to (8).

[0063] The term "antigen-binding fragment (antibody fragment)" refers to a fragment of an antibody that retains at least a portion of its specific binding activity with an antigen. Examples of binding fragments include Fab, Fab', F(ab')2, variable region (Fv), heavy chain variable region (V), and H ) and the light chain variable region (V L ) linked with an appropriate linker, and a single-chain antibody (scFv) H ) and the light chain variable region (V L ) and scFv heavy chains (H chains) containing a portion of the constant region (C H Examples of antigen-binding fragments include minibodies, which are dimers of the antibody fragments fused to the antibody fragment 3), and other minibodies. Antigen-binding fragments also include heavy chain antibodies, light chain antibodies, VHHs, VNARs, and fragments lacking portions of these. However, they are not limited to these molecules as long as they have the ability to bind to antigens.

[0064] In the present invention, the term "single-chain antibody" refers to a protein that can specifically bind to a specific antigen, comprising an amino acid sequence containing all or part of the variable region of an immunoglobulin light chain, to which a linker sequence is attached at the C-terminus, and to which an amino acid sequence containing all or part of the variable region of an immunoglobulin heavy chain is further attached at the C-terminus. Furthermore, a protein that can specifically bind to a specific antigen, comprising an amino acid sequence containing all or part of the variable region of an immunoglobulin heavy chain, to which a linker sequence is attached at the C-terminus, and to which an amino acid sequence containing all or part of the variable region of an immunoglobulin light chain is further attached at the C-terminus, is also considered a "single-chain antibody" in the present invention. For example, the antibodies described in (2) and (3) above are included in single-chain antibodies. In single-chain antibodies in which an immunoglobulin light chain is attached to the C-terminus of an immunoglobulin heavy chain via a linker sequence, the immunoglobulin heavy chain usually lacks an Fc region. The variable region of an immunoglobulin light chain has three complementarity-determining regions (CDRs) that are involved in the antigen specificity of the antibody. Similarly, the variable region of an immunoglobulin heavy chain also has three CDRs. These CDRs are the main regions that determine the antigen specificity of an antibody. Therefore, a single-chain antibody preferably contains all three CDRs of an immunoglobulin heavy chain and all three CDRs of an immunoglobulin light chain. However, as long as the antigen-specific affinity of the anti-transferrin receptor antibody is maintained, a single-chain antibody can also be produced by deleting one or more CDRs.

[0065] In single-chain antibodies, the linker disposed between the immunoglobulin light chain and heavy chain is a peptide chain composed of preferably 2 to 50, more preferably 8 to 50, even more preferably 10 to 30, and even more preferably 12 to 18 or 15 to 25, for example, 15 or 25 amino acid residues. The amino acid sequence of such a linker is not limited, as long as the anti-transferrin receptor antibody formed by linking both chains retains affinity for the transferrin receptor. Preferably, the linker is composed of glycine alone or glycine and serine, such as the amino acid sequence Gly-Ser, Gly-Gly-Ser, Gly-Gly-Gly, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or sequences in which any of these amino acid sequences are repeated 2 to 10 times or 2 to 5 times. For example, when an ScFV is prepared by linking the variable region of an immunoglobulin light chain via a linker to the C-terminus of an amino acid sequence consisting of the entire variable region of an immunoglobulin heavy chain, a linker having the sequence of SEQ ID NO: 4 is preferably used.

[0066] In one embodiment of the present invention, the antibody is derived from a camelid (including alpaca). Some camelid antibodies consist of two heavy chains linked by disulfide bonds. Such antibodies are referred to as heavy-chain antibodies. VHHs are antibodies consisting of a single heavy chain containing the variable region of the heavy chain constituting the heavy-chain antibody, or antibodies consisting of a single heavy chain lacking the constant region (CH) constituting the heavy-chain antibody. VHHs are also an antibody within an embodiment of the present invention. Antibodies derived from camelids (including VHHs) with mutations added to the amino acid sequence to reduce antigenicity when administered to humans are also antibodies within an embodiment of the present invention. When adding mutations to the amino acids of camelid antibodies, the same mutations that can be added to antibodies described herein can be used. Another antibody within an embodiment of the present invention is an antibody consisting of two light chains linked by disulfide bonds. Such antibodies with two light chains are referred to as light-chain antibodies.

[0067] An antibody in one embodiment of the present invention is a shark-derived antibody. Shark antibodies consist of two heavy chains linked by disulfide bonds. An antibody consisting of these two heavy chains is called a heavy-chain antibody. VNAR is an antibody consisting of a single heavy chain that includes the variable region of the heavy chain that constitutes a heavy-chain antibody, or an antibody consisting of a single heavy chain that lacks the constant region (CH) that constitutes a heavy-chain antibody. VNAR is also one of the antibodies in an embodiment of the present invention. Antibodies in one embodiment of the present invention include shark antibodies (including VNARs) in which mutations have been made to the amino acid sequence of the shark antibody to reduce antigenicity when administered to humans. When mutations are made to the amino acids of a shark antibody, the same mutations that can be made to the antibodies described herein can be made. Humanized shark antibodies are also one of the antibodies in an embodiment of the present invention.

[0068] In one embodiment of the present invention, a single domain antibody refers to an antibody that has the property of specifically binding to an antigen through a single variable region. Single domain antibodies include antibodies whose variable region consists only of the variable region of a heavy chain (heavy chain single domain antibodies) and antibodies whose variable region consists only of the variable region of a light chain (light chain single domain antibodies). VHH and VNAR are types of single domain antibodies.

[0069] In one embodiment of the present invention, an anti-TfR antibody has specific affinity for TfR and can bind to TfR present on the surface of cerebrovascular endothelial cells. Anti-TfR antibodies bound to TfR can cross the blood-brain barrier (BBB). Therefore, by binding a protein with desired physiological activity to TfR to form a fusion protein, the fusion protein can cross the blood-brain barrier (BBB) ​​and exert its physiological activity in the brain. In other words, such a fusion protein can be used as a pharmaceutical agent that exerts its efficacy in the brain.

[0070] In the present invention, the term "human transferrin receptor" or "hTfR" refers to a membrane protein having the amino acid sequence shown in SEQ ID NO: 12. In one embodiment, the anti-transferrin receptor antibody of the present invention specifically binds to the portion of the amino acid sequence shown in SEQ ID NO: 12 from the 89th cysteine ​​residue from the N-terminus to the C-terminal phenylalanine (extracellular domain of hTfR), but is not limited to this.

[0071] In one embodiment, the light chain of the anti-hTfR antibody Fab has the amino acid sequence of light chain CDR1 shown in SEQ ID NO: 40 or 41, the amino acid sequence of light chain CDR2 shown in SEQ ID NO: 42 or 43, and the amino acid sequence of light chain CDR3 shown in SEQ ID NO: 44. In one embodiment, the heavy chain of the anti-hTfR antibody Fab has the amino acid sequence of heavy chain CDR1 shown in SEQ ID NO: 45 or 46, the amino acid sequence of heavy chain CDR2 shown in SEQ ID NO: 47 or 48, and the amino acid sequence of heavy chain CDR3 shown in SEQ ID NO: 49 or 50.

[0072] In one embodiment, the anti-hTfR antibody is a Fab and comprises a light chain having the amino acid sequence shown in SEQ ID NO:15 and a heavy chain having the amino acid sequence shown in SEQ ID NO:16.

[0073] The binding modes of the anti-TfR antibody and the physiologically active protein in a fusion protein of an anti-TfR antibody and a physiologically active protein include the following (a) to (h): (a) the anti-TfR antibody is a single-chain antibody in which the antibody light chain is bound to the C-terminus of the antibody heavy chain, and the physiologically active protein is bound to the N-terminus of the single-chain antibody directly or via a linker; (b) the anti-TfR antibody is a single-chain antibody in which the antibody light chain is bound to the C-terminus of the antibody heavy chain, and the physiologically active protein is bound to the C-terminus of the single-chain antibody directly or via a linker; (c) the anti-TfR antibody is a single-chain antibody in which the antibody heavy chain is bound to the C-terminus of the antibody light chain, and the physiologically active protein is bound to the N-terminus of the single-chain antibody directly or via a linker; (d) the anti-TfR antibody is a single-chain antibody in which the antibody heavy chain is bound to the C-terminus of the antibody light chain, and a physiologically active protein is bound to the C-terminus of the single-chain antibody directly or via a linker; (e) the anti-TfR antibody is an antibody comprising at least one light chain and at least one heavy chain, and a physiologically active protein is bound to the C-terminus of the antibody light chain, directly or via a linker; (f) the anti-TfR antibody is an antibody comprising at least one light chain and at least one heavy chain, and a physiologically active protein is bound to the N-terminus of the antibody light chain, directly or via a linker; (g) the anti-TfR antibody is an antibody comprising at least one light chain and at least one heavy chain, and a physiologically active protein is bound to the C-terminus of the antibody heavy chain, directly or via a linker; (h) the anti-TfR antibody is an antibody comprising at least one light chain and at least one heavy chain, and a physiologically active protein is bound to the N-terminus of the antibody heavy chain, directly or via a linker. In the above (a) to (h), the light chain can be appropriately interpreted as the light chain variable region, and the heavy chain can be appropriately interpreted as the heavy chain variable region.

[0074] In the above (a) to (h), when a linker sequence is disposed between the anti-TfR antibody and the fusion protein of a physiologically active protein, the sequence preferably consists of 1 to 50 amino acids, more preferably 1 to 17, even more preferably 1 to 10, and even more preferably 1 to 5 amino acids, but the number of amino acids constituting the linker sequence can be appropriately adjusted to 1, 2, 3, 1 to 17, 1 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, etc., depending on the protein to be bound to the anti-TfR antibody. The amino acid sequence of such a linker sequence is not particularly limited, as long as the anti-TfR antibody linked thereto retains its affinity for TfR and the protein linked via the linker sequence can exert the physiological activity of the protein under physiological conditions. The amino acid sequence is preferably composed of glycine and serine, and examples thereof include those consisting of a single amino acid of either glycine or serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or a sequence consisting of 1 to 50 amino acids consisting of 1 to 10 or 2 to 5 consecutive amino acids of these amino acid sequences, or a sequence consisting of 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, or 25 to 29 amino acids. For example, those having the amino acid sequence Gly-Ser, those having the amino acid sequence shown in SEQ ID NO: 4, and those consisting of 17 amino acids with Gly-Ser added to the C-terminus of the amino acid sequence shown in SEQ ID NO: 4 can be suitably used as linker sequences.

[0075] For convenience, in the present invention, a linker sequence (linker) that links the light chain or heavy chain of an anti-TfR antibody to a physiologically active protein is referred to as a linker sequence (linker) or a first linker sequence (first linker), and a linker sequence (linker) that links the light chain and heavy chain of an anti-TfR antibody, which is a single-chain antibody, is referred to as a second linker sequence (second linker).

[0076] In one embodiment of the present invention, a nucleic acid molecule contains, between a first inverted terminal repeat (ITR) and a second inverted terminal repeat (ITR), a gene encoding a fusion protein of an anti-TfR antibody and a physiologically active protein. The following (1) to (4) are examples of such nucleic acid molecules: (1) a fusion protein comprising a conjugate formed by binding a physiologically active protein to the C-terminus or N-terminus of the heavy chain of an anti-TfR antibody, directly or via a linker, and the light chain of an anti-TfR antibody, and a nucleotide sequence encoding an internal ribosome binding site downstream of the gene encoding the light chain and a nucleotide sequence encoding the conjugate downstream of that; (2) a fusion protein comprising a conjugate formed by binding a physiologically active protein to the C-terminus or N-terminus of the heavy chain of an anti-TfR antibody, directly or via a linker, and the light chain of an anti-TfR antibody, and a nucleotide sequence encoding an internal ribosome binding site downstream of the gene encoding the conjugate, and a nucleotide sequence encoding the light chain downstream of that; (3) The fusion protein comprises a conjugate formed by directly or via a linker linking a physiologically active protein to the C- or N-terminus of the anti-TfR antibody light chain and an anti-TfR antibody heavy chain, and further comprises a nucleotide sequence encoding an internal ribosome binding site downstream of the gene encoding the heavy chain, and further comprises a nucleotide sequence encoding the conjugate downstream of that gene; (4) The fusion protein comprises a conjugate formed by directly or via a linker linking a physiologically active protein to the C- or N-terminus of the anti-TfR antibody light chain and an anti-TfR antibody heavy chain, and further comprises a nucleotide sequence encoding an internal ribosome binding site downstream of the gene encoding the conjugate, and further comprises a nucleotide sequence encoding the heavy chain downstream of that gene. In (1) to (4) above, the nucleic acid molecule may comprise a nucleotide sequence comprising a gene expression regulatory site between the first inverted terminal repeat (ITR) and the gene encoding the fusion protein. In (1) to (4) above, the nucleotide sequence encoding the internal ribosome binding site may be replaced with a nucleotide sequence comprising a gene expression regulatory site. However, without being limited to this, when a nucleic acid molecule has two gene expression control sites, for convenience they are referred to as the first gene expression control site and the second gene expression control site, in that order from the first inverted terminal repeat (ITR) side.In addition, in the above (1) to (4), the nucleotide sequence encoding the internal ribosome binding site may be replaced with a nucleotide sequence encoding a 2A self-cleaving peptide. The 2A peptide derived from porcine teschovirus is a suitable example of a 2A self-cleaving peptide. In addition, in the above (1) to (4), the light chain can be appropriately interpreted as the light chain variable region, and the heavy chain can be appropriately interpreted as the heavy chain variable region.

[0077] In one embodiment of the present invention, when the anti-TfR antibody is a single-chain antibody, the nucleic acid molecule comprises, between the first and second inverted terminal repeats (ITRs), a gene encoding a fusion protein of the single-chain anti-TfR antibody and a physiologically active protein. The following (1) to (4) are examples of such nucleic acid molecules: (1) A molecule containing a gene encoding a fusion protein in which a light chain is linked to the C-terminus of the heavy chain of an anti-TfR antibody via a second linker, and a physiologically active protein is further linked to the C-terminus of the light chain, either directly or via a linker; (2) A molecule containing a gene encoding a fusion protein in which a heavy chain is linked to the C-terminus of the light chain of an anti-TfR antibody via a second linker, and a physiologically active protein is further linked to the C-terminus of the light chain, either directly or via a linker; (3) A molecule containing a gene encoding a fusion protein in which a heavy chain of an anti-TfR antibody is linked to the C-terminus of a physiologically active protein, either directly or via a linker, and a light chain is further linked to the C-terminus of the heavy chain; (4) A molecule containing a gene encoding a fusion protein in which a light chain of an anti-TfR antibody is linked to the C-terminus of a physiologically active protein, either directly or via a linker, and a heavy chain is further linked to the C-terminus of the heavy chain. In the above (1) to (4), the nucleic acid molecule may contain a base sequence containing a gene expression control site between the first inverted terminal repeat (ITR) and the gene encoding the fusion protein. In addition, in the above (1) to (4), the light chain can be appropriately interpreted as the light chain variable region, and the heavy chain can be appropriately interpreted as the heavy chain variable region.

[0078] In one embodiment of the present invention, the fusion protein comprises a heavy chain Fab region of an anti-TfR antibody to which human iduronate-2-sulfatase (hI2S) is linked via a linker downstream, and a light chain of the anti-TfR antibody. A specific example of this fusion protein is one in which the heavy chain Fab region of the anti-TfR antibody comprises the amino acid sequence shown in SEQ ID NO: 13, the linker comprises the amino acid sequence shown in SEQ ID NO: 4, the hI2S comprises the amino acid sequence shown in SEQ ID NO: 14, and the light chain of the anti-TfR antibody comprises the amino acid sequence shown in SEQ ID NO: 15. Here, the conjugate as a whole comprises the amino acid sequence shown in SEQ ID NO: 51.

[0079] In one embodiment of the present invention, the nucleic acid molecule comprises a gene encoding a fusion protein of an anti-TfR antibody and a biologically active protein between a first long terminal repeat (LTR) and a second long terminal repeat (LTR). The following (1) to (4) are examples of such nucleic acid molecules: (1) a fusion protein comprising a conjugate in which a physiologically active protein is bound to the C-terminal or N-terminal side of the heavy chain of an anti-TfR antibody, and the light chain of an anti-TfR antibody, and comprising a base sequence encoding an internal ribosome binding site downstream of the gene encoding the light chain, and a base sequence encoding the conjugate downstream of that; (2) a fusion protein comprising a conjugate in which a physiologically active protein is bound to the C-terminal or N-terminal side of the heavy chain of an anti-TfR antibody, and the light chain of an anti-TfR antibody, and comprising a base sequence encoding an internal ribosome binding site downstream of the gene encoding the conjugate, and a base sequence encoding the light chain downstream of that; (3) a fusion protein comprising a conjugate in which a physiologically active protein is bound to the C-terminal or N-terminal side of the light chain of an anti-TfR antibody, and the heavy chain of an anti-TfR antibody, and comprising a base sequence encoding an internal ribosome binding site downstream of the gene encoding the heavy chain, and a base sequence encoding the conjugate downstream of that; (4) The fusion protein comprises a conjugate in which a physiologically active protein is bound to the C-terminus or N-terminus of the light chain of an anti-TfR antibody and the heavy chain of the anti-TfR antibody, and further comprises a nucleotide sequence encoding an internal ribosome binding site downstream of the gene encoding the conjugate, and a nucleotide sequence encoding the heavy chain downstream of that. In (1) to (4) above, the nucleic acid molecule may contain a nucleotide sequence containing a gene expression regulatory site between the first inverted terminal repeat (LTR) and the gene encoding the fusion protein. Furthermore, in (1) to (4) above, the nucleotide sequence encoding the internal ribosome binding site may be replaced with a nucleotide sequence containing a gene expression regulatory site. When the nucleic acid molecule has two gene expression regulatory sites, for convenience, they are referred to as the first gene expression regulatory site and the second gene expression regulatory site, in that order from the first inverted terminal repeat (LTR). Furthermore, in (1) to (4) above, the nucleotide sequence encoding the internal ribosome binding site may be replaced with a nucleotide sequence encoding a 2A self-cleaving peptide. The 2A peptide derived from porcine teschovirus is a suitable example of a 2A self-cleaving peptide.In addition, in the above (1) to (4), the light chain can be appropriately interpreted as the light chain variable region, and the heavy chain can be appropriately interpreted as the heavy chain variable region.

[0080] In one embodiment of the present invention, when the anti-TfR antibody is a single-chain antibody, the nucleic acid molecule comprises, between a first long terminal repeat (LTR) and a second long terminal repeat (LTR), a gene encoding a fusion protein of the single-chain anti-TfR antibody and a biologically active protein. The following (1) to (4) are examples of such nucleic acid molecules: (1) A molecule containing a gene encoding a fusion protein in which a light chain is linked to the C-terminus of the heavy chain of an anti-TfR antibody via a second linker, and a physiologically active protein is further linked to the C-terminus of the light chain, either directly or via a linker; (2) A molecule containing a gene encoding a fusion protein in which a heavy chain is linked to the C-terminus of the light chain of an anti-TfR antibody via a second linker, and a physiologically active protein is further linked to the C-terminus of the light chain, either directly or via a linker; (3) A molecule containing a gene encoding a fusion protein in which a heavy chain of an anti-TfR antibody is linked to the C-terminus of a physiologically active protein, either directly or via a linker, and a light chain is further linked to the C-terminus of the heavy chain; (4) A molecule containing a gene encoding a fusion protein in which a light chain of an anti-TfR antibody is linked to the C-terminus of a physiologically active protein, either directly or via a linker, and a heavy chain is further linked to the C-terminus of the heavy chain. In the above (1) to (4), the nucleic acid molecule may contain a base sequence containing a gene expression control site between the first long terminal repeat (LTR) and the gene encoding the fusion protein. In addition, in the above (1) to (4), the light chain can be appropriately interpreted as the light chain variable region, and the heavy chain can be appropriately interpreted as the heavy chain variable region.

[0081] In the case of a nucleic acid molecule containing a base sequence encoding the internal ribosome binding site, the expression of one peptide chain constituting the fusion protein is controlled by the gene expression control site, and the expression of the other peptide chain is controlled by the base sequence encoding the internal ribosome binding site. The two peptide chains pair up in cells to form a fusion protein of an anti-TfR antibody and a physiologically active protein.

[0082] In one embodiment of the present invention, the affinity (binding activity) of a fusion protein of an anti-TfR antibody and a physiologically active protein for TfR can be adjusted by appropriately selecting the amino acid sequence of the anti-TfR antibody portion constituting the fusion protein and / or by appropriately selecting the binding mode between the anti-TfR antibody and the physiologically active protein. Here, appropriately selecting the binding mode between the anti-TfR antibody and the physiologically active protein means, for example, (1) if the original fusion protein has a physiologically active protein bound to the heavy chain of the anti-TfR antibody, binding the physiologically active protein to the light chain instead of the heavy chain of the anti-TfR antibody, (2) if the original fusion protein has a physiologically active protein bound to the light chain of the anti-TfR antibody, binding the physiologically active protein to the heavy chain instead of the light chain of the anti-TfR antibody, or (3) if the original fusion protein has a physiologically active protein bound to the N-terminus of the heavy or light chain of the anti-TfR antibody. (3) When a biologically active protein is bound to the heavy or light chain of an anti-TfR antibody, the biologically active protein is bound to the C-terminus of the heavy or light chain of the anti-TfR antibody. (4) When a biologically active protein is bound to the C-terminus of the heavy or light chain of an anti-TfR antibody, the biologically active protein is bound to the N-terminus of the heavy or light chain of the anti-TfR antibody. (5) When a biologically active protein is bound to the heavy or light chain of an anti-transferrin receptor antibody via a linker, the amino acid sequence of the linker (including changing the length of the linker) or the linker is deleted. The binding mode can also be changed by combining (1) to (5). These changes in the binding mode can, for example, create steric hindrance, inhibiting the binding between the anti-TfR antibody portion of the fusion protein and TfR, resulting in a decrease in the affinity (binding activity) of the fusion protein as a whole for TfR.

[0083] Fusion proteins whose overall affinity (binding activity) for TfR is adjusted to a certain value suppress the effect of binding to reticulocytes, which express a high level of TfR on their cell surface, impairing reticulocyte function. Impaired reticulocyte function reduces the number of red blood cells in the blood, resulting in anemia. Fusion proteins with a certain affinity for TfR suppress the anemia symptoms that occur when administered to the body. The severity of anemia can be objectively assessed by comparing the hemoglobin concentration in the blood with that of a normal individual. Hemoglobin concentration and the severity of anemia are inversely correlated; the lower the hemoglobin concentration, the greater the severity of anemia.

[0084] Furthermore, if the affinity (binding activity) of the fusion protein of an anti-TfR antibody and a physiologically active protein for TfR as a whole is very high, it may strongly inhibit the binding of endogenous transferrin to TfR, inhibiting the transport of transferrin into cells via TfR and causing iron deficiency in the cells. The fusion protein of one embodiment of the present invention can reduce the risk of cells becoming iron deficient when an anti-TfR antibody is administered in vivo.

[0085] When a fusion protein of an anti-TfR antibody and a physiologically active protein is administered as a drug by subcutaneous injection, intramuscular injection, intravenous injection, or other means, if anemia symptoms are observed as a side effect, the administration can be discontinued. However, when gene therapy is performed using a gene encoding the fusion protein, the fusion protein is continuously released into the body from the administered gene over the long term. Therefore, even if a side effect occurs, its expression cannot be interrupted. One embodiment of the present invention suppresses the occurrence of side effects expected to occur due to the continuous expression of the fusion protein in the patient's body by appropriately adjusting the affinity (binding activity) for TfR.

[0086] When a gene encoding a fusion protein of an anti-TfR antibody and a physiologically active protein is used in gene therapy, the affinity (binding activity) of the fusion protein as a whole for TfR becomes particularly problematic. Therefore, a gene encoding a fusion protein with an adjusted affinity (binding activity) for TfR as a whole can be used by appropriately adjusting the amino acid sequence and / or the binding mode between TfR and the physiologically active protein, for example, by adding mutations to the anti-TfR antibody portion of the fusion protein. Use of such a gene can suppress anemia symptoms caused by the expression of the fusion protein in vivo, and enable the development of safe gene therapy therapeutic agents that can translocate the fusion protein into cells via TfR on the cells and exert their physiological activity. In other words, a gene encoding such a fusion protein can be used as part of a safe gene therapy drug. In particular, if the fusion protein is designed to cross the blood-brain barrier (BBB) ​​by binding to TfR present on the surface of cerebrovascular endothelial cells, the gene encoding the fusion protein can be used as part of a highly safe gene therapy drug for disorders of the brain and central nervous system.

[0087] When a gene encoding the fusion protein is used as part of a drug for gene therapy, the binding activity of the anti-TfR antibody or the fusion protein of the anti-TfR antibody and a physiologically active protein to TfR should be measured using, for example, the 50% effective concentration (EC 50The 50% effective concentration can be measured, for example, by the method described in Example 15. In one embodiment of the present invention, the binding activity of a fusion protein of an anti-TfR antibody and a physiologically active protein to TfR, when measured by the measurement method, is such that the 50% effective concentration is preferably 3 nM to 10 μM, and more preferably 3 nM to 5 μM, for example, 3 nM to 2 μM, 3 nM to 1 μM, 3 nM to 500 nM, 3 nM to 200 nM, 3 nM to 100 nM, 3.5 nM to 2 μM, 3.5 nM to 1 μM, 3.5 nM to 500 nM, 3.5 nM to 200 nM, 3.5 nM to 100 nM, 4 nM to 2 μM, 4 nM to 1 μM, 4 nM to 500 nM, 4 nM to 200 nM, 4 nM to 100 nM, 5 nM to 5 μM, 5 nM to 1 μM, 5 nM to 500 nM, 5 nM to 5 μM, The 50% effective concentration (EC ) of the anti-TfR antibody is 10 nM to 200 nM, 5 nM to 100 nM, 10 nM to 5 μM, 10 nM to 1 μM, 5 nM to 500 nM, 5 nM to 200 nM, 5 nM to 100 nM, and 10 to 100 nM. When the anti-TfR antibody is an anti-human TfR antibody, the 50% effective concentration (EC ) of the anti-TfR antibody is determined by using, for example, a human transferrin receptor extracellular domain having the amino acid sequence from positions 87 to 763 of the amino acid sequence set forth in SEQ ID NO: 12 in place of the mouse transferrin receptor extracellular domain in the method described in Example 15. 50 ) may be obtained.

[0088] In one embodiment of the present invention, a nucleic acid molecule can be used as part of a therapeutic agent for AAV-based gene therapy. When wild-type AAV infects a human host cell alone, the viral genome is site-specifically integrated into chromosome 19 via the inverted terminal repeats (ITRs) present at both ends of the viral genome. Although the genes of the viral genome integrated into the host cell genome are rarely expressed, when the cell is infected with a helper virus, AAV is excised from the host genome and infectious viral replication begins. When the helper virus is an adenovirus, the genes responsible for the helper function are E1A, E1B, E2A, VA1, and E4. Here, when the host cell is 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 cell. In one embodiment of the present invention, an AAV vector refers to a vector used to produce rAAV virions, which has a base sequence including a first inverted terminal repeat (ITR) and a second inverted terminal repeat (ITR) derived from the AAV genome, and which can incorporate a foreign gene. The site in the AAV vector where a foreign gene can be incorporated can be located between the first and second inverted terminal repeats (ITR). An AAV vector into which a foreign gene has been incorporated is also an AAV vector.

[0089] The wild-type AAV genome 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 chromosomal integration of the viral genome. The cap gene is responsible for the production of three capsid proteins (VP1, VP2, and VP3).

[0090] In the wild-type AAV genome, ITRs are present at both ends, with the rep gene and the cap gene present between the ITRs. This genome is encapsulated in a capsid to form an AAV virion. In one embodiment, a recombinant AAV virion (rAAV virion) is a wild-type AAV genome in which the region containing the rep gene and the cap gene has been replaced with a gene encoding a foreign protein, and the resulting virion is encapsulated in a capsid encoded by the cap gene. However, the recombinant AAV virion (rAAV virion) is not limited to this, and any wild-type AAV genome in which a portion of the wild-type AAV genome has been substituted with a gene encoding a foreign protein and then encapsulated in a capsid.

[0091] rAAV virions can be used for therapeutic purposes as vectors to deliver foreign genes to cells. In a specific embodiment of the present invention, a pharmaceutical composition is provided comprising an rAAV virion and a pharmaceutically acceptable carrier.

[0092] Three types of plasmids are typically used to produce recombinant AAV virions used to introduce foreign genes into cells, tissues, or living organisms: (1) a plasmid (plasmid 1) having a structure containing a nucleotide sequence including a first inverted terminal repeat (ITR) and a nucleotide sequence including a second inverted terminal repeat (ITR) derived from a virus such as AAV, and a gene encoding a desired protein located between these two ITRs; (2) a plasmid (plasmid 2) containing an AAV Rep gene that has the function necessary to integrate the nucleotide sequence of the region flanked by the ITR sequences (including the ITR sequences) into the genome of a host cell, and a gene encoding an AAV capsid protein; and (3) a plasmid (plasmid 3) containing the adenovirus E2A region, E4 region, and VA1 RNA region. However, this is not limited to this, and two types of plasmids can also be used: one formed by linking two of plasmids 1 to 3 and the other by linking the remaining plasmid. Furthermore, one formed by linking plasmids 1 to 3 can also be used. The desired protein in the present invention is a fusion protein of a ligand and a physiologically active protein.

[0093] Generally, to produce recombinant AAV virions, these three types of plasmids are first introduced into host cells, such as HEK293 cells, containing adenovirus E1a and E1b genes integrated into their genomes, by a standard transfection technique. Then, the region containing the nucleotide sequence including the first and second inverted terminal repeats (ITRs), as well as the gene encoding the desired protein located between these two ITRs, is replicated in the host cells, and the resulting single-stranded DNA is packaged into AAV capsid proteins to form recombinant AAV virions. These recombinant AAV virions are infectious and can be used to introduce foreign genes into cells, tissues, or living organisms. In one embodiment of the present invention, the foreign gene is a gene encoding a fusion protein of an anti-TfR antibody and a physiologically active protein. The fusion protein is expressed from this gene in the host cells.

[0094] The Rep protein of adeno-associated virus (AAV) is encoded by the AAV rep gene. The Rep protein has a function necessary for integrating, for example, the AAV genome into the genome of a host cell via the ITRs present in the genome. There are multiple subtypes of Rep proteins, but two types, Rep68 and Rep78, are required for integration of the AAV genome into the genome of a host cell. Rep68 and Rep78 are translation products of two types of mRNA transcribed by alternative splicing from the same gene. In the present invention, the term "AAV Rep protein" refers to at least the two types of proteins, Rep68 and Rep78.

[0095] In one embodiment of the present invention, the nucleotide sequence encoding the Rep protein of an adeno-associated virus (nucleotide sequence of the Rep region) refers to a nucleotide sequence encoding at least Rep68 and Rep78, or a nucleotide sequence obtained by adding a mutation thereto. The Rep protein is preferably that of AAV serotype 2, but is not limited thereto and may be that of any of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, or 11. A preferred example of a nucleotide sequence encoding the Rep protein of wild-type AAV serotype 2 is one having the nucleotide sequence shown in SEQ ID NO: 32.

[0096] Furthermore, as long as Rep68 exerts its function, it may be a mutant in which 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 has been modified by substitution, deletion, addition, or the like. In the present invention, Rep68 with these mutations is also included in Rep68.

[0097] When amino acids in the amino acid sequence of wild-type Rep68 are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of wild-type Rep68 are deleted, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Rep68 obtained by adding mutations that combine these amino acid substitutions and deletions is also included in Rep68. When amino acids are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus of wild-type Rep68. Rep68 obtained by adding mutations that combine these amino acid additions, substitutions, and deletions is also included in Rep68. The amino acid sequence of the mutated Rep68 preferably exhibits 80% or more identity to the amino acid sequence of wild-type Rep68, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.

[0098] Furthermore, as long as Rep78 exerts its function, it may be a mutant in which 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 has been modified by substitution, deletion, addition, or the like. In the present invention, Rep78 with these mutations is also included in Rep78.

[0099] When amino acids in the amino acid sequence of wild-type Rep78 are substituted with other amino acids, the number of substituted amino acids is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of wild-type Rep78 are deleted, the number of deleted amino acids is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Rep78 obtained by adding mutations that combine these amino acid substitutions and deletions is also included in Rep78. When amino acids are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus of wild-type Rep78. Rep78 obtained by adding mutations that combine these amino acid additions, substitutions, and deletions is also included in Rep78. The amino acid sequence of the mutated Rep78 preferably exhibits 80% or more identity to the amino acid sequence of wild-type Rep78, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.

[0100] The 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 functional equivalent of a Rep protein may be a mutant of a wild-type Rep protein.

[0101] The base sequence shown in SEQ ID NO: 32 can be modified by substitution, deletion, addition, or the like, so long as it encodes functional Rep68 and Rep78. When bases in the base sequence shown in SEQ ID NO: 32 are substituted with other bases, the number of substituted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence shown in SEQ ID NO: 32 are deleted, the number of deleted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Furthermore, a base sequence encoding a Rep protein can also be obtained by adding mutations that combine these base substitutions and deletions. When bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence shown in SEQ ID NO: 32 or to the 5' or 3' end. A base sequence encoding a Rep protein can also be obtained by adding mutations that combine these base additions, substitutions, and deletions. The mutated base sequence preferably exhibits 80% or more identity, more preferably 85% or more identity, even 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 shown in SEQ ID NO: 32. However, when these mutations are added, it is preferable that the start codons of the genes encoding the Rep proteins Rep68 and Rep78 are ACG.

[0102] In one embodiment of the present invention, the nucleotide sequence encoding the Cap protein of an adeno-associated virus (nucleotide sequence of the Cap region) refers to a nucleotide sequence encoding at least VP1, one of the proteins that constitute the AAV capsid, or a nucleotide sequence containing a mutated nucleotide sequence thereof. VP1 is preferably that of AAV serotype 8, but is not limited thereto and may be any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. A preferred example of a nucleotide sequence of the Cap region of serotype 8 is one containing the nucleotide sequence shown in SEQ ID NO: 31.

[0103] Furthermore, as long as VP1 exhibits its function, it may be modified by substitution, deletion, addition, or the like in the amino acid sequence of wild-type VP1 of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In one embodiment of the present invention, VP1 with these mutations is also included in VP1.

[0104] When bases in the base sequence shown in SEQ ID NO: 31 are substituted with other bases, the number of substituted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence shown in SEQ ID NO: 31 are deleted, the number of deleted bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Furthermore, a mutation that combines these base substitutions and deletions can also be used as a base sequence encoding a Cap protein. When bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence shown in SEQ ID NO: 31 or to the 5' end or 3' end. A mutation that combines these base additions, substitutions, and deletions can also be used as a nucleic acid molecule encoding a Cap protein. The mutated base sequence preferably exhibits 80% or more identity to the base sequence shown in SEQ ID NO: 31, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.

[0105] In one embodiment of the present invention, recombinant AAV virions can be obtained using an AAV vector, in which a nucleic acid molecule comprising a foreign gene is packaged between the first and second ITRs. Because recombinant AAV virions are infective, they can be used to introduce foreign genes into cells, tissues, or living organisms. The foreign gene in the present invention is a gene encoding a fusion protein of an anti-TfR antibody and a physiologically active protein. In cells, etc., into which the gene is introduced, the fusion protein is expressed from this gene. Recombinant virions in which a nucleic acid molecule comprising a foreign gene is packaged between the first and second ITRs and can be used to introduce genes into cells, etc. include those that use AAV (AAV vector systems) and those that use adenovirus (adenovirus vector systems). In the adenovirus vector system, recombinant adenovirus virions can be obtained in which a nucleic acid molecule comprising a foreign gene is packaged between the first and second ITRs. Because recombinant adenovirus virions are infective, they can be used to introduce foreign genes into cells, tissues, or living organisms.

[0106] When a gene encoding a fusion protein of an anti-TfR antibody and a physiologically active protein is used as a component of a pharmaceutical for gene therapy using an AAV virus, preferred embodiments of the binding activity of the fusion protein to TfR are described in detail below. These embodiments can also be applied to those using adenovirus, lentivirus, and retrovirus. That is, the binding activity of the fusion protein to TfR can be measured, for example, by the 50% effective concentration (EC 50) is preferably 0.5 nM to 10 μM, more preferably 3 nM to 5 μM, for example, 0.5 nM to 5 μM, 0.5 nM to 1 μM, 1 nM to 2 μM, 1 nM to 1 μM, 3 nM to 2 μM, 3 nM to 1 μM, 3 nM to 1 μM, 3 to 500 nM, 3 to 200 nM, 3 to 100 nM, 3.5 nM to 2 μM, 3.5 nM to 1 μM, 3.5 to 500 nM, 3.5 to 200 nM, 3.5 to 100 nM, 4 nM to 2 μM, 4 nM to 1 μM, 4 to 500 nM, 4 to 200 nM, 4 to 100 nM, 5 nM to 5 μM, 5 nM to 1 μM, 5 to 500 nM, 5 to 200 nM, 5 to 100 nM, 10 nM to 5 μM, 10 nM to 1 μM, 5 to 500 nM, 5 to 200 nM, 5 to 100 nM, and 10 to 100 nM. 50 This value is particularly preferred when the fusion protein is a fusion protein of an anti-TfR antibody and a lysosomal enzyme, for example, a fusion protein of a TfR antibody and human I2S, or a fusion protein of a TfR antibody and human GAA. When the anti-TfR antibody is anti-human TfR, the 50% effective concentration (EC ) can be determined by using, for example, a human transferrin receptor extracellular domain having the amino acid sequence from positions 87 to 763 of the amino acid sequence shown in SEQ ID NO: 12 in place of the mouse transferrin receptor extracellular domain in the method described in Example 15. 50 ) may be obtained. The rAAV virion, in which a DNA fragment containing a gene encoding a fusion protein is packaged, is one embodiment of a pharmaceutical for gene therapy using the AAV virus.

[0107] The dosage of the drug should be adjusted appropriately according to the patient's symptoms, etc., but 50 For rAAV virions packaged with a gene encoding a fusion protein of an anti-TfR antibody and a physiologically active protein, the dose is preferably 1.0 × 10 10 ~1.0×10 14 vg / kg, e.g., 1.0 × 10 10 ~5.0×10 13 vg / kg, 1.0×10 10~2.0×10 13 vg / kg, 1.0×10 10 ~1.0×10 13 vg / kg, 1.0×10 11 ~1.0×10 13 vg / kg, 1.0×10 11 vg / kg, 1.0×10 12 vg / kg, 2.0×10 12 vg / kg, 5.0×10 12 vg / kg, 6.0×10 12 vg / kg, 1.0×10 13 vg / kg. In other words, the dosage can be safely increased or decreased over a wide range without causing anemia. Furthermore, there is no particular limitation on the administration interval of the pharmaceutical; for example, if the blood concentration of the fusion protein falls below a predetermined concentration, the pharmaceutical can be administered again. Here, falling below a predetermined concentration means that the blood concentration of the fusion protein is 5% or less, 10% or less, 20% or less, or 50% or less compared to the blood concentration measured on any day between 1 week and 10 years after administration, for example, between 3 and 6 weeks after administration. As long as the blood concentration of the fusion protein does not decrease, the pharmaceutical can be administered only once.

[0108] When a gene encoding a fusion protein of an anti-TfR antibody and a physiologically active protein is used as a component of a pharmaceutical for gene therapy using the AAV virus, the binding activity of the fusion protein to TfR can be controlled by limiting the dose per administration, which is applicable to all fusion proteins of anti-TfR antibodies and physiologically active proteins, as well as the 50% effective concentration (EC ) measured by the ELISA method described in Example 15. 50 ) can be less than 3 nM, for example, 0.5 to 3 nM, 0.8 to 3 nM, or 1 to 3 nM. In this case, the dose per administration is 1.0 × 10 13 Not to exceed 7.5 × 10 vg / kg, preferably 7.5 × 10 12 vg / kg or less, preferably 6.0×10 12 vg / kg or less, more preferably 5.0 × 10 12 vg / kg or less, 3.0×10 12 vg / kg or less, or 2.5 x 1012 The dose is adjusted to 1.0 × 10 10 ~7.5×10 12 vg / kg, 1.0×10 11 ~7.5×10 12 vg / kg, 5.0×10 11 ~7.5×10 12 vg / kg, 1.0×10 10 ~6.0×10 12 vg / kg, 1.0×10 11 ~6.0×10 12 vg / kg, 5.0×10 11 ~6.0×10 12 vg / kg, 1.0×10 10 ~5.0×10 12 vg / kg, 1.0×10 11 ~5.0×10 12 vg / kg, 5.0×10 11 ~5.0×10 12 vg / kg, 1.0×10 10 ~3.0×10 12 vg / kg, 1.0×10 11 ~3.0×10 12 vg / kg, 5.0×10 11 ~3.0×10 12 vg / kg, 1.0×10 10 ~2.5×10 12 vg / kg, 1.0×10 11 ~2.5×10 12 vg / kg, 5.0×10 11 ~2.5×10 12 vg / kg, 1.0×10 10 ~2.0×10 12 vg / kg, 1.0×10 11 ~2.0×10 12 vg / kg, or 5.0 × 10 11 ~2.0×10 12 vg / kg. However, EC 50 Administration of drugs with EC values ​​of less than 3 nM may cause anemia, so the patient's physical condition must be carefully monitored after administration. 50A drug with an EC value of 3 nM or higher can be considered to be excellent for gene therapy. 50 Administration of drugs with a value of less than 3 nM may be clinically valuable because it may have a beneficial effect.

[0109] Furthermore, when a gene encoding a fusion protein of an anti-TfR antibody and a physiologically active protein is used as a component of a pharmaceutical for gene therapy using the AAV virus, and the physiologically active protein is human I2S, the binding activity of the fusion protein with TfR can be measured by the EC200 / EC2010 (EC2010 / EC2010) assayed by the ELISA method described in Example 15 in addition to the above. 50 The value can be less than 3 nM, for example, 0.5 to 3 nM, 0.8 to 3 nM, or 1 to 3 nM (0.5 to 4 nM, 0.8 to 4 nM, or 1 to 4 nM). In this case, the dose per administration is 1.0 × 10 13 Not to exceed 7.5 × 10 vg / kg, preferably 7.5 × 10 12 vg / kg or less, preferably 6.0×10 12 vg / kg or less, more preferably 5.0 × 10 12 vg / kg or less, and even more preferably 3.0 × 10 12 vg / kg or less, 2.5×10 12 The dose is adjusted to 1.0 × 10 10 ~7.5×10 12 vg / kg, 1.0×10 11 ~7.5×10 12 vg / kg, 5.0×10 11 ~7.5×10 12 vg / kg, 1.0×10 10 ~6.0×10 12 vg / kg, 1.0×10 11 ~6.0×10 12 vg / kg, 5.0×10 11 ~6.0×10 12 vg / kg, 1.0×10 10 ~5.0×10 12 vg / kg, 1.0×10 11 ~5.0×10 12vg / kg, 5.0×10 11 ~5.0×10 12 vg / kg, 1.0×10 10 ~3.0×10 12 vg / kg, 1.0×10 11 ~3.0×10 12 vg / kg, 5.0×10 11 ~3.0×10 12 vg / kg, 1.0×10 10 ~2.5×10 12 vg / kg, 1.0×10 11 ~2.5×10 12 vg / kg, 5.0×10 11 ~2.5×10 12 vg / kg, 1.0×10 10 ~2.0×10 12 vg / kg, 1.0×10 11 ~2.0×10 12 vg / kg, or 5.0 × 10 11 ~2.0×10 12 Adjusted to vg / kg.

[0110] That is, when a gene encoding a fusion protein of an anti-TfR antibody and human I2S is used, the dose per administration is preferably 7.5 × 10 12 vg / kg or less, preferably 6.0×10 12 vg / kg or less, more preferably 5.0 × 10 12 vg / kg or less, and even more preferably 3.0 × 10 12 vg / kg or less, 2.5×10 12 The EC value of the fusion protein and TfR is adjusted to 0.5 vg / kg or less. 50 Values ​​of 0.5 nM or more, 0.8 nM or more, or 1 nM or more can also be used. For example, EC 50The value of is preferably 0.5 nM to 1 μM, more preferably 0.5 to 100 nM, even more preferably 0.5 to 10 nM, and still more preferably 0.5 to 5 nM, for example, 0.5 to 4 nM, 0.5 to 3 nM, 0.8 nM to 1 μM, 0.8 to 500 nM, 0.8 to 100 nM, 0.8 to 10 nM, 0.8 to 5 nM, 0.8 to 4 nM, 0.8 to 3 nM, 1.0 nM to 1 μM, 1.0 to 500 nM, 1.0 to 100 nM, 1.0 to 10 nM, 1.0 to 5 nM, 1.0 to 4 nM, 1.0 to 3 nM, 1 nM to 5 μM, 1 nM to 1 μM, 1 to 500 nM, 1 to 200 nM, 1 to 100 nM, 1 nM to 5 μM, 1 nM to 1 μM, 1 to 500 nM, 1 to 200 nM, or 1 to 100 nM.

[0111] However, when using a gene encoding a fusion protein of an anti-TfR antibody and human I2S, the EC of the fusion protein with TfR is 50 Administration of drugs with an EC value of less than 3 nM (less than 4 nM) may cause anemia, so there is a drawback in that the dosage setting is limited. In addition, the patient's physical condition after administration must be carefully monitored. From this perspective, 50 A drug with an EC value of 3 nM or higher (or 4 nM or higher) can be considered excellent as a drug for gene therapy. 50 Administration of a drug with a value of less than 3 nM (less than 4 nM) may be clinically valuable because it may have a beneficial effect.

[0112] When a gene encoding a fusion protein of an anti-TfR antibody and human I2S is used, there is no particular limitation on the administration interval. For example, the fusion protein can be administered again when the blood concentration of the fusion protein falls below a predetermined concentration. Here, "falling below a predetermined concentration" refers to the fusion protein concentration falling to 5% or less, 10% or less, 20% or less, or 50% or less compared to the blood concentration measured any day between 1 week and 10 years after administration, for example, between 3 and 6 weeks after administration. As long as the blood concentration of the fusion protein does not decrease, the administration frequency can be limited to one time.

[0113] The dosage described above when using a gene encoding a fusion protein of an anti-TfR antibody and human I2S can also be applied to a fusion protein of an anti-TfR antibody and another physiologically active substance, such as a fusion protein of an anti-TfR antibody and another lysosomal enzyme.

[0114] Lentiviruses are viruses belonging to the Lentivirus genus of the Orthoretrovirinae subfamily of the Retroviridae family. They possess a single-stranded (+)-strand RNA genome (ssRNA). The lentivirus genome contains essential genes, gag (encoding structural proteins including capsid protein), pol (encoding enzymes including reverse transcriptase), and env (encoding envelope proteins required for host cell binding), flanked by two long terminal repeats (5'LTR and 3'LTR). In addition, the lentivirus genome contains accessory genes, Rev (encoding a protein that binds to the RRE (rev responsive element) present in the viral RNA and transports the viral RNA from the nucleus to the cytoplasm), tat (encoding a protein that binds to the TAR in the 5'LTR and enhances the promoter activity of the LTR), and other genes, such as vif, vpr, vpu, and nef.

[0115] Lentiviruses are enveloped viruses that infect cells by fusing the envelope with the cell membrane. Furthermore, lentiviruses are RNA viruses, and reverse transcriptase is present in virions. After lentivirus infection, single-stranded plus-strand DNA is replicated from the (+)-strand RNA genome by the reverse transcriptase, and double-stranded DNA is then synthesized. Proteins, which are components of virions, are expressed from this double-stranded DNA, and the (+)-strand RNA genome is packaged into these proteins, resulting in proliferation of virions. In one embodiment of the present invention, the lentivirus vector system has been developed based on the genome of HIV-1, a type of lentivirus, but is not limited thereto.

[0116] First-generation lentiviral vector systems consist of three plasmids: a packaging plasmid, an Env plasmid, and a transfer plasmid. The packaging plasmid contains the gag and pol genes under the control of a CMV promoter or the like. The Env plasmid contains the env gene under the control of a CMV promoter. The transfer plasmid contains a 5'LTR, an RRE, a gene encoding a desired protein under the control of a CMV promoter, and a 3'LTR. By introducing these into host cells using a standard transfection technique, recombinant virions can be obtained in which a nucleic acid molecule containing a foreign gene between the first and second ITRs is packaged in the capsid protein. The packaging plasmid of the first-generation lentiviral vector system also contains the virus-derived auxiliary genes rev, tat, vif, vpr, vpu, and nef. Here, the desired protein in the present invention is a fusion protein of an anti-TfR antibody and a physiologically active protein. Preferably, the promoter controlling the gene encoding the desired protein is a promoter other than the CMV promoter, such as the SV40 early promoter, the human elongation factor-1α (EF-1α) promoter, the human ubiquitin C promoter, the retroviral Rous sarcoma virus LTR promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, the mouse albumin promoter, the human albumin promoter, and the human α-1 antitrypsin promoter. For example, a synthetic promoter having the nucleotide sequence shown in SEQ ID NO: 9, which includes the mouse albumin promoter downstream of the mouse α-fetoprotein enhancer (mouse α-fetoprotein enhancer / mouse albumin promoter), can also be used.

[0117] Like the first-generation lentiviral vectors, second-generation lentiviral vectors consist of three plasmids: a packaging plasmid, an Env (envelope) plasmid, and a transfer plasmid. However, the non-essential accessory genes vif, vpr, vpu, and nef have been deleted from the packaging plasmid.

[0118] The third-generation lentiviral vector system consists of four plasmids: a packaging plasmid, an Env plasmid (envelope plasmid), a Rev plasmid, and a transfer plasmid. In the third generation, the rev gene present in the packaging plasmid in the second generation has been separated into the Rev plasmid. Furthermore, the tat gene has been deleted from the packaging plasmid. Furthermore, the TAR gene in the 5'LTR of the transfer plasmid has been replaced with a CMV promoter.

[0119] Because recombinant lentiviral virions have infectivity, they can be used to introduce foreign genes into cells, tissues, or living organisms. The foreign gene in the present invention is a gene encoding a fusion protein of an anti-transferrin receptor antibody and a physiologically active protein. In cells, etc., into which the gene has been introduced, the fusion protein is expressed from this gene.

[0120] Retroviruses have a single-stranded (+) strand RNA genome (ssRNA). The viral genome encodes the gag (encoding structural proteins including capsid protein), pol (encoding a group of enzymes including reverse transcriptase), env (encoding envelope proteins required for binding to host cells), and packaging signal (Ψ) genes, which are flanked by two long terminal repeats (LTR, 5'LTR, and 3'LTR). When a retrovirus infects a host cell, the (+) strand RNA and reverse transcriptase are translocated into the cell and reverse transcribed into double-stranded DNA.

[0121] Retroviral vector systems, primarily based on murine leukemia viruses, have been developed by segmenting the viral genome to eliminate its infectivity while deleting its self-replication ability, thereby eliminating pathogenicity and enhancing safety. First-generation retroviral vector systems consist of a packaging plasmid (the viral genome excluding the packaging signal) and a transfer plasmid (containing the packaging signal, a portion of gag, and a foreign gene flanked by the 5'LTR and 3'LTR). Therefore, homologous recombination at the gag sequence shared by both the packaging and transfer plasmids results in the emergence of a self-replicating retrovirus, i.e., a replication-competent (RC) virus. In second-generation retroviral vector systems, the 3' LTR of the first-generation packaging plasmid is replaced with a poly(A) addition signal. This requires simultaneous homologous recombination at two sites, the gag sequence and upstream of the poly(A) addition signal, for RC virus to be generated. This extremely low probability of this occurs, enhancing safety. Third-generation vectors consist of three plasmids, with the second-generation packaging plasmid further segmented into a plasmid encoding gag / pol and a plasmid encoding env. This means that for RC virus to be generated, homologous recombination must occur simultaneously at three locations, making the probability of this occurring extremely low, further increasing safety.

[0122] Generally, to produce recombinant retroviral virions, these packaging plasmids and transfer plasmids are first introduced into host cells by a common transfection technique. Then, the region containing the 5'LTR and 3'LTR and the gene encoding the desired protein located between these two LTRs is replicated in the host cell, and the resulting single-stranded (+)-strand RNA is packaged into retroviral capsid proteins to form recombinant retroviral virions. Because these recombinant retroviral virions are infectious, they can be used to introduce foreign genes into cells, tissues, or living organisms. The foreign gene in the present invention is a gene encoding a fusion protein of an anti-transferrin receptor antibody and a physiologically active protein. In cells, etc., into which the gene has been introduced, the fusion protein is expressed from this gene.

[0123] In one embodiment of the present invention, nucleic acid molecules can be encapsulated in liposomes, lipid nanoparticles (LNPs), or the like. Liposomes are spherical vesicles with a lipid bilayer and are primarily composed of phospholipids, particularly phosphatidylcholine. However, liposomes are not limited to this and may contain other lipids, such as egg yolk phosphatidylethanolamine, as long as they form a lipid bilayer. Because cell membranes are primarily composed of phospholipid bilayers, liposomes have the advantage of being highly biocompatible. Lipid nanoparticles are particles with a diameter of 10 nm to 1000 nm, typically less than approximately 200 nm, primarily composed of lipids. They can encapsulate hydrophobic (lipophilic) molecules and are primarily composed of biocompatible lipids, such as triglycerides, diglycerides, monoglycerides, fatty acids, and steroids. When a gene encapsulated in liposomes or lipid nanoparticles is administered into a living body, it is believed to fuse directly with the cell membrane or be taken up by the cell via endocytosis, then translocate to the nucleus and be introduced into the cell. Compared to gene transfer using viral vectors, gene transfer methods using liposomes or lipid nanoparticles are superior in that there is no limit to the size of the gene to be transferred and that they are highly safe. Liposomes, lipid nanoparticles, etc. encapsulating the nucleic acid molecules of the present invention can be used to transfer genes for fusion proteins of ligands and physiologically active proteins into cells, tissues, or living organisms. The fusion protein is expressed in cells, etc., into which the gene has been introduced. In this specification, the term "liposomes, lipid nanoparticles, etc." includes not only the liposomes and lipid nanoparticles described above, but also polymer nanoparticles, micelles, emulsions, nanoemulsions, microspheres, nanospheres, microcapsules, nanocapsules, dendrimers, nanogels, metal nanoparticles, and any other nano- or microparticles that can be used as drug delivery systems (DDS).

[0124] In the present invention, the behavior of nucleic acid molecules introduced into cells, tissues, or living organisms in the form of a plasmid, encapsulated in a recombinant viral virion, or encapsulated in a liposome, lipid nanoparticle, or the like is exemplified below in (1) to (6). However, the behavior of nucleic acid molecules is not limited to these. (1) The nucleic acid molecule is single-stranded (+) strand RNA. When introduced into a cell, a gene encoding a fusion protein of an anti-transferrin receptor antibody and a physiologically active protein contained in the nucleic acid molecule is translated, resulting in the expression of the fusion protein. (2) The nucleic acid molecule is single-stranded (+) strand RNA. When introduced into a cell, the nucleic acid molecule is reverse transcribed to form single-stranded (+) strand DNA, which is then transcribed and translated to express the fusion protein. (3) The nucleic acid molecule is single-stranded (+) strand RNA or (-) strand RNA. When introduced into a cell, the nucleic acid molecule is reverse transcribed to form double-stranded DNA, which is then transcribed and translated to express the fusion protein. (4) The nucleic acid molecule is single-stranded (+) or (-) RNA, and when introduced into a cell, the nucleic acid molecule is reverse transcribed to become double-stranded DNA, which then undergoes random or homologous recombination with the genome of the host cell and is integrated into the genome, where the integrated DNA is transcribed and translated to express the fusion protein. (5) The nucleic acid molecule is single-stranded (+) DNA, and when introduced into a cell, the nucleic acid molecule is transcribed and translated to express the fusion protein. (6) The nucleic acid molecule is double-stranded DNA, and when introduced into a cell, the nucleic acid molecule is transcribed and translated to express the fusion protein.

[0125] Nucleic acid molecules in the form of plasmids, encapsulated in recombinant viral virions, or encapsulated in liposomes, lipid nanoparticles, etc. can be introduced into cells, tissues, or organisms.

[0126] When the nucleic acid molecule is to be introduced into a living body, the nucleic acid molecule is administered parenterally, such as by subcutaneous injection, intramuscular injection, or intravenous injection, together with a pharmaceutically acceptable excipient, i.e., as a pharmaceutical composition, in the form of a plasmid, encapsulated in a recombinant viral virion, or encapsulated in a liposome, lipid nanoparticle, etc. Such pharmaceutical compositions are provided to medical institutions, for example, as aqueous liquid preparations packed in vials, or as prefilled syringe-type or cartridge-type aqueous liquid preparations that are pre-filled in syringes.

[0127] Nucleic acid molecules in the form of plasmids, encapsulated in recombinant virus virions, or encapsulated in liposomes, lipid nanoparticles, etc. can be used as various medicines. The use of nucleic acid molecules in which the fusion protein encoded by the nucleic acid molecule specifically recognizes the transferrin receptor and the physiologically active protein is a human lysosomal enzyme is described in detail below.

[0128] When the human lysosomal enzyme is α-L-iduronidase, it can be used as a therapeutic agent for central nervous system disorders in Hurler syndrome or Hurler-Scheie syndrome; when it is iduronate-2-sulfatase, it can be used as a therapeutic agent for central nervous system disorders in Hunter syndrome; when it is acid α-glucosidase, it can be used as a therapeutic agent for central nervous system disorders in Pompe disease; when it is glucocerebrosidase, it can be used as a therapeutic agent for central nervous system disorders in Gaucher disease; and when it is β-galactosidase, it can be used as a therapeutic agent for central nervous system disorders in GM1-gangliosidosis types 1 to 3. If it is GM2 activator protein, it can be used as a therapeutic agent for central nervous system disorders in GM2-gangliosidosis AB variant; if it is β-hexosaminidase A, it can be used as a therapeutic agent for central nervous system disorders in Sandhoff disease and Tisachs disease; if it is β-hexosaminidase B, it can be used as a therapeutic agent for central nervous system disorders in Sandhoff disease; if it is N-acetylglucosamine-1-phosphotransferase, it can be used as a therapeutic agent for central nervous system disorders in I-cell disease; and if it is α-mannosidase, it can be used as a therapeutic agent for central nervous system disorders in α-mannosidosis. If it is β-mannosidase, it is used as a therapeutic agent for central nervous system disorders in β-mannosidosis; if it is galactosylceramidase, it is used as a therapeutic agent for central nervous system disorders in Krabbe disease; if it is saposin C, it is used as a therapeutic agent for central nervous system disorders in Gaucher-like storage disease; if it is arylsulfatase A, it is used as a therapeutic agent for central nervous system disorders in metachromatic leukodystrophy; if it is α-L-fucosidase, it is used as a therapeutic agent for central nervous system disorders in fucosidosis; and if it is aspartylglucosaminidase, it is used as a therapeutic agent for central nervous system disorders in As a therapeutic agent for central nervous system disorders in aspartylglucosaminuria, if it is α-N-acetylgalactosaminidase, it can be used as a therapeutic agent for central nervous system disorders in Schindler disease and Kawasaki disease, if it is acid sphingomyelinase, it can be used as a therapeutic agent for central nervous system disorders in Niemann-Pick disease, if it is α-galactosidase A, it can be used as a therapeutic agent for central nervous system disorders in Fabry disease, and if it is β-glucuronidase, it can be used as a therapeutic agent for central nervous system disorders in Sly syndrome,When the enzyme is either acetyl-CoA α-glucosaminide N-acetyltransferase or N-acetylglucosamine-6-sulfatase, it can be used as a therapeutic agent for central nervous system disorders in Sanfilippo syndrome (particularly, when the enzyme is heparan N-sulfatase, it can be used as Sanfilippo syndrome A; when the enzyme is α-N-acetylglucosaminidase, it can be used as Sanfilippo syndrome B; when the enzyme is acetyl-CoA α-glucosaminide N-acetyltransferase, it can be used as Sanfilippo syndrome C; and when the enzyme is N-acetylglucosamine-6-sulfatase, it can be used as Sanfilippo syndrome D). When the enzyme is acid ceramidase, it can be used as a therapeutic agent for central nervous system disorders in Farber disease. When the enzyme is amylo-1,6-glucosidase, it can be used as a therapeutic agent for central nervous system disorders in Cori's disease (Forbes-Coli disease). If it is sialidase, it can be used as a therapeutic agent for central nervous system disorders caused by sialidase deficiency; if it is aspartylglucosaminidase, it can be used as a therapeutic agent for central nervous system disorders caused by aspartylglucosaminuria; if it is palmitoyl protein thioesterase-1 (PPT-1), it can be used as a therapeutic agent for central nervous system disorders caused by neuronal ceroid lipofuscinosis or Santavuori-Haltias disease; if it is tripeptidyl peptidase-1 (TPP-1), it can be used as a therapeutic agent for central nervous system disorders caused by neuronal ceroid lipofuscinosis or Jansky-Bielschowsky disease; if it is hyaluronidase-1, it can be used as a therapeutic agent for central nervous system disorders caused by hyaluronidase deficiency; and if it is either CLN1 or CLN2, it can be used as a therapeutic agent for central nervous system disorders caused by Batten disease.

[0129] When the nucleic acid molecule is introduced into a cell, the type of cell is not particularly limited, but examples include mesenchymal stem cells, dental pulp-derived stem cells, hematopoietic stem cells, embryonic stem cells, endothelial stem cells, mammary stem cells, intestinal stem cells, hepatic stem cells, pancreatic stem cells, neural stem cells, and iPS cells.

[0130] The cells transfected with the nucleic acid molecule express the fusion protein and can then be transplanted into a patient for therapeutic purposes. For example, cells transfected with a nucleic acid molecule in which the biologically active protein is a human lysosomal enzyme can be used for the above-mentioned purposes.

[0131] The present invention will be described in more detail below with reference to examples, but it is not intended that the present invention be limited to these examples.

[0132] Example 1: Construction of pAAV-mMAP-mscFv-GS3-hI2S Vector A DNA fragment was synthesized containing, from the 5' end, a ClaI site, a mouse α-fetoprotein enhancer / mouse albumin promoter, a chicken β-actin / MVM chimeric intron, a gene encoding a conjugate of a mouse anti-mouse transferrin receptor single-chain antibody (mouse anti-mTfRscFv antibody) to which human iduronate-2-sulfatase (hI2S) is linked via a linker sequence shown in SEQ ID NO: 4 at the C-terminus, the conjugate having the amino acid sequence shown in SEQ ID NO: 19 (mscFv-GS3-hI2S), a bovine growth hormone poly(A) sequence, and the nucleotide sequence shown in SEQ ID NO: 18, including a BglII site. This DNA fragment was digested with ClaI and BglII. The pAAV-CMV vector (Takara Bio Inc.) was digested with ClaI and BglII, and the above-mentioned restriction enzyme-treated DNA fragment was inserted into it. The resulting plasmid was designated the pAAV-mMAP-mscFv-GS3-hI2S vector (Figure 1). The pAAV-mMAP-mscFv-GS3-hI2S vector contains, from upstream to downstream, a functional equivalent of the first AAV-ITR (SEQ ID NO: 7), a mouse α-fetoprotein enhancer / mouse albumin promoter (SEQ ID NO: 9), a chicken β-actin / MVM chimeric intron (SEQ ID NO: 10), a gene encoding mscFv-GS3-hI2S (SEQ ID NO: 20), a bovine growth hormone polyA signal (SEQ ID NO: 17), and a functional equivalent of the second AAV-ITR (SEQ ID NO: 8). Furthermore, the pAAV-mMAP-mscFv-GS3-hI2S vector contains an ampicillin resistance gene and an origin of replication (ColE1 ori). The mouse anti-mTfRscFv antibody is composed of the mouse anti-mTfR heavy chain variable region linked to the C-terminus of the mouse anti-mTfR heavy chain variable region via a linker having the amino acid sequence shown in SEQ ID NO:4.

[0133] Example 2: Construction of pAAV-mMAP-mscFv2-GS3-hI2S Vector A DNA fragment was synthesized containing, from the 5' end, a ClaI site, a mouse α-fetoprotein enhancer / mouse albumin promoter, a chicken β-actin / MVM chimeric intron, a gene encoding a conjugate (mscFv2-GS3-hI2S) having the amino acid sequence shown in SEQ ID NO:22, in which hI2S was linked via the linker sequence shown in SEQ ID NO:4 to the C-terminus of a mouse anti-mouse transferrin receptor single-chain antibody (mouse anti-mTfRscFv antibody 2) with amino acid substitutions introduced at two positions in the CDR of the heavy chain and two positions in the CDR of the light chain of mscFv, a bovine growth hormone poly(A) sequence, and the nucleotide sequence shown in SEQ ID NO:21, including a BglII site. This DNA fragment was digested with ClaI and BglII. The pAAV-CMV vector (Takara Bio) was digested with ClaI and BglII, and the above-mentioned restriction enzyme-treated DNA fragment was inserted into it. The resulting plasmid was designated the pAAV-mMAP-mscFv2-GS3-hI2S vector (Figure 2). The pAAV-mMAP-mscFv2-GS3-hI2S vector contains, from upstream to downstream, a functional equivalent of the first AAV-ITR (SEQ ID NO: 7), a mouse α-fetoprotein enhancer / mouse albumin promoter (SEQ ID NO: 9), a chicken β-actin / MVM chimeric intron (SEQ ID NO: 10), a gene encoding mscFv2-GS3-hI2S (SEQ ID NO: 23), a bovine growth hormone polyA signal (SEQ ID NO: 17), and a functional equivalent of the second AAV-ITR (SEQ ID NO: 8). Furthermore, the pAAV-mMAP-mscFv2-GS3-hI2S vector contains an ampicillin resistance gene and an origin of replication (ColE1 ori).

[0134] Example 3: Construction of pAAV-mMAP-hI2S-mscFv2 Vector. A DNA fragment containing, from the 5' end, a ClaI site, a mouse α-fetoprotein enhancer / mouse albumin promoter, a chicken β-actin / MVM chimeric intron, a gene encoding a conjugate (hI2S-mscFv2) in which mouse anti-mTfRscFv antibody 2 is linked to the C-terminus of hI2S and has the amino acid sequence shown in SEQ ID NO: 25, a bovine growth hormone polyA sequence, and the nucleotide sequence shown in SEQ ID NO: 24, including a BglII site, was synthesized. This DNA fragment was digested with ClaI and BglII. The pAAV-CMV vector (Takara Bio Inc.) was digested with ClaI and BglII, and the above-mentioned restriction enzyme-treated DNA fragment was inserted into it. The resulting plasmid was designated pAAV-mMAP-hI2S-mscFv2 vector (Figure 3). The pAAV-mMAP-hI2S-mscFv2 vector has a structure containing, from upstream to downstream, a functional equivalent of the first AAV-ITR (SEQ ID NO: 7), a mouse α-fetoprotein enhancer / mouse albumin promoter (SEQ ID NO: 9), a chicken β-actin / MVM chimeric intron (SEQ ID NO: 10), a gene encoding hI2S-mscFv2 (SEQ ID NO: 26), a bovine growth hormone polyA signal (SEQ ID NO: 17), and a functional equivalent of the second AAV-ITR (SEQ ID NO: 8). The pAAV-mMAP-hI2S-mscFv2 vector further contains an ampicillin resistance gene and an origin of replication (ColE1 ori).

[0135] Example 4: Construction of pAAV-mMAP-hI2S Vector A DNA fragment was synthesized containing, from the 5' end, a ClaI site, a mouse α-fetoprotein enhancer / mouse albumin promoter, a chicken β-actin / MVM chimeric intron, a gene encoding hI2S having the amino acid sequence shown in SEQ ID NO:14, a bovine growth hormone polyA sequence, and the nucleotide sequence shown in SEQ ID NO:27, which contains a BglII site. This DNA fragment was digested with ClaI and BglII. The pAAV-CMV vector (Takara Bio Inc.) was digested with ClaI and BglII, and the above-mentioned restriction enzyme-treated DNA fragment was inserted into it. The resulting plasmid was designated pAAV-mMAP-hI2S vector (Figure 4). The pAAV-mMAP-hI2S vector has a structure containing, from upstream to downstream, a functional equivalent of the first AAV-ITR (SEQ ID NO: 7), a mouse α-fetoprotein enhancer / mouse albumin promoter (SEQ ID NO: 9), a chicken β-actin / MVM chimeric intron (SEQ ID NO: 10), a gene encoding hI2S (SEQ ID NO: 28), a bovine growth hormone polyA signal (SEQ ID NO: 17), and a functional equivalent of the second AAV-ITR (SEQ ID NO: 8). The pAAV-mMAP-hI2S vector further contains an ampicillin resistance gene and an origin of replication (ColE1 ori).

[0136] Example 5: Construction of pR2(mod)C6 Vector A DNA fragment containing the nucleotide sequence shown in SEQ ID NO:29, which includes from the 5' end an AfeI site, an RsrII site, a BsrGI site, an AvrII site, an ampicillin resistance gene, an origin of replication (ColE1 ori), an RsrII site, the 5' portion of the AAV2 Rep region (including the p5 promoter), and a SacII site, was synthesized. This DNA fragment was digested with AfeI and SacII. The pRC6 vector (Takara Bio Inc.) was digested with AfeI and SacII, and the above-mentioned restricted synthetic gene was inserted into it. The resulting plasmid was designated pR2(mod)C6 vector.

[0137] Example 6: Construction of pR2(mod)C8 Vector A DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 30, which includes, from the 5' end, a HindIII site, the 3' portion of the AAV2 Rep region, the AAV8 Cap region, the p5 promoter, an RsrII site, and a BsrGI site, was synthesized. This DNA fragment was digested with HindIII and BsrGI. The pR2(mod)C6 vector was digested with HindIII and BsrGI, and the above-mentioned restriction enzyme-treated synthetic gene was inserted into it. The resulting plasmid was designated pR2(mod)C8 vector (Figure 5).

[0138] [Example 7] Production of rAAV virions HEK293 cells, a cell line derived from human embryonic kidney cells, were used as host cells. HEK293 cells were placed in a 10-layer CellStack chamber (Corning) at a density of 1.59 x 10 per CellStack chamber. 8The cells were seeded and cultured in 10% FBS-containing DMEM medium (Thermo Fisher Scientific) at 37°C under 5% CO2 for 3 days. The following DNA solutions containing the following combinations of plasmids were prepared for transfection: (1) pHelper vector (Takara Bio), pR2(mod)C8 vector, and pAAV-mMAP-mscFv-GS3-hI2S vector; (2) pHelper vector (Takara Bio), pR2(mod)C8 vector, and pAAV-mMAP-mscFv2-GS3-hI2S vector; (3) pHelper vector (Takara Bio), pR2(mod)C8 vector, and pAAV-mMAP-hI2S-mscFv2 vector; and (4) pHelper vector (Takara Bio), pR2(mod)C8 vector, and pAAV-mMAP-hI2S vector. Each solution contained three plasmids at equimolar concentrations, with a total DNA concentration of 1.0 mg / mL and a total volume of 2.2 mL. Polyethylenimine was added to this solution at a weight ratio of DNA (μg):polyethylenimine (μg) = 1:2, and DMEM medium was added to adjust the volume to 1.0 L to create the transfection solution. After 3 days of culture, all of the culture supernatant was removed from the 10-layer cell stack chamber, and the entire 1.0 L of transfection solution was added. The cells were cultured at 37°C in the presence of 5% CO2 for 3 days to produce rAAV virions. Some of the rAAV virions produced in the host cells were released into the culture supernatant, while others remained within the host cells. The resulting rAAV virions were designated rAAV-mscFv-GS3-hI2S, rAAV-mscFv2-GS3-hI2S, rAAV-hI2S-mscFv2, and rAAV-hI2S, respectively.rAAV-mscFv-GS3-hI2S is expected to express mscFv-GS3-hI2S when infected into cells, rAAV-mscFv2-GS3-hI2S is expected to express mscFv2-GS3-hI2S when infected into cells, rAAV-hI2S-mscFv2 is expected to express hI2S-mscFv2 when infected into cells, and rAAV-hI2S is expected to express hI2S when infected into cells.

[0139] Example 8: Cell Lysis and Released Nucleic Acid Removal Step After the completion of the culture in Example 7, one-ninth the volume of an aqueous solution containing 0.5 M HEPES, 20 mM MgCl2, and 10% (w / v) polysorbate 20 was added to each cell culture solution. Benzonase (Merck) endonuclease was then added to a concentration of 50 U / mL, and the mixture was 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 3 hours at room temperature using a stirrer and a stirrer. One-ninth the volume of the solution was then added to a 5 M NaCl aqueous solution containing 10% (w / v) sucrose, and the mixture was stirred to obtain a cell lysate.

[0140] Example 9 Preparation of Affinity Step Loading Solution Each cell lysate obtained in Example 8 was aliquoted 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 μm pore size Nalgene Rapid-Flow PES filter unit (Thermo Fisher Scientific) to obtain a cell lysate containing rAAV virions. A quarter volume of 20 mmol / L Tris buffer (pH 7.5) containing 2 mmol / L MgCl2 and 2000 mmol / L arginine was added to the lysate, which was used as the affinity step loading solution.

[0141] Example 10: Chromatographic Purification of rAAV Virions Using Affinity Resin A column (1.6 cm diameter, 10 cm height (approximately 20 mL column volume)) packed with POROS Capture Select AAVX resin (Thermo Fisher Scientific) was equilibrated with 20 mmol / L Tris buffer (pH 7.5) containing 2 mmol / L MgCl2 and 150 mmol / L NaCl. The affinity step loading solution obtained in Example 9 was passed through the column at a flow rate of 5 mL / min to adsorb rAAV virions to the resin. The column was then washed with at least five column volumes of 20 mmol / L Tris buffer (pH 7.5) containing 2 mmol / L MgCl2 and 400 mmol / L arginine, followed by at least three column volumes of 20 mmol / L Tris buffer (pH 7.5) containing 2 mmol / L MgCl2 and 150 mmol / L NaCl. Next, rAAV virions adsorbed to the resin were eluted by passing at least three column volumes of 10 mM citrate buffer (pH 3.5) containing 2 mM MgCl2 through the column to obtain a fraction containing rAAV virions. This fraction was adjusted to pH 7.0 with 500 mmol / L Tris buffer (pH 8.5) containing 2 mM MgCl2, and used as the affinity step elution fraction.

[0142] Example 11: Concentration of rAAV virions using an ultrafiltration membrane. Spectrum MicroKros Hollow Fiber Modules 100 kDa, 75 cm, equilibrated with 20 mmol / L Tris buffer (pH 7.5) containing 2 mmol / L MgCl and 150 mmol / L NaCl, were used. 2 The affinity step elution fractions obtained in Example 10 were transferred to a column chromatography column (Repligen) at a flow rate of 7 L / min / ml. 2The solution was concentrated to approximately 8 mL by circulating at the following flux: Next, the ultrafiltration membrane was washed twice with 2 mL of 20 mmol / L Tris buffer (pH 7.5) containing 2 mmol / L MgCl2 and 150 mmol / L NaCl, and this washing solution was combined with the previously collected solution to obtain a total of approximately 12 mL of concentrated solution.

[0143] Example 12: Purification of rAAV virions by ultracentrifugation using iodixanol. 60% iodixanol (multipurpose density gradient centrifugation medium OptiPrep TM A 45% 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 (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 1M NaCl / PBS-MK buffer (PBS-MK buffer containing 1 mol / L sodium chloride) to 60% iodixanol.

[0144] 39 mL, Quick-Seal TMA round-top polypropylene tube, 25 x 89 mm (Beckman Coulter, Inc.), was layered from the bottom with 5 mL, 8 mL, 5 mL, and 8 mL of 60% iodixanol with phenol red, followed by 45% iodixanol, 25% iodixanol with phenol red, and 15% iodixanol, respectively. Approximately 12 mL of the concentrate obtained in Example 11 was added to each tube, and the tube was then filled with 1M NaCl / PBS-MK buffer. The tube's inlet was then sealed using a cordless tube topping sealer kit (Beckman Coulter, Inc.), and the tube was placed in the 70Ti rotor of an Optima XPN ultracentrifuge (Beckman Coulter, Inc.) and centrifuged at 63,000 rpm (408,500 × g) for 2 hours. After centrifugation, a Tygon E-LFL tube with a needle was connected to the bottom of the tube using a fraction recovery system (Beckman Coulter). An 18 G syringe needle was inserted into the top of the tube to create an air hole, and the solvent was fractionated in 0.5 mL increments from the bottom of the tube using a peristaltic pump. The 9th to 13th or 10th to 14th fractions were collected to obtain fractions containing purified rAAV virions.

[0145] Example 13: Concentration and buffer exchange process of rAAV virions using an ultrafiltration membrane Each rAAV virion-containing fraction obtained in Example 12 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, 2.7 mmol / L potassium chloride, and 0.001% F-68), and this was passed through Spectrum MicroKros Hollow fiber Modules 100 kDa, 75 cm, equilibrated with 350 mmol / L NaCl / PBS buffer. 2 (Repligen), 7 L / min / m 2 The solution was concentrated to approximately 15 mL by circulating at a flow rate of 7 L / min / m. Then, a 10 DV buffer exchange was performed with 350 mmol / L NaCl / PBS buffer.2 The ultrafiltration membrane was then concentrated to approximately 8 mL by circulating the solution at the following flow rate, and the concentrate was collected. The ultrafiltration membrane was then washed twice with 2 mL of 350 mmol / L NaCl / PBS buffer, and this wash was combined with the previously collected concentrate to obtain a total of approximately 12 mL of concentrate. This concentrate was then further concentrated by centrifugation using an Amicon Ultra 15 Centrifugal Filter Unit (50 kDa, Merck) at ≤3200 rpm (2100 × g) for ≤20 minutes, yielding a rAAV virion solution in which the solvent had been replaced with 350 mmol / L NaCl / PBS buffer. Each of the resulting rAAV virion solutions was used in the following experiments.

[0146] [Example 14] Measurement of the amount of rAAV genome contained in an rAAV virion solution The amount of viral genome in the rAAV virion solution obtained in Example 13 was measured by droplet digital PCR. The measurement method is described in detail below.

[0147] To an 8-tube tube (TOHO), 5 μL of rAAV virion solution was added 2 μL of Recombinant DNase I (Takara Bio), 5 μL of 10× DNase I Buffer (supplied with Recombinant DNase I, Takara Bio), 5 μL of 10% F-68-containing water for injection, and 33 μL of water for injection (Otsuka Distilled Water, Otsuka Pharmaceutical Factory), and the mixture was incubated at 37°C for 30 minutes to digest DNA not encapsulated in the rAAV virion.

[0148] Each DNase I-treated rAAV virion solution was diluted appropriately with DNA suspension buffer (10 mM Tris buffer (pH 8.0) containing 0.1 mM ethylenediaminetetraacetic acid, 100 μg / mL polyA, and 0.001% F-68). The diluted rAAV virion solution was then incubated at 95°C for 10 minutes to thermally disrupt the rAAV virions, and the sample solution for droplet digital PCR was prepared.

[0149] A FAM-labeled 20x primer / probe mix was prepared containing 1.0 μM forward primer (primer SI-1, SEQ ID NO:33), 1.0 μM reverse primer (primer SI-2, SEQ ID NO:34), and 0.25 μM probe (probe SI-1, SEQ ID NO:35, modified at the 5' end with FAM as a reporter dye and at the 3' end with BHQ1 as a quencher dye). A HEX-labeled 20x primer / probe mix was also prepared containing 1.0 μM forward primer (primer SI-3, SEQ ID NO:36), 1.0 μM reverse primer (primer SI-4, SEQ ID NO:37), and 0.25 μM probe (probe SI-2, SEQ ID NO:38, modified at the 5' end with HEX as a reporter dye and at the 3' end with BHQ1 as a quencher dye).

[0150] A PCR reaction mixture was prepared by adding 12 μL of ddPCR Supermix for probes (no dUTP) (BioRad), 1.2 μL of FAM-labeled 20x primer / probe mix, 1.2 μL of HEX-labeled 20x primer / probe mix, 0.3 μL of MspI (NEB), and 5.3 μL of water for injection to 4 μL of droplet digital PCR sample solution. A 20 μL suspension of PCR reaction solution and 70 μL of Droplet Generator Oil for Probes (BioRad) was prepared using a droplet generator (BioRad). These droplets were then used in a QX200 Droplet Digital PCR System (BioRad). The PCR conditions were denaturation (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 (98°C, 10 minutes). Droplets positive for both FAM and HEX were defined as rAAV-positive droplets, and the rAAV genome quantity (vg: viral genome) was calculated using QuantaSoft Version 1.7 (BioRad). The DNA regions amplified in this PCR were the bovine growth hormone polyA and the internal region of the ITR.

[0151] Example 15: Evaluation of mouse TfR binding activity of conjugates of mouse anti-mTfRscFv antibody and hI2S Genes encoding three types of conjugates of mouse anti-mTfRscFv antibody and hI2S, mscFv-GS3-hI2S, mscFv2-GS3-hI2S, and hI2S-mscFv2, were each incorporated into an expression vector. The resulting expression vectors were introduced into CHO cells to obtain expression cells for each of the three types of conjugates. The resulting expression cells were cultured to express the conjugates as recombinant proteins, and each recombinant protein was recovered from the culture supernatant.

[0152] The binding activity of the resulting three proteins, mscFv-GS3-hI2S, mscFv2-GS3-hI2S, and hI2S-mscFv2, to the mouse TfR receptor was measured by ELISA. The ELISA was performed roughly as follows: 100 μL of the mouse transferrin receptor extracellular domain, which has the amino acid sequence from positions 87 to 763 of the amino acid sequence set forth in SEQ ID NO: 39, diluted in 0.05 M bicarbonate buffer (pH 9.6) to a concentration of 10 μg / mL, was added to each well of a 96-well microtiter plate (Nunc). The plate was then left to stand at room temperature for at least 1 hour to allow the antibody to adsorb to the plate. Next, 300 μL of TBS containing 1% BSA and 0.05% Tween 20 was added to each well, and the plate was left to stand at room temperature for 1 hour. After washing each well three times with TBS containing 0.05% Tween 20 (TBS-T), mscFv-GS3-hI2S was diluted to 300, 100, 33.33, 11.11, 3.70, 1.23, 0.41, and 0.14 nM, mscFv2-GS3-hI2S was diluted to 600, 200, 66.67, 22.22, 7.41, 2.47, 0.82, and 0.27 nM, and hI2S-mscFv2 was diluted to 1200, 400, 133.33, 44.44, 14.81, 4.94, 1.65, and 0.55 nM with TBS containing 0.1% BSA and 0.05% Tween 20. 100 μL of each diluted solution was added to each well, and the plate was left to stand at room temperature for at least 1 hour. After washing the plate three times with TBS-T, 100 μL of anti-hI2S monoclonal antibody diluted to 0.5 μg / mL in TBS containing 0.1% BSA and 0.05% Tween 20 was added to each well and the plate was left to stand at room temperature for at least 1 hour. After washing three times with TBS-T, 100 μL of HRP-labeled anti-mouse IgG polyclonal antibody (Bethyl) diluted to 50 ng / mL in TBS containing 0.1% BSA and 0.05% Tween 20 was added to each well and the plate was left to stand at room temperature for at least 1 hour.After washing each well three times with TBS-T, 50 μL of TMB Stabilized Substrate for Horseradish Peroxidase (Promega) was added to each well and allowed to stand at room temperature for 5-10 minutes. Next, 100 μL of stop solution (1N hydrochloric acid) was added to each well, and the absorbance at 450 nm of each well was measured using a plate reader (SpectraMax iD3, Molecular Devices). The EC values ​​were calculated from the measured values. 50 The binding activity of each protein to mTfR was evaluated.

[0153] EC of mscFv-GS3-hI2S, mscFv2-GS3-hI2S, and hI2S-mscFv2 against mouse TfR 50 were 2.4 nM, 13.2 nM, and 96.7 nM, respectively.

[0154] Example 16: Evaluation of the efficacy of each rAAV virion using I2S-KO mice. The efficacy of each rAAV when administered to a living body was evaluated using I2S gene knockout mice (I2S-KO mice). The I2S-KO mice used were male mice aged 14 to 15 weeks (at the start of administration). The rAAV virions, rAAV-mscFv-GS3-hI2S, rAAV-mscFv2-GS3-hI2S, rAAV-hI2S-mscFv2, and rAAV-hI2S, were each administered at a dose of 1.0 × 10 to male I2S-KO mice. 11 vg / kg, 1.0×10 12 vg / kg, and 1.0×10 13 A single dose of 1000mg / kg was administered into the tail vein (N=3 per group). A normal control group consisting of male wild-type mice and a pathological control group consisting of male I2S-KO mice were also included (N=3 per group). The normal and pathological control groups were administered saline (Otsuka Pharmaceutical Factory).

[0155] One and six weeks after administration, mice were anesthetized with a triple-drug mixture of Betolfar (Meiji Seika Pharma), midazolam (Sandoz), and Domitor (Nippon Zenyaku Kogyo). The thoracotomy was performed and cardiac blood was collected. Blood was collected in EDTA-2K-coated blood collection tubes (Capiject, Terumo), anticoagulated, and then used for hemoglobin concentration measurement. The remaining anticoagulated blood was placed on ice and centrifuged (2000 × g, 20 minutes, 4°C) to recover plasma. After cardiac blood collection, the mice were perfused with saline (Otsuka Pharmaceutical Factory), and the brains were removed. A portion of the brain tissue was removed for quantification of heparan sulfate in the brain tissue and quickly immersed in liquid nitrogen for rapid freezing. Separately, a portion of the brain tissue was weighed and then rapidly frozen by immersion in liquid nitrogen. The frozen brain tissue was homogenized in RIPA Buffer (Wako Pure Chemical Industries, Ltd.) containing 0.025% Protease Inhibitor Cocktail (Sigma-Aldrich), and then centrifuged to recover the supernatant.

[0156] Example 17: Measurement of rAAV-derived protein concentrations in plasma and brain tissue. The plasma prepared in Example 16 and the supernatant obtained by homogenizing brain tissue were quantified for the rAAV-derived proteins rAAV-mscFv-GS3-hI2S, rAAV-mscFv2-GS3-hI2S, rAAV-hI2S-mscFv2, and rAAV-hI2S. Quantification was generally performed as follows: 150 μL 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. Next, each standard curve standard containing known concentrations of mscFv-GS3-hI2S, hI2S-mscFv2, or hI2S, each expressed in CHO cells by standard methods, and each sample were added to a mixed solution of biotinylated anti-hI2S monoclonal antibody and sulfo-modified anti-hI2S monoclonal antibody, and the mixture was shaken for 1 hour to prepare antibody reaction solutions. The anti-hI2S monoclonal antibody was obtained by culturing an anti-hI2S-producing hybridoma prepared by standard methods using splenocytes obtained from a mouse immunized with hI2S having the amino acid sequence set forth in SEQ ID NO: 14. One of the obtained monoclonal antibodies was modified using Biotin Labeling Kit-NH2 (Dojindo Laboratories) according to the attached protocol to obtain a biotinylated anti-hI2S monoclonal antibody. Another monoclonal antibody obtained was modified with MSD GOLD SULFO-TAG NHS-Ester (Mesoscale Diagnostics) according to the attached protocol to obtain a sulfo-modified anti-hI2S monoclonal antibody. After removing the blocking solution from each well and washing with PBS containing 0.05% Tween 20 (PBST, Sigma-Aldrich), 25 μL of antibody reaction solution was added to each well and shaken for at least 1 hour. The antibody reaction solution was then removed, washed with PBST, and 150 μL of 4X Read Buffer (Mesoscale Diagnostics) diluted with an equal volume of water for injection (Otsuka Pharmaceutical Factory) was added. Sector TMThe luminescence intensity from each well was measured using an Imager 6000 (Mesoscale Diagnostics). A calibration curve was created from the measurements of each standard sample, and the amount of rAAV-derived protein contained per mL of sample (each rAAV-derived protein concentration) was calculated by interpolating the measurements of each sample. Note that when measuring mscFv2-GS3-hI2S, mscFv-GS3-hI2S was used as the standard sample for the calibration curve.

[0157] The results of measuring the plasma concentrations of the above rAAV-derived proteins are shown in Figure 6. Plasma concentrations of each rAAV-derived protein increased dose-dependently in all rAAV virion-administered groups. Furthermore, in all groups, expression levels remained similar or slightly increased 1 and 6 weeks after administration. These results indicate that rAAV virions injected intravenously into mice were taken up into cells, and that at least the genes encoded by rAAV were transcribed and translated within the cells, resulting in the expression of rAAV-derived proteins, which were then released into the blood.

[0158] Next, the results of measuring the concentration of rAAV-derived proteins in brain tissue are shown in Figure 7. The concentration of expressed proteins in brain tissue increased dose-dependently in all rAAV virion-administered groups. The expression concentration of mscFv-GS3-hI2S in brain tissue in the rAAV-mscFv-GS3-hI2S-administered group was significantly higher than that in the same dose groups of rAAV-mscFv2-GS3-hI2S, rAAV-hI2S-mscFv2, and rAAV-hI2S, and the concentration did not fluctuate significantly between 1 week and 6 weeks after administration. The 1.0 × 10 13 In the 1.0 × 10 vg / kg group, brain tissue concentrations increased approximately 3.4-fold and 2.1-fold, respectively, from 1 week to 6 weeks after administration. 13 The brain tissue concentrations in the vg / kg group did not fluctuate significantly between 1 week and 6 weeks after administration, and were less than half of those in the other groups at the same dose.

[0159] The above results demonstrate that administration of rAAV-mscFv-GS3-hI2S, rAAV-mscFv2-GS3-hI2S, and rAAV-hI2S-mscFv2 resulted in the expected expression of the anti-mTfR antibody scFv-hI2S conjugate in the mouse body, as expected, and that the anti-mTfR antibody scFv crossed the BBB and reached central nervous system tissues by binding to mTfR.

[0160] Example 18 Quantitation of Heparan Sulfate in Brain Tissue Quantitation of heparan sulfate (HS) in the brain was generally carried out by the following method. Note that heparan sulfate is a substrate for the enzyme I2S.

[0161] The solutions (a) to (l) used in the test were prepared as follows: (a) MeCN / water: 2 mL of water for injection (Otsuka Pharmaceutical Co., Ltd.) and 18 mL of acetonitrile (Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed to prepare MeCN / water. (b) Deuterium-labeled solvent: 240 μL of acetyl chloride (Sigma-Aldrich Co., Ltd.) was added dropwise to 1.5 mL of methanol-d4 (Sigma-Aldrich Co., Ltd.) in an ice bath to prepare the deuterium-labeled solvent. (c) Mobile phase A: 475 mL of water for injection and 25 mL of 1 M ammonium formate aqueous solution (Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed to prepare mobile phase A. (d) Mobile phase B: A 93:7 (v / v) mixture of acetonitrile and mobile phase A was used to prepare mobile phase B. (e) Heparan sulfate standard stock solution (HS standard stock solution): Heparan sulfate (Iduron) was dissolved in water for injection to prepare a 5.0 mg / mL solution. This solution was used as the HS standard stock solution. (f) Heparan sulfate internal standard solution (HS internal standard solution): 40 μL of the HS standard stock solution was measured and placed in a borosilicate screw-cap test tube, and the solvent was evaporated under a nitrogen stream. 400 μL of deuterium-labeled solvent was added to the dried product, stirred, and then reacted at 65°C for 75 minutes to perform deuterated methanolysis. After the reaction, the solvent was evaporated under a nitrogen stream. 500 μL of MeCN / water was added to the dried product, and the mixture was sonicated for 30 minutes. This solution was used as the heparan sulfate internal standard solution (HS internal standard solution). (i) Internal standard solution: 1 μL of the HS internal standard solution was added to 1 mL of methanol, stirred, and then sonicated for 30 minutes. This solution was used as the internal standard solution. (j) Calibration curve sample: 980 μL of water for injection was measured out, and 10 μL of the HS standard stock solution was added to each solution to prepare a solution containing 50 μg / mL of heparan sulfate. This solution was diluted with water for injection to prepare a solution containing 5000 ng / mL of heparan sulfate. This solution was serially diluted with water for injection to prepare solutions containing heparan sulfate at concentrations of 25, 50, 100, 250, 500, 1000, 2500, and 5000 ng / mL. 20 μL of this solution was measured out and dispensed into borosilicate screw-cap test tubes. This solution was used as the calibration curve sample.(k) Tissue extraction solution: 1 mL of polyoxyethylene (10) octylphenyl ether was added to physiological saline to make a total volume of 500 mL. This solution was used as the tissue extraction solution. (l) 10% ammonium carbonate solution: 5 g of ammonium carbonate was dissolved in 50 mL of water for injection. This solution was used as the 10% ammonium carbonate solution.

[0162] 20 μL of plasma was measured and dispensed into a borosilicate screw-cap test tube, which was used as the blood sample solution.

[0163] The brain tissue fractionated in Example 16 was freeze-dried and the dry weight was measured. The freeze-dried tissue was disrupted in tissue extraction solution, and the supernatant was centrifuged. 20 μL of this was measured and dispensed into a borosilicate screw-cap test tube. This was used as the sample solution.

[0164] The solvent in each sample solution and calibration curve sample was evaporated under a nitrogen stream. 20 μL of 2,2-dimethoxypropane (Tokyo Chemical Industry Co., Ltd.) and 200 μL of 3 mol / L HCl-methanol (Sigma-Aldrich) were added to the dried product and stirred. The methanolysis reaction was carried out at 70°C for 90 minutes. The reaction was stopped by cooling on ice, and 200 μL of 10% ammonium carbonate solution and 50 μL of the internal standard solution were added. After the solvent was evaporated under a nitrogen stream, 250 μL of water for injection was added to the dried product. The resulting mixture was loaded onto a solid-phase cartridge (OASIS HLB (1 cc, 30 mg, Waters)) previously conditioned with methanol and water for injection. After washing with water for injection, 500 μL of methanol was added and eluted by centrifugation. After distilling off the solvent under a nitrogen stream, 2 mL of water for injection and 18 mL of acetonitrile were added and redissolved by ultrasonic treatment. After centrifuging, the supernatant was filled into an LC vial.

[0165] 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) was a QTRAP5500 (AB Sciex), and the HPLC device was a Nexera X2 (Shimadzu). The LC column was an Acquity UPLC. TM BEH Amide 1.7 μm (2.1 × 150 mm, Waters) was used. Mobile phases A and B were used. The column temperature was set to 60°C.

[0166] After equilibrating the column with mobile phase B, 5 μL of sample was injected, and chromatography was performed under the mobile phase gradient conditions shown in Table 1. The mobile phase flow rate was 0.4 mL / min.

[0167]

[0168] The ion source parameters of the MS / MS instrument were set as shown in Table 2 according to the instruction manual for QTRAP5500 (AB Sciex).

[0169]

[0170] Table 3 shows the MS internal parameters.

[0171]

[0172] LC / MS / MS analysis was performed on the calibration curve samples, and the areas of the peaks on the chromatogram chart corresponding to the product ions derived from heparan sulfate in the calibration curve samples (HS detection peak areas) were calculated. In addition, the areas of the detection peaks corresponding to the product ions derived from the HS internal standard solution (HS-IS detection peak areas) were calculated.

[0173] For each calibration curve sample, the area of ​​the detection peak derived from heparan sulfate relative to the detection peak area derived from the HS internal standard solution (HS detection peak area / HS-IS detection peak area) was plotted on the vertical axis, and the heparan sulfate concentration of each calibration curve sample was plotted on the horizontal axis. A regression equation was obtained using quadratic discriminant analysis.

[0174] The brain tissue sample solution was subjected to LC / MS / MS analysis, and the heparan sulfate contained in the sample solution was quantified by interpolation into a regression equation.

[0175] The results of measuring HS concentrations (μg / mg dry weight) in brain tissue are shown in Figure 8. HS concentrations in brain tissue decreased dose-dependently in all rAAV virion-administered groups. Six weeks after administration, HS concentrations in brain tissue decreased in a dose-dependent manner in 1.0 × 10 13 The HS concentration in the rAAV-mscFv-GS3-hI2S group was significantly reduced, reaching a value similar to that in the normal control group. 12 vg / kg and 1.0×10 11 The HS-reducing effect in the rAAV virion group administered at the same dose was also higher than that in the other rAAV virion groups administered at the same dose. These results indicate that after intravenous administration of rAAV virions, the conjugate between the anti-mTfR antibody scFv expressed in the liver and hI2S is distributed to the brain, where I2S exerts its activity and reduces HS accumulated in brain tissue. Furthermore, the effect of reducing HS accumulated in the brain tissue persists for at least 6 weeks.

[0176] Example 19 Measurement of Hemoglobin Concentration in Blood Hemoglobin concentration was measured using the anticoagulated blood obtained in Example 16. The hemoglobin concentration was measured by the SLS hemoglobin method using a research-grade automated blood cell counter (Procyte Dx Research, Sysmex Corporation). Blood hemoglobin concentration is a commonly used index of anemia. The results are shown in Figure 9.

[0177] Six weeks after administration, 1.0 × 10 13 A decrease in blood hemoglobin concentration was confirmed only in the vg / kg administration group. In the other groups, no significant differences were observed compared with the normal control group and the pathological control group at 1 week and 6 weeks after administration. These results indicate that anemia can be avoided by adjusting the binding activity to TfR, for example, to 10-100 nM.

[0178] [Example 20] Summary 1 The above results demonstrate that a single intravenous administration of rAAV virions carrying DNA encoding anti-TfR antibody-fused hI2S can induce long-term expression of anti-TfR antibody-fused hI2S in vivo, and that the expressed anti-TfR antibody-fused hI2S can degrade substrates distributed and accumulated in the central nervous system tissues of pathological model mice. Furthermore, the anti-TfR-mediated binding activity to TfR (EC 50 By producing rAAV virions containing a gene encoding a fusion protein of an anti-TfR antibody with hI2S, the concentration of which is adjusted to 10 nM or more, for example, 10-100 nM, and the anti-TfR antibody, it is possible to maintain a high level of expression of the fusion protein in the blood and to provide a safe therapeutic rAAV virion that does not induce anemia while exerting the medicinal effect of reducing HS accumulated in the central nervous system through the enzymatic activity of hI2S. Furthermore, it is possible to provide a safe therapeutic rAAV virion that does not induce anemia while maintaining ... 50 This indicates that by producing rAAV virions containing a gene encoding a fusion protein of an anti-TfR antibody with an adjusted EC value of 10 nM or more, for example, 10-100 nM, and a desired physiologically active protein, it is possible to provide safe therapeutic rAAV virions that do not induce anemia while maintaining a high level of expression of the fusion protein in the blood and allowing the activity of the physiologically active protein to be exerted in the central nervous system. Furthermore, in the case of a fusion protein of an anti-TfR antibody and hI2S, the anti-TfR-mediated binding activity to TfR (EC 50 Even if the rAAV virion contains a gene encoding a fusion protein of an anti-TfR antibody and hI2S, the concentration of which has been adjusted to 10 nM or less, for example, 0.5 to 4 nM, the dose per administration should be 1.0 × 10 13 Doses less than vg / kg, e.g., 7.5 × 10 12 This indicates that the rAAV virion can be used as a safe therapeutic rAAV virion that does not induce anemia by using a dose of 1000 vg / kg.

[0179] Example 21: Construction of pAAV-mMAP-mscFv3-GS3-hI2S Vector To verify the conclusions presented in Examples 19 and 20, the following experiment was further performed. A DNA fragment was synthesized containing, from the 5' end, a ClaI site, a mouse α-fetoprotein enhancer / mouse albumin promoter, a chicken β-actin / MVM chimeric intron, a mouse anti-mouse transferrin receptor single-chain antibody (mscFv3) with two amino acid substitutions introduced into the CDR of the heavy chain of mscFv, to which human iduronate-2-sulfatase (hI2S) was linked via the linker sequence shown in SEQ ID NO: 4 at the C-terminus, the conjugate having the amino acid sequence shown in SEQ ID NO: 56 (mscFv3-GS3-hI2S), a bovine growth hormone polyA sequence, and the nucleotide sequence shown in SEQ ID NO: 57, including a BglII site. This DNA fragment was digested with ClaI and BglII. The pAAV-CMV vector (Takara Bio) was digested with ClaI and BglII, and the above-mentioned restriction enzyme-treated DNA fragment was inserted into it. The resulting plasmid was designated the pAAV-mMAP-mscFv3-GS3-hI2S vector. The pAAV-mMAP-mscFv3-GS3-hI2S vector contains, from upstream to downstream, a functional equivalent of the first AAV-ITR (SEQ ID NO: 7), a mouse α-fetoprotein enhancer / mouse albumin promoter (SEQ ID NO: 9), a chicken β-actin / MVM chimeric intron (SEQ ID NO: 10), a gene encoding mscFv3-GS3-hI2S, a bovine growth hormone polyA signal (SEQ ID NO: 17), and a functional equivalent of the second AAV-ITR (SEQ ID NO: 8). The pAAV-mMAP-mscFv3-GS3-hI2S vector has a structure in which the gene encoding I2S in the vector map shown in Figure 4 has been replaced with the gene encoding mscFv3-GS3-hI2S.

[0180] Example 22: Production of rAAV virions HEK293 cells, a cell line derived from human embryonic kidney cells, were used as host cells. HEK293 cells were placed in a 10-layer CellStack chamber (Corning) at 1.59 x 10 cells per CellStack chamber. 8The cells were seeded and cultured in 10% FBS-containing DMEM medium (Thermo Fisher Scientific) at 37°C in the presence of 5% CO for 3 days. DNA solutions containing the following plasmids were prepared for transfection: pHelper vector (Takara Bio Inc.), pR2(mod)C8 vector constructed in Example 6, and pAAV-mMAP-mscFv3-GS3-hI2S vector constructed in Example 21.

[0181] The solution contained three plasmids at equimolar concentrations, with a total DNA concentration of 1.0 mg / mL and a total volume of 2.2 mL. Polyethylenimine was added to this solution at a weight ratio of DNA (μg):polyethylenimine (μg) = 1:2, and DMEM medium was added to adjust the volume to 1.0 L. This was used as the transfection solution. After 3 days of culture, all of the culture supernatant was removed from the 10-layer cell stack chamber, and the entire 1.0 L of transfection solution was added. The cells were cultured at 37°C with 5% CO2 for 3 days to produce rAAV virions. Some of the rAAV virions produced by the host cells were released into the culture supernatant, while others remained within the host cells. The resulting rAAV virions were designated rAAV-mscFv3-GS3-hI2S. rAAV-mscFv3-GS3-hI2S is expected to express mscFv3-GS3-hI2S when it is infected into cells.

[0182] Example 23 Preparation of an rAAV virion solution containing rAAV-mscFv3-GS3-hI2S and measurement of the rAAV genome amount After completion of the culture in Example 22, an rAAV virion solution containing rAAV-mscFv3-GS3-hI2S (rAAV-mscFv3-GS3-hI2S solution) was prepared from the culture supernatant by the methods described in Examples 8 to 13. The rAAV genome amount contained in the rAAV virion solution was measured by the method described in Example 14.

[0183] Example 24: Evaluation of mTfR binding activity of mscFv3-GS3-hI2S. The binding activity of the recombinant mscFv3-GS3-hI2S protein obtained by standard methods using CHO cells to mouse transferrin receptor was measured by ELISA. The ELISA was performed roughly as follows: 100 μL of the mouse transferrin receptor extracellular domain having the amino acid sequence from positions 87 to 763 of the amino acid sequence set forth in SEQ ID NO: 39 diluted with 0.05 M bicarbonate buffer (pH 9.6) to a concentration of 10 μg / mL was added to each well of a 96-well microtiter plate (Nunc). The plate was then left to stand at room temperature for at least 1 hour to allow the antibody to adsorb to the plate. Next, 300 μL of TBS containing 1% BSA and 0.05% Tween 20 was added to each well, and the plate was left to stand at room temperature for 1 hour. After washing each well three times with TBS containing 0.05% Tween 20 (TBS-T), 100 μL of mscFv3-GS3-hI2S diluted to 200, 66.7, 22.2, 7.41, 2.47, 0.82, and 0.27 nM in TBS containing 0.1% BSA and 0.05% Tween 20 was added to each well. The plate was then incubated at room temperature for at least 1 hour. After washing the plate three times with TBS-T, 100 μL of anti-I2S monoclonal antibody diluted to 0.5 μg / mL in TBS containing 0.1% BSA and 0.05% Tween 20 was added to each well. The plate was then incubated at room temperature for at least 1 hour. After washing three times with TBS-T, 100 μL of HRP-labeled anti-mouse IgG polyclonal antibody (Bethyl) diluted to 50 ng / mL in TBS containing 0.1% BSA and 0.05% Tween 20 was added to each well, and the plate was left to stand at room temperature for at least 1 hour. After washing three times with TBS-T, 50 μL of TMB Stabilized Substrate for Horseradish Peroxidase (Promega) was added to each well and the plate was left to stand at room temperature for 5 to 10 minutes.Next, 100 μL of stop solution (1 N hydrochloric acid) was added to each well, and the absorbance of each well at 450 nm was measured using a plate reader (MOLECULAR DEVICES). 50 The EC value of mscFv3-GS3-hI2S for mouse TfR was calculated to evaluate its binding activity to mTfR. 50 was 4.4 nM.

[0184] Example 25: Evaluation of the efficacy of rAAV-mscFv3-GS3-hI2S using I2S-KO mice. The efficacy of each rAAV administered to a living body was evaluated using I2S gene knockout mice (I2S-KO mice). The I2S-KO mice used were male mice aged 14 to 15 weeks (at the start of administration). rAAV virions, rAAV-mscFv3-GS3-hI2S, were administered to male I2S-KO mice at a dose of 1.0 × 10 11 vg / kg, 1.0×10 12 vg / kg, and 1.0×10 13 A single dose of 1000mg / kg was administered into the tail vein (N=5 or 6 per group). A normal control group consisting of male wild-type mice and a pathological control group consisting of male I2S-KO mice were also included (N=2 or 5 per group). The normal and pathological control groups were administered saline (Otsuka Pharmaceutical Factory).

[0185] Six weeks after administration, cerebrospinal fluid (CSF) was collected from the cisterna magna of mice under triple anesthesia with Betolfar (Meiji Seika Pharma), midazolam (Sandoz), and Domitor (Nippon Zenyaku Kogyo). The mice were then thoracically opened and cardiac blood was collected. Blood was collected into EDTA-2K-coated blood collection tubes (Capiject, Terumo), anticoagulated, and subjected to hemoglobin concentration measurement. Hemoglobin concentration was measured using the SLS hemoglobin method described in Example 19. The remaining anticoagulated blood was placed on ice and centrifuged (2000 × g, 20 minutes, 4°C) to recover plasma. After cardiac blood collection, the mice were systemically perfused with saline (Otsuka Pharmaceutical Factory), and the brains were removed. A portion of the brain tissue was removed for quantification of heparan sulfate in the brain tissue and quickly immersed in liquid nitrogen for flash-frozen storage. Separately, a portion of the brain tissue was weighed and then rapidly frozen by immersion in liquid nitrogen. The frozen brain tissue was homogenized in RIPA Buffer (Wako Pure Chemical Industries, Ltd.) containing 0.025% Protease Inhibitor Cocktail (Sigma-Aldrich), and then centrifuged to recover the supernatant.

[0186] Example 26: Measurement of rAAV-Derived Protein Concentrations in CSF, Plasma, and Brain Tissue. The amount of mscFv3-GS3-hI2S derived from rAAV-mscFv3-GS3-hI2S contained in the plasma prepared in Example 25 and in the supernatant obtained by homogenizing brain tissue was measured using the method described in Example 17. The measurement results for mscFv3-GS3-hI2S contained in plasma are shown in Figure 10. The concentration of mscFv3-GS3-hI2S in plasma increased dose-dependently, indicating that rAAV virions injected intravenously into mice were internalized into cells, and at least the mscFv3-GS3-hI2S expressed in the cells through transcription and translation of the gene encoding mscFv3-GS3-hI2S was released into the blood. Next, the measurement results for the concentration of mscFv3-GS3-hI2S contained in brain tissue are shown in Figure 11. The concentration of mscFv3-GS3-hI2S in plasma increased in a dose-dependent manner, indicating that mscFv3-GS3-hI2S expressed in mouse cells was released into the blood, and at least a portion of it crossed the BBB and reached central nervous system tissues by binding to mouse TfR.

[0187] Example 27: Quantification of heparan sulfate in brain tissue and CSF The brain tissue frozen in Example 25 was freeze-dried and then its dry weight was measured. The freeze-dried tissue was disrupted in tissue extraction solution, and the supernatant was centrifuged. 20 μL of this was measured and dispensed into a borosilicate screw-cap test tube. This was used as the sample solution. For CSF measurement, 20 μL of CSF was measured and dispensed into a borosilicate screw-cap test tube. This was used as the sample solution. Using the prepared sample solution, heparan sulfate (HS) in brain tissue and CSF was quantified by the method described in Example 18.

[0188] The measurement results of HS concentration in brain tissue (μg / mg dry weight) and CSF (μg / mL) are shown in Figures 12 and 13, respectively. HS concentrations in both brain tissue and CSF decreased in a dose-dependent manner. 11Even in the 500mg / kg group, HS concentrations in both brain tissue and CSF were significantly reduced. These results indicate that intravenous administration of rAAV-mscFv3-GS3-hI2S results in intracellularly expressed mscFv3-GS3-hI2S being released into the bloodstream, where at least a portion of it binds to mouse TfR, crossing the BBB and reaching central nervous system tissues, where it exerts its enzymatic activity by reducing HS concentrations. Furthermore, the effect of reducing HS accumulation in brain tissue persists for at least 6 weeks.

[0189] [Example 28] Measurement of hemoglobin concentration in blood Hemoglobin concentration was measured using the anticoagulated blood obtained in Example 25. The hemoglobin concentration was measured by the SLS hemoglobin method described in Example 19. Blood hemoglobin concentration is an item commonly used as an indicator of anemia. The results are shown in Figure 14.

[0190] Six weeks after administration, no significant differences were observed in blood hemoglobin levels in any of the rAAV-mscFv3-GS3-hI2S administration groups compared with the normal control group or the pathological control group. These results suggest that the EC of the fusion protein of anti-TfR antibody and a physiologically active protein on the transferrin receptor via the anti-TfR antibody is significant. 50 is approximately 4.4 nM, it indicates that intravenous administration of rAAV virions encoding the fusion protein can allow the physiologically active protein to exert its function in the central nervous system without reducing hemoglobin concentration in the blood, i.e., while avoiding anemia.

[0191] [Example 29] Summary 2 Considering the above results together with the description of Summary 1 in Example 20, it is possible to conclude that the anti-TfR-mediated binding activity to TfR (EC 50By producing rAAV virions containing a gene encoding a fusion protein of an anti-TfR antibody with hI2S, the concentration of which is adjusted to 3 nM or more, for example, 3-100 nM, and the anti-TfR antibody, a high level of expression of the fusion protein in the blood can be maintained for a long period of time, and the enzyme activity of hI2S can be used to reduce HS accumulated in the central nervous system, while providing a safe therapeutic rAAV virion that does not induce anemia. Furthermore, it is possible to provide rAAV virions that can maintain a high level of expression of the fusion protein in the blood for a long period of time, and that exhibit the medicinal effect of reducing HS accumulated in the central nervous system through the enzymatic activity of hI2S, without causing anemia. Furthermore, it is possible to provide rAAV virions that can maintain a high level of expression of the fusion protein in the blood for a long period of time, without causing anemia, and that exhibit the medicinal effect of reducing HS accumulated in the central nervous system through the enzymatic activity of hI2S. 50 This indicates that by producing rAAV virions containing a gene encoding a fusion protein of an anti-TfR antibody with a desired physiologically active protein, the EC value of which has been adjusted to 3 nM or more, for example, 3-100 nM, it is possible to provide safe therapeutic rAAV virions that maintain high blood expression levels of the fusion protein and allow the physiologically active protein to exert its activity in the central nervous system without inducing anemia. Furthermore, in the case of a fusion protein of an anti-TfR antibody and hI2S, the anti-TfR-mediated binding activity to TfR (EC 50 Even if the rAAV virion contains a gene encoding a fusion protein of an anti-TfR antibody and hI2S, the concentration of which has been adjusted to 4 nM or less, for example, 0.5 to 4 nM, the dose per administration should be 1.0 × 10 13 Doses less than vg / kg, e.g., 7.5 × 10 12 This indicates that the rAAV virion can be used as a safe therapeutic rAAV virion that does not induce anemia by using a dose of 1000 vg / kg.

[0192] Example 30: Construction of pAAV-mMAP-mscFv-GS3-hGAA vector A DNA fragment was synthesized containing, from the 5' end, a BamHI site, a gene encoding hGAA having the amino acid sequence shown in SEQ ID NO:58, and the nucleotide sequence shown in SEQ ID NO:59, including a NotI site. This DNA fragment was digested with BamHI and NotI. pAAV-mMAP-mscFv-GS3-hI2S constructed in Example 1 was digested with BamHI and NotI, and the above-mentioned restriction enzyme-treated DNA fragment was inserted into it. The resulting plasmid was designated pAAV-mMAP-mscFv-GS3-hGAA vector. The pAAV-mMAP-mscFv-GS3-hGAA vector contains, from upstream to downstream, a functional equivalent of the first AAV-ITR (SEQ ID NO: 7), a mouse α-fetoprotein enhancer / mouse albumin promoter (SEQ ID NO: 9), a chicken β-actin / MVM chimeric intron (SEQ ID NO: 10), a gene encoding a conjugate (mscFv-GS3-hGAA) comprising a mouse anti-mouse transferrin receptor single-chain antibody (mouse anti-mTfRscFv antibody) linked to the C-terminus of the antibody via a linker shown in SEQ ID NO: 4, the conjugate having the amino acid sequence shown in SEQ ID NO: 60, a bovine growth hormone polyA signal (SEQ ID NO: 17), and a functional equivalent of the second AAV-ITR (SEQ ID NO: 8). The pAAV-mMAP-mscFv-GS3-hGAA vector has a structure in which the gene encoding I2S in the vector map shown in Figure 4 has been replaced with a gene encoding mscFv-GS3-hGAA.

[0193] Example 31: Construction of pAAV-mMAP-hGAA(Pro)-GS-mscFv2 Vector A DNA fragment was synthesized containing, from the 5' end, an MluI site, a gene encoding hGAA(Pro) having the amino acid sequence shown in SEQ ID NO:61, a gene encoding a GS linker having the amino acid sequence shown in SEQ ID NO:1, and the nucleotide sequence shown in SEQ ID NO:62, which includes a Bsu36I site. This DNA fragment was digested with MluI, ScaI, and Bsu36I. pAAV-mMAP-hI2S-mscFv2 constructed in Example 3 was digested with MluI and Bsu36I, and the above-mentioned restriction enzyme-treated DNA fragment was inserted into it. The resulting plasmid was designated pAAV-mMAP-hGAA(Pro)-GS-mscFv2 vector. The pAAV-mMAP-hGAA(Pro)-GS-mscFv2 vector contains, from upstream to downstream, a functional equivalent of the first AAV-ITR (SEQ ID NO: 7), a mouse α-fetoprotein enhancer / mouse albumin promoter (SEQ ID NO: 9), a chicken β-actin / MVM chimeric intron (SEQ ID NO: 10), a gene encoding a conjugate of hGAA(Pro) and mouse anti-mTfRscFv antibody 2 linked to the C-terminus of hGAA(Pro) via the linker shown in SEQ ID NO: 1, the conjugate having the amino acid sequence shown in SEQ ID NO: 63 (hGAA(Pro)-GS-mscFv2), a bovine growth hormone polyA signal (SEQ ID NO: 17), and a functional equivalent of the second AAV-ITR (SEQ ID NO: 8). The pAAV-mMAP-hGAA(Pro)-GS-mscFv2 vector has a structure in which the gene encoding I2S in the vector map shown in Figure 4 has been replaced with a gene encoding hGAA(Pro)-GS-mscFv2.

[0194] Example 32: Construction of pAAV-mMAP-hGAA Vector A DNA fragment containing the nucleotide sequence shown in SEQ ID NO:64, including an MluI site, a gene encoding hGAA, and a Not1 site from the 5' end, was synthesized. This DNA fragment was digested with MluI and NotI. pAAV-mMAP-hI2S-mscFv2 constructed in Example 3 was digested with MluI and NotI, and the above-mentioned restriction enzyme-treated DNA fragment was inserted into it. The resulting plasmid was designated pAAV-mMAP-hGAA vector. The pAAV-mMAP-hGAA vector has a structure containing, from upstream to downstream, a functional equivalent of the first AAV-ITR (SEQ ID NO:7), a mouse α-fetoprotein enhancer / mouse albumin promoter (SEQ ID NO:9), a chicken β-actin / MVM chimeric intron (SEQ ID NO:10), a gene encoding hGAA, a bovine growth hormone polyA signal (SEQ ID NO:17), and a functional equivalent of the second AAV-ITR (SEQ ID NO:8). The pAAV-mMAP-hGAA vector has a structure in which the gene encoding I2S in the vector map shown in Figure 4 has been replaced with a gene encoding hGAA.

[0195] [Example 33] Production of rAAV virions HEK293 cells, a cell line derived from human embryonic kidney cells, were used as host cells. HEK293 cells were placed in a 10-layer CellStack chamber (Corning) at 1.59 x 10 per CellStack chamber. 8 The cells were seeded and cultured in 10% FBS-containing DMEM medium (Thermo Fisher Scientific) at 37°C in the presence of 5% CO2 for 3 days. DNA solutions containing the following combinations of plasmids (1) to (3) were prepared for transfection: (1) pHelper vector (Takara Bio), pR2(mod)C8 vector, and pAAV-mMAP-mscFv-GS3-hGAA, (2) pHelper vector (Takara Bio), pR2(mod)C8 vector, and pAAV-mMAP-hGAA(Pro)-GS-mscFv2, and (3) pHelper vector (Takara Bio), pR2(mod)C8 vector, and pAAV-mMAP-hGAA vector.

[0196] The solutions (1) to (3) above each contained three plasmids at equimolar concentrations, with a total DNA concentration of 1.0 mg / mL and a total volume of 2.2 mL. Polyethyleneimine was added to these solutions at a weight ratio of DNA (μg):polyethyleneimine (μg) = 1:2, and DMEM medium was added to adjust the volume to 1.0 L to form the transfection solution. After 3 days of culture, all of the culture supernatant was removed from the 10-layer cell stack chamber, and the entire 1.0 L of transfection solution was added. The cells were cultured at 37°C in the presence of 5% CO2 for 3 days to produce rAAV virions. Some of the rAAV virions produced in the host cells were released into the culture supernatant, while others remained within the host cells. The resulting rAAV virions were designated rAAV-mscFv-GS3-hGAA, rAAV-hGAA(Pro)-GS-mscFv2, and rAAV-hGAA, respectively. For example, rAAV-mscFv-GS3-hGAA is expected to express mscFv-GS3-hGAA when infected into cells.

[0197] Example 34 Preparation of rAAV virion solutions and measurement of rAAV genome content After completion of the culture in Example 33, rAAV virion solutions containing rAAV-mscFv-GS3-hGAA, rAAV-hGAA(Pro)-GS-mscFv2, and rAAV-hGAA, respectively (rAAV-mscFv-GS3-hGAA solution, rAAV-hGAA(Pro)-GS-mscFv2 solution, and rAAV-hGAA solution) were prepared from the culture supernatant by the methods described in Examples 8 to 13. The rAAV genome content of each rAAV virion solution was measured by the method described in Example 14.

[0198] Example 35: Evaluation of mTfR binding activity of mouse anti-mTfRscFv and hGAA conjugates. The binding activity of two recombinant proteins, mscFv-GS3-hGAA and hGAA(Pro)-GS-mscFv2, obtained by standard methods using CHO cells, to mouse transferrin receptor was measured by ELISA. The ELISA was performed roughly as follows: 100 μL of the mouse transferrin receptor extracellular domain, represented by the amino acid sequence from positions 87 to 763 from the N-terminus of SEQ ID NO: 39, diluted with 0.05 M bicarbonate buffer (pH 9.6) to a concentration of 10 μg / mL, was added to each well of a 96-well microtiter plate (Nunc). The plate was then left to stand at room temperature for at least 1 hour to allow the antibody to adsorb to the plate. Next, 300 μL of TBS containing 1% BSA and 0.05% Tween 20 was added to each well, and the plate was left to stand at room temperature for 1 hour. After washing each well three times with TBS containing 0.05% Tween 20 (TBS-T), mscFv-GS3-hGAA was diluted with TBS containing 0.1% BSA and 0.05% Tween 20 to the following concentrations: 33.33, 11.11, 3.70, 1.23, 0.41, 0.14, and 0.046 nM. hGAA(Pro)-GS-mscFv2 was diluted with TBS containing 0.1% BSA and 0.05% Tween 20 to the following concentrations: 200, 66.7, 133.33, 22.2, 7.41, 2.47, 0.82, and 0.27 nM. 100 μL of each diluted solution was added to each well. The plate was then incubated at room temperature for at least 1 hour. After washing the plate three times with TBS-T, 100 μL of anti-hGAA monoclonal antibody diluted to 0.5 μg / mL in TBS containing 0.1% BSA and 0.05% Tween 20 was added to each well and the plate was left to stand at room temperature for at least 1 hour. After washing three times with TBS-T, 100 μL of HRP-labeled anti-mouse IgG polyclonal antibody (Bethyl) diluted to 50 ng / mL in TBS containing 0.1% BSA and 0.05% Tween 20 was added to each well and the plate was left to stand at room temperature for at least 1 hour.After washing each well three times with TBS-T, 50 μL of TMB Stabilized Substrate for Horseradish Peroxidase (Promega) was added to each well and allowed to stand at room temperature for 5-10 minutes. Next, 100 μL of stop solution (1 N hydrochloric acid) was added to each well, and the absorbance at 450 nm of each well was measured using a plate reader (MOLECULAR DEVICES). The EC was calculated from the measured values. 50 The binding activity of each recombinant protein to mTfR was evaluated.

[0199] EC values ​​of binding affinity of mscFv-GS3-hGAA and hGAA(Pro)-GS-mscFv2 to mouse TfR 50 were 0.173 nM and 83.7 nM, respectively.

[0200] Example 36: Generation of GAA KO Mice GAA KO mice are mice homozygously deficient in the acid α-glucosidase gene (Gaa gene). GAA KO mice were generated generally as follows. Using ES cell genomic DNA as a template, two fragments, the 5' homologous region and the 3' homologous region, were amplified by PCR. Exon 6 of the Gaa gene was then constructed by inserting a stop codon along with a loxP sequence and a PGK-neo region. This fragment was then inserted by standard methods to construct a homologous recombination vector for the Gaa gene. This targeting vector was introduced into C57NBL / 6N ES cells by electroporation, and ES cells that showed positive results in the primary screening were obtained after drug selection. Homologous recombinants were selected from the resulting ES cells that showed positive results in the primary screening, and homologous recombinant ES cell clones were obtained. Chimeric embryos were generated by aggregation using the resulting homologous recombinant ES cell clones and 8-cell ICR mouse embryos (host embryos). The resulting chimeric embryos were transplanted into pseudopregnant mice (recipient mice) obtained by mating with vasoligated mice. The resulting offspring (chimeric mice) were assessed for coat color, and individuals in which 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 then crossed with ICR mice to obtain F1 mice. White F1 mice were selected, and DNA extracted from their tail tissue was analyzed. Mice with a heterozygous Gaa gene on the chromosome were designated Gaa heterozygotes. Based on these mice, mice with a homozygous deficiency of the Gaa gene (GAA KO mice) were generated.

[0201] Example 37: Evaluation of the efficacy of each rAAV virion using GAA KO mice The efficacy of each rAAV when administered to a living body was evaluated using the GAA KO mice prepared in Example 36. The GAA KO mice used were male mice aged 10 to 11 weeks (at the start of administration). The rAAV-mscFv-GS3-hGAA solution, rAAV-hGAA(Pro)-GS-mscFv2 solution, and rAAV-hGAA solution obtained in Example 34 were each administered to male GAA KO mice at a dose of 1.0 × 10 13A single dose of 1000mg / kg was administered into the tail vein (N=5 per group). A normal control group consisting of male wild-type mice and a pathological control group consisting of male GAA KO mice (N=5 per group) were also included. Physiological saline (Otsuka Pharmaceutical Factory) was administered to the normal and pathological control groups.

[0202] Four weeks after administration, mice were subjected to cardiac blood collection via chest opening under isoflurane inhalation anesthesia. Blood was collected into EDTA-2K-coated blood collection tubes (Capiject, Terumo Corporation), anticoagulated, and then used for hemoglobin concentration measurement. The remaining anticoagulated blood was placed on ice and centrifuged (2000 × g, 20 minutes) to recover plasma. After cardiac blood collection, the mice were perfused whole body with saline (Otsuka Pharmaceutical Factory), and the brain and tibialis anterior muscle were removed. The brain was immersed in liquid nitrogen 3 minutes after the start of perfusion. The wet weight of the skeletal muscle was measured, and then it was quickly immersed in liquid nitrogen and flash-frozen.

[0203] Example 38: Measurement of rAAV-derived protein concentrations in plasma and tissues The rAAV-derived proteins rAAV-mscFv-GS3-hGAA, rAAV-hGAA(Pro)-GS-mscFv2, and rAAV-hGAA contained in the plasma prepared in Example 37 and in the supernatant obtained by homogenizing brain tissue were quantified. Quantification was generally performed as follows: 150 μL 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. Next, a standard sample for the calibration curve (GAA standard sample for the calibration curve) containing known concentrations of mscFv-GS3-hGAA, hGAA(Pro)-GS-mscFv2, and hGAA, respectively, and each sample were added to the mixture of biotinylated anti-hGAA polyclonal antibody and sulfonated anti-hGAA polyclonal antibody, and the mixture was shaken for at least 1 hour to prepare an antibody reaction solution. The anti-hGAA polyclonal antibody was obtained by affinity purification of antiserum obtained from whole blood of rabbits immunized multiple times with human acid α-glucosidase having the amino acid sequence shown in SEQ ID NO: 58. One of the obtained polyclonal antibodies was modified using Biotin Labeling Kit-NH2 (Dojindo Laboratories) according to the attached protocol to obtain a biotinylated anti-hGAA polyclonal antibody. Another polyclonal antibody obtained was modified with MSD GOLD SULFO-TAG NHS-Ester (Mesoscale Diagnostics) according to the attached protocol to obtain a sulfo-modified anti-hGAA polyclonal antibody. After removing the blocking solution from each well and washing with PBS containing 0.05% Tween 20 (PBST, Sigma-Aldrich), 25 μL of antibody reaction solution was added to each well and shaken for at least 1 hour. The antibody reaction solution was then removed, washed with PBST, and 150 μL of 4X Read Buffer (Mesoscale Diagnostics) diluted 1 / 2 with water for injection (Otsuka Pharmaceutical Factory) was added. Sector 1 was then inserted. TMThe luminescence intensity from each well was measured using an Imager 6000 (Mesoscale Diagnostics). A calibration curve was created from the measurements of the standard sample for the GAA calibration curve, and the amount of each protein contained per mL of sample was calculated by interpolating the measurements of each sample. Note that mscFv-GS3-hGAA and hGAA(Pro)-GS-mscFv2 contained in the standard sample for the calibration curve were obtained by expressing them using CHO cells transfected with expression vectors incorporating the genes encoding the respective proteins using standard methods.

[0204] The results of measuring the plasma concentrations of the above rAAV-derived proteins are shown in Figure 15. Plasma concentrations of each rAAV-derived protein increased by 4 weeks after administration in all rAAV virion-administered groups. These results indicate that rAAV virions injected intravenously into mice were taken up into cells, and that at least the rAAV-encoded genes were transcribed and translated within the cells, resulting in the expression of rAAV-derived proteins, which were then released into the blood.

[0205] Next, the results of measuring rAAV-derived protein concentrations in brain tissue are shown in Figure 16. It was demonstrated that the fusion proteins encoded by each rAAV virion were detected in brain tissue 4 weeks after administration in all rAAV virion-administered groups. Expression levels in brain tissue were particularly high in the rAAV-mscFv-GS3-hGAA-administered group. Although expression levels in brain tissue were low in the rAAV-hGAA(Pro)-GS-mscFv2-administered group, as shown in Table 4, glycogen concentrations in brain tissue of the rAAV-hGAA(Pro)-GS-mscFv2-administered group were dramatically reduced. This indicates that rAAV-hGAA(Pro)-GS-mscFv2 administered to mice crossed the BBB and exerted its enzymatic activity as hGAA in brain tissue, degrading glycogen.

[0206] The above results demonstrate that administration of rAAV-mscFv-GS3-hGAA and rAAV-hGAA(Pro)-GS-mscFv2 resulted in the expression of the anti-mTfR antibody scFv-hGAA conjugate in mice as expected, and that GAA conjugated to the anti-mTfR antibody scFv crossed the BBB and reached the central nervous system tissues.

[0207] Example 39: Measurement of Glycogen Concentration in Tissues The glycogen concentration in the supernatant obtained by homogenizing brain and tibialis anterior muscle was measured using the following method. Measurements were performed using a Glycogen Assay Kit (Biovision) according to the attached protocol. 50 μL each of a calibration curve standard sample containing known glycogen concentrations and tissue homogenate supernatant was added to a 96F Nontreated Black microwell (Thermo Scientific). Next, 1 μL of Hydrolysis Enzyme Mix, a hydrolysis reaction reagent, was added to each well, mixed, and incubated at room temperature for 30 minutes. Next, 50 μL of Development Enzyme Mix containing OxiRed Probe was added to each well, mixed, and incubated at room temperature for 30 minutes in the dark. Fluorescence intensity from each well was measured using a SpectraMax iD3 (Molecular Devices) (excitation wavelength: 535 nm, detection wavelength: 587 nm). A calibration curve was created from the measurement values ​​of each calibration standard sample, and the measurement values ​​of each sample were interpolated to calculate the glycogen concentration in each sample, and further the amount of glycogen contained per mg of tissue (mg / g tissue).

[0208] The reduction ratio (%), which represents the glycogen reduction effect in each tissue, was used as an index of drug efficacy. The reduction ratio (%) was defined as {([KO] - [WT]) - ([Test article] - [WT])} × 100 / ([KO] - [WT]). Here, [WT] represents the average glycogen concentration in the normal control group, [KO] represents the average glycogen concentration in the pathological control group, and [Test article] represents the average glycogen concentration in each test substance administration group.

[0209] The measurement results of brain glycogen concentration (μg / g wet weight) and reduction ratio (%) are shown in Table 4. Brain glycogen concentration was reduced in both the rAAV-mscFv-GS3-hGAA and rAAV-hGAA(Pro)-GS-mscFv2 groups compared to the pathological control group (reduction ratios (%): 101% and 23.9%, respectively). On the other hand, in the rAAV-hGAA group expressing hGAA not fused to the anti-mTfR antibody scFv, brain glycogen concentration was reduced, but the reduction ratio (%) was only 5.47%.

[0210]

[0211] The measurement results of glycogen concentration (μg / g wet weight) and reduction ratio (%) in the tibialis anterior muscle are shown in Table 5. Glycogen concentration in the tibialis anterior muscle was significantly reduced in all treatment groups compared to the pathological control group.

[0212]

[0213] These results indicate that mscFv-GS3-hGAA and hGAA-GS-mscFv2, expressed in vivo by intravenous injection of rAAV-mscFv-GS3-hGAA and rAAV-hGAA-GS-mscFv2 into mice, crossed the BBB, reached the central nervous system tissues, and exerted their enzymatic activity as hGAA in the central nervous system tissues, thereby degrading glycogen accumulated in the central nervous system. [Example 40] Measurement of Hemoglobin Concentration in Blood Hemoglobin concentration was measured using the anticoagulated blood obtained in Example 37. Hemoglobin concentration was measured using the SLS hemoglobin method described in Example 19. Blood hemoglobin concentration is a commonly used indicator of anemia. The results are shown in Figure 17.

[0214] Four weeks after administration, a decrease in blood hemoglobin concentration was confirmed only in the rAAV-mscFv-GS3-hGAA administration group. No clear difference was observed in the rAAV-hGAA(Pro)-GS-mscFv2 and rAAV-hGAA administration groups compared with the normal control group and the pathological control group. These results suggest that anti-TfR-mediated TfR binding activity (EC 50 This is consistent with the description in Summary 1 of Example 20 and Summary 2 of Example 29 that an rAAV virion containing a gene encoding a fusion protein of an anti-TfR antibody, the concentration of which has been adjusted to 0.5 nM, for example, 3 to 100 nM, and a desired physiologically active protein, can maintain high expression levels of the fusion protein in the blood, and allow the activity of the physiologically active protein to be exerted in the central nervous system, while being able to provide a safe rAAV virion for therapeutic use that does not induce anemia.

[0215] These results demonstrate that intravenous injection of rAAV-hGAA(Pro)-GS-mscFv2 can induce in vivo expression of hGAA(Pro)-GS-mscFv2 for at least one month, and that the expressed hGAA-GS(Pro)-mscFv2 exerts hGAA activity in the central nervous system and muscle tissue of pathological model mice, thereby degrading accumulated glycogen. Furthermore, these results demonstrate that by regulating the activity of anti-TfR antibodies, high blood concentrations are maintained, demonstrating efficacy, and that this safe, non-anemic agent is effective in treating Pompe syndrome.

[0216] According to the present invention, when a fusion protein of an anti-transferrin receptor antibody and a physiologically active protein is used in gene therapy, by appropriately adjusting the affinity or binding activity with the transferrin receptor, it is possible to provide a transferrin receptor antibody and a physiologically active protein that are less likely to cause anemia symptoms, and a viral vector incorporating a gene encoding such a fusion protein.

[0217] 1. Functional equivalent of the first AAV-ITR 2. Mouse α-fetoprotein enhancer / mouse albumin promoter 3. Chicken β-actin / MVM chimeric intron 4. Gene encoding a conjugate (mscFv-GS3-hI2S) in which hI2S is linked to the C-terminal side of a mouse anti-mouse transferrin receptor single-chain antibody (mouse anti-mTfRscFv antibody) via a linker sequence consisting of three repeats of the amino acid sequence of SEQ ID NO: 1 5. Bovine growth hormone polyA signal 6. Functional equivalent of the second AAV-ITR 7. Ampicillin resistance gene 8. Origin of replication (ColE1 ori) 9. A gene encoding a conjugate (mscFv2-GS3-hI2S) in which hI2S is linked to the C-terminus of a mouse anti-mouse transferrin receptor single-chain antibody (mouse anti-mTfRscFv antibody 2) having amino acid mutations introduced into two positions in the CDR of the heavy chain and two positions in the CDR of the light chain of mscFv via a linker sequence comprising three repeats of the amino acid sequence of SEQ ID NO: 1. 10. A gene encoding a conjugate (hI2S-mscFv2) in which mouse anti-mTfRscFv antibody 2 is linked to the C-terminus of hI2S. 11. A gene encoding hI2S. 12. p5 promoter. 13. Rep region of AAV2. 14. Cap region of AAV8. 15. p5 promoter functioning as an enhancer.

[0218] SEQ ID NO: 1: Example of the amino acid sequence of a linker 1 SEQ ID NO: 2: Example of the amino acid sequence of a linker 2 SEQ ID NO: 3: Example of the amino acid sequence of a linker 3 SEQ ID NO: 4: Example of the amino acid sequence of a linker 4 SEQ ID NO: 5: Nucleotide sequence of the first inverted terminal repeat (first ITR) of AAV of serotype 2, wild type SEQ ID NO: 6: Nucleotide sequence of the first inverted terminal repeat (second ITR) of AAV of serotype 2, wild type SEQ ID NO: 7: A preferred example of the nucleotide sequence of the first inverted terminal repeat (first ITR), synthetic sequence SEQ ID NO: 8: A preferred example of the nucleotide sequence of the second inverted terminal repeat (second ITR), synthetic sequence SEQ ID NO: 9: Nucleotide sequence of mouse α-fetoprotein enhancer / mouse albumin promoter, synthetic sequence SEQ ID NO: 10: Nucleotide sequence of chicken β-actin / MVM chimeric intron, synthetic sequence SEQ ID NO: 11: An example of the nucleotide sequence of an internal ribosome binding site derived from the 5' untranslated region of murine encephalomyocarditis virus SEQ ID NO: 12: Amino acid sequence of human transferrin receptor SEQ ID NO: 13: An example of the amino acid sequence of the Fab region of the heavy chain of an anti-hTfR antibody SEQ ID NO: 14: Amino acid sequence of human I2S SEQ ID NO: 15: An example of the amino acid sequence of the light chain of an anti-hTfR antibody SEQ ID NO: 16: An example of the amino acid sequence of the heavy chain of an anti-hTfR antibody SEQ ID NO: 17: Nucleotide sequence of the bovine growth hormone polyA signal SEQ ID NO: 18: Nucleotide sequence of the DNA fragment synthesized to prepare the pAAV-mMAP-mscFv-GS3-hI2S vector, synthetic sequence SEQ ID NO: 19: Amino acid sequence of mscFv-GS3-hI2S SEQ ID NO: 20: Nucleotide sequence encoding mscFv-GS3-hI2S, synthetic sequence SEQ ID NO: 21: Nucleotide sequence of the DNA fragment synthesized to prepare the pAAV-mMAP-mscFv2-GS3-hI2S vector, synthetic sequence SEQ ID NO: 22: Amino acid sequence of mscFv2-GS3-hI2S SEQ ID NO: 23: Nucleotide sequence encoding mscFv2-GS3-hI2S, synthetic sequence SEQ ID NO: 24: Nucleotide sequence of a DNA fragment synthesized to prepare the pAAV-mMAP-hI2S-mscFv2 vector, synthetic sequence SEQ ID NO: 25: Amino acid sequence of hI2S-mscFv2 SEQ ID NO: 26: Nucleotide sequence encoding hI2S-mscFv2, synthetic sequence SEQ ID NO: 27: Nucleotide sequence of a DNA fragment synthesized to prepare the pAAV-mMAP-hI2S vectorSynthetic sequence SEQ ID NO: 28: Nucleotide sequence encoding hI2S SEQ ID NO: 29: Nucleotide sequence of DNA fragment synthesized to prepare pR2(mod)C6 vector, synthetic sequence SEQ ID NO: 30: Nucleotide sequence of DNA fragment synthesized to prepare pR2(mod)C8 vector, synthetic sequence SEQ ID NO: 31: Nucleotide sequence of the Cap region of AAV8 SEQ ID NO: 32: Nucleotide sequence including the Rep region of AAV2, synthetic sequence SEQ ID NO: 33: Nucleotide sequence of primer SI-1, synthetic sequence SEQ ID NO: 34: Nucleotide sequence of primer SI-2, synthetic sequence SEQ ID NO: 35: Nucleotide sequence of probe SI-1, synthetic sequence SEQ ID NO: 36: Nucleotide sequence of primer SI-3, synthetic sequence SEQ ID NO: 37: Nucleotide sequence of primer SI-4, synthetic sequence SEQ ID NO: 38: Nucleotide sequence of probe SI-2, synthetic sequence SEQ ID NO: 39: Amino acid sequence of mouse transferrin receptor SEQ ID NO: 40: Amino acid sequence 1 of light chain CDR1 of anti-hTfR antibody SEQ ID NO: 41: Amino acid sequence 2 of light chain CDR1 of anti-hTfR antibody SEQ ID NO: 42: Amino acid sequence 1 of light chain CDR2 of anti-hTfR antibody SEQ ID NO: 43: Amino acid sequence 2 of light chain CDR2 of anti-hTfR antibody SEQ ID NO: 44: Amino acid sequence 1 of light chain CDR3 of anti-hTfR antibody SEQ ID NO: 45: Amino acid sequence 1 of heavy chain CDR1 of anti-hTfR antibody SEQ ID NO: 46: Amino acid sequence 2 of heavy chain CDR1 of anti-hTfR antibody SEQ ID NO: 47: Amino acid sequence 1 of heavy chain CDR2 of anti-hTfR antibody SEQ ID NO: 48: Amino acid sequence 2 of heavy chain CDR2 of anti-hTfR antibody SEQ ID NO: 49: Amino acid sequence 1 of heavy chain CDR3 of anti-hTfR antibody SEQ ID NO: 50: Amino acid sequence 2 of heavy chain CDR3 of anti-hTfR antibody SEQ ID NO: 51: Amino acid sequence of a complex of the Fab region of the heavy chain of an anti-hTfR antibody and human I2S SEQ ID NO: 52: A preferred example of the base sequence of the first long terminal repeat (first LTR), a synthetic sequence SEQ ID NO: 53: A suitable example of the nucleotide sequence of the second long terminal repeat (second LTR), a synthetic sequence SEQ ID NO: 54: The nucleotide sequence of the leader of the Sendai virus genome SEQ ID NO: 55: The nucleotide sequence of the trailer of the Sendai virus genome SEQ ID NO: 56: The amino acid sequence of mscFv3-GS3-hI2S SEQ ID NO: 57: The nucleotide sequence of the DNA fragment synthesized to prepare the pAAV-mMAP-mscFv3-GS3-hI2S vectorSynthetic sequence SEQ ID NO: 58: Amino acid sequence of hGAA SEQ ID NO: 59: Nucleotide sequence of DNA fragment synthesized to prepare pAAV-mMAP-mscFv-GS3-hGAA vector, synthetic sequence SEQ ID NO: 60: Amino acid sequence of mscFv-GS3-hGAA SEQ ID NO: 61: Amino acid sequence of hGAA(Pro) SEQ ID NO: 62: Nucleotide sequence of DNA fragment synthesized to prepare pAAV-mMAP-hGAA(Pro)-GS-mscFv2 vector, synthetic sequence SEQ ID NO: 63: Amino acid sequence of hGAA(Pro)-GS-mscFv2 SEQ ID NO: 64: Nucleotide sequence of DNA fragment synthesized to prepare pAAV-mMAP-hGAA vector, synthetic sequence

Claims

1. A nucleic acid molecule comprising any one of the base sequences selected from the group consisting of (1) to (6) below, wherein the fusion protein of an anti-transferrin receptor antibody encoded by the nucleic acid molecule and a physiologically active protein has a binding activity (EC ５０ ) (1) A nucleotide sequence containing a first inverted terminal repeat (ITR) or a functional equivalent thereof, followed downstream by a nucleotide sequence encoding a fusion protein of an anti-transferrin receptor antibody and a protein having physiological activity, and further downstream by a nucleotide sequence containing a second inverted terminal repeat (ITR) or a functional equivalent thereof; (2) A nucleotide sequence containing a first inverted terminal repeat (ITR) or a functional equivalent thereof, a nucleotide sequence containing a gene expression regulatory site downstream thereof, a nucleotide sequence encoding a fusion protein of an anti-transferrin receptor antibody and a physiologically active protein further downstream thereof, and a nucleotide sequence containing a second inverted terminal repeat (ITR) or a functional equivalent thereof further downstream thereof; (3) a nucleotide sequence containing a first long terminal repeat (LTR) or a functional equivalent thereof, followed downstream by a nucleotide sequence encoding a fusion protein of an anti-transferrin receptor antibody and a protein having physiological activity, and further downstream by a nucleotide sequence containing a second long terminal repeat (LTR) or a functional equivalent thereof; (4) A nucleotide sequence containing a first long terminal repeat (LTR) or a functional equivalent thereof, a nucleotide sequence containing a gene expression control site downstream thereof, a nucleotide sequence encoding a fusion protein of an anti-transferrin receptor antibody and a physiologically active protein further downstream thereof, and a nucleotide sequence containing a second long terminal repeat (LTR) or a functional equivalent thereof further downstream thereof; (5) A nucleotide sequence containing a leader or a functional equivalent thereof, a nucleotide sequence downstream thereof encoding a fusion protein of an anti-transferrin receptor antibody and a protein having physiological activity, and a nucleotide sequence further downstream thereof containing a trailer or a functional equivalent thereof; and (6) A base sequence containing a leader or a functional equivalent thereof, a base sequence containing a gene expression control site downstream thereof, a base sequence encoding a fusion protein of an anti-transferrin receptor antibody and a physiologically active protein further downstream thereof, and a base sequence containing a trailer or a functional equivalent thereof further downstream thereof.

2. The nucleic acid molecule of claim 1, which is selected from the group consisting of (1) to (4) below: (1) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein comprises a nucleotide sequence encoding a conjugate in which the physiologically active protein is bound to the C-terminus of the heavy chain of the anti-transferrin receptor antibody, and a nucleotide sequence encoding the light chain of the anti-transferrin receptor antibody; (2) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a nucleotide sequence encoding a conjugate in which the physiologically active protein is bound to the N-terminus of the heavy chain of the anti-transferrin receptor antibody, and a nucleotide sequence encoding the light chain of the anti-transferrin receptor antibody; (3) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a nucleotide sequence encoding a conjugate in which the physiologically active protein is bound to the C-terminus of the light chain of the anti-transferrin receptor antibody, and a nucleotide sequence encoding the heavy chain of the anti-transferrin receptor antibody; and (4) The base sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a base sequence encoding a conjugate in which the physiologically active protein is bound to the N-terminus of the light chain of the anti-transferrin receptor antibody, and a base sequence encoding the heavy chain of the anti-transferrin receptor antibody.

3. The nucleic acid molecule of claim 1, which is selected from the group consisting of (1) to (4) below: (1) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein comprises a nucleotide sequence encoding a conjugate in which the physiologically active protein is linked to the C-terminus of the heavy chain of the anti-transferrin receptor antibody via a linker, and a nucleotide sequence encoding the light chain of the anti-transferrin receptor antibody; (2) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a nucleotide sequence encoding a conjugate in which the physiologically active protein is linked to the N-terminus of the heavy chain of the anti-transferrin receptor antibody via a linker, and a nucleotide sequence encoding the light chain of the anti-transferrin receptor antibody; (3) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a nucleotide sequence encoding a conjugate in which the physiologically active protein is linked to the C-terminus of the light chain of the anti-transferrin receptor antibody via a linker, and a nucleotide sequence encoding the heavy chain of the anti-transferrin receptor antibody; and (4) The base sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a base sequence encoding a conjugate in which the physiologically active protein is linked to the N-terminus of the light chain of the anti-transferrin receptor antibody via a linker, and a base sequence encoding the heavy chain of the anti-transferrin receptor antibody.

4. The nucleic acid molecule of claim 2, which is selected from the group consisting of the following (1) to (4): (1) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a nucleotide sequence encoding a conjugate in which the physiologically active protein is bound to the C-terminus of the heavy chain of the anti-transferrin receptor antibody, a nucleotide sequence downstream thereof encoding an internal ribosome binding site, and a nucleotide sequence further downstream thereof encoding the light chain of the anti-transferrin receptor antibody; (2) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a nucleotide sequence encoding a conjugate in which the physiologically active protein is bound to the N-terminus of the heavy chain of the anti-transferrin receptor antibody, a nucleotide sequence downstream thereof encoding an internal ribosome binding site, and a nucleotide sequence further downstream thereof encoding the light chain of the anti-transferrin receptor antibody; (3) The nucleotide sequence encoding the fusion protein of the anti-transferrin receptor antibody and a physiologically active protein includes a nucleotide sequence encoding a conjugate in which the physiologically active protein is bound to the C-terminus of the light chain of the anti-transferrin receptor antibody, a nucleotide sequence downstream thereof encoding an internal ribosome binding site, and a nucleotide sequence further downstream thereof encoding the heavy chain of the anti-transferrin receptor antibody; and (4) The base sequence encoding the fusion protein of the anti-transferrin receptor antibody and a protein having physiological activity includes a base sequence encoding a conjugate in which the protein having physiological activity is bound to the N-terminus of the light chain of the anti-transferrin receptor antibody, a base sequence downstream of which encodes an internal ribosome binding site, and a base sequence further downstream of which encodes the heavy chain of the anti-transferrin receptor antibody.

5. 5. The nucleic acid molecule of claim 4, wherein the internal ribosome binding site is derived from the 5' untranslated region of a virus or gene selected from the group consisting of Picornaviridae viruses, foot-and-mouth disease virus, hepatitis A virus, hepatitis C virus, coronavirus, bovine enterovirus, Theiler's murine encephalomyelitis virus, Coxsackie B virus, human immunoglobulin heavy chain binding protein gene, Drosophila antennapedia gene, and Drosophila ultravithorax gene.

6. The nucleic acid molecule of claim 1, wherein the gene expression control site is selected from the group consisting of a promoter derived from cytomegalovirus, an SV40 early promoter, a human elongation factor-1α (EF-1α) promoter, a human ubiquitin C promoter, a Rous sarcoma virus LTR promoter (a retrovirus), a dihydrofolate reductase promoter, a β-actin promoter, a phosphoglycerate kinase (PGK) promoter, a mouse albumin promoter, a human albumin promoter, a human α-1 antitrypsin promoter, and a mouse α-fetoprotein enhancer / mouse albumin promoter.

7. The physiologically active protein is selected from the group consisting of growth hormone, lysosomal enzyme, somatomedin, insulin, glucagon, cytokine, lymphokine, blood coagulation factor, anti-transferrin receptor antibody, fusion protein of anti-transferrin receptor antibody with other 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 (FSH), gonadotropin-releasing hormone (GnRH), gonadotropin, nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GLN), and the like. The nucleic acid molecule of claim 1, which is selected from the group consisting of trophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor α receptor (TNF-α receptor), enzymes having beta-amyloid degrading activity, etanercept, pegvisomant, metreleptin, abatacept, asfotase, GLP-1 receptor agonists, and antibody drugs.

8. The physiologically active protein is selected from the group consisting of α-L-iduronidase, iduronate-2-sulfatase, acid α-glucosidase, glucocerebrosidase, β-galactosidase, GM2-activating protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid 2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is selected from the group consisting of erythrocyte sphingomyelinase, α-galactosidase A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2.

9. The binding activity (EC ５０ 2. The nucleic acid molecule of claim 1, wherein the ATP concentration is 3 to 500 nM, 3 to 100 nM, 4 nM to 1 μM, 4 to 500 nM, 4 to 100 nM, 5 nM to 1 μM, 5 to 500 nM, or 5 to 100 nM.

10. A cell, tissue or animal into which the nucleic acid molecule of claim 1 has been introduced.

11. A plasmid comprising the nucleic acid molecule of claim 1.

12. A viral virion comprising the nucleic acid molecule of claim 1.

13. A pharmaceutical composition comprising the viral virion of claim 12 and a carrier.