Nuecleic acid molecule, vector, recombinant cells, and drug for treating central nervous system diseases

Abstract

The purpose of the present invention is to provide a nucleic acid molecule, a vector, recombinant cells, and a drug for treating a central nervous system disease which is likely to migrate into the central nervous system. The nucleic acid molecule according to the present invention comprises a base sequence encoding a fusion protein of: an anti-transferrin receptor (TfR) antibody or an antigen-binding fragment thereof; and a protein which functions in the central nervous system.

Description

Nucleic acid molecules, vectors, recombinant cells and agents for the treatment of central nervous system disorders

[0001] The present invention relates to nucleic acid molecules, vectors, recombinant cells and agents for the treatment of central nervous system disorders.

[0002] Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disease caused by a deficiency of iduronate-2-sulfatase (IDS). The deficiency of IDS leads to the accumulation of its substrate, glycosaminoglycan (GAG), which results in a variety of systemic symptoms, including central nervous system symptoms, characteristic facial features, joint contractures, hepatosplenomegaly, and valvular heart disease.

[0003] Treatments for MPS II include enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT). However, conventional ERT, HSCT, or BMT have not been effective enough for central nervous system lesions, bone lesions, etc. (see, for example, Non-Patent Document 1). In 2021, ERT, which utilizes central nervous system-delivering IDS and intraventricular enzyme administration, was approved in Japan. While ERT is highly effective, it requires weekly infusion and is extremely expensive, making the establishment of a cheaper and more effective treatment an important challenge.

[0004] The present inventors have investigated the pretreatment methods and transplantation rates of HSCT or BMT using MPS II mice, but none of them were effective on the central nervous system (Non-Patent Documents 4 and 5).On the other hand, Non-Patent Documents 2 and 3 report the effect of hematopoietic stem cell gene therapy on MPS II mice as a method other than ERT, HSCT, or BMT.

[0005] The present inventors have succeeded in reducing GAG in the brain of MPS II model mice by expressing the IDS gene in hematopoietic stem cells using a (B6 / MPS II) viral promoter (MND promoter: Moloney murine leukemia virus long terminal repeat / myeloproliferative sarcoma virus enhancer) in model mice, a feat that could not be achieved by ERT or HSCT (Non-Patent Document 6). The present inventors have also developed a lentiviral vector system for gene therapy of mucopolysaccharidosis type II (Patent Document 1). The present inventors further subsequently reported that hematopoietic stem cell gene therapy improves central nervous system lesions in a mouse GM1-gangliosidosis model (Non-Patent Document 7).

[0006] JP 2019-122371 A JP 2018-134095 A WO2018 / 124121 JP 2022-017258 A

[0007] Akiyama K, et al., Mol Genet Metab. 2014, 111(2):139-146.Miwa S, et al., Mol Genet Metab. 2020, 130(4): 262-273Wada M, et al., Mol Ther Methods Clin Dev. 2020, 19:261-274Yokoi T, et al., Mol Genet Metab. 2016, 119(3): 232-238Yokoi K, et al., J Inherit Metab Dis. 2015, 38(2):333-340Wakabayashi T, et al., Hum Gene Ther. 2015, 26(6):357-366Tsunogai T, et al., Mol Ther Methods Clin Dev. 2022, 25:448-460Kyosen SO, et al. Gene Ther. 2010 Apr;17(4):521-30Matsuda J, et al., Glycoconj J. 1997;14(6):729-36Toya Ohashi et al., Mol Ther. 2012 Oct;20(10):1924-31

[0008] In IDS gene therapy targeting hematopoietic stem cells, such as that described in Non-Patent Document 6, cells derived from transplanted IDS gene-introduced hematopoietic stem cells migrate to the central nervous system, whereby IDS is secreted in the central nervous system and is recognized to have a certain therapeutic effect. On the other hand, IDS secreted into the blood outside the central nervous system cannot pass through the blood-brain barrier, and its contribution to the therapeutic effect in the central nervous system is limited.

[0009] Therefore, an object of the present invention is to provide nucleic acid molecules, vectors, recombinant cells and drugs for treating central nervous system diseases that tend to spread to the central nervous system.

[0010] The present inventors have used a lentiviral vector system (Patent Document 1) previously developed for gene therapy of mucopolysaccharidosis type II and a nucleic acid molecule capable of expressing a fusion protein of an IDS and an antibody against the transferrin receptor (TfR), and have found that it is possible to significantly reduce the accumulation of GAG not only in the internal organs but also in the central nervous system. They have also found that it is possible to replace the IDS in this nucleic acid molecule with another protein that should function in the central nervous system, thereby making it possible to treat various central nervous system diseases, which has led to the completion of the present invention.

[0011] That is, the present invention includes the following: [1] A nucleic acid molecule comprising a base sequence encoding a fusion protein comprising an anti-transferrin receptor (TfR) antibody or an antigen-binding fragment thereof and a protein to be functional in the central nervous system. [2] The nucleic acid molecule according to [1], wherein the protein to be functional in the central nervous system is a lysosomal enzyme. [3] The nucleic acid molecule according to [2], wherein the lysosomal enzyme is acid α-glucosidase (GAA) or β-galactosidase (GLB1). [4] The nucleic acid molecule according to [1], wherein the protein to be functional in the central nervous system is iduronate-2-sulfatase (IDS). [5] The nucleic acid molecule according to [1], wherein the protein to be functional in the central nervous system is a neurotrophic factor. [6] The nucleic acid molecule according to [1], wherein the protein to be functional in the central nervous system is an antibody. [7] A vector comprising the nucleic acid molecule according to any of [1] to [6]. [8] The vector according to [7], which is a vector that stably expresses the fusion protein when the nucleic acid molecule is introduced into a host cell using the vector. [9] The vector according to [8], which is a lentiviral vector.

[10] A recombinant cell obtained by introducing the nucleic acid molecule into a host cell using the vector according to [7] or [8].

[11] The recombinant cell according to

[10] , wherein the host cell is a hematopoietic stem cell, a T cell, or a B cell.

[12] A drug for diagnosing, preventing, or treating a central nervous system disease, comprising the recombinant cell according to

[10] .

[13] A method for diagnosing, preventing, or treating a central nervous system disease, comprising transplanting the recombinant cell according to

[10] into a subject in need thereof.

[14] Use of the recombinant cell according to

[10] for diagnosing, preventing, or treating a central nervous system disease.

[15] The recombinant cell according to

[10] for use in diagnosing, preventing, or treating a central nervous system disease.

[16] Use of the recombinant cell according to

[10] in the manufacture of a drug for diagnosing, preventing, or treating a central nervous system disease.

[0012] The present invention provides nucleic acid molecules, vectors, recombinant cells, and drugs for treating central nervous system diseases, such as diseases caused by IDS deficiency and diseases related to proteins other than IDS that should function in the central nervous system. According to the present invention, transplantation of host cells, such as hematopoietic stem cells, expressing a fusion protein of an anti-TfR antibody or its antigen-binding fragment with a protein, such as IDS, that should function in the central nervous system increases the translocation of proteins, such as IDS, to the central nervous system, thereby enabling the treatment of central nervous system diseases, such as mucopolysaccharidosis type II. Because the present invention can use the patient's own autologous hematopoietic stem cells, there is no need to search for a compatible donor, as is the case with conventional allogeneic hematopoietic stem cell transplants, and the risk of transplant-associated graft-versus-host disease (GVHD) or graft failure due to rejection is extremely low. Furthermore, because sustained effects can be maintained with a single transplant, lifetime medical costs are expected to be lower than with enzyme replacement therapy, and the therapeutic effect is also high.

[0013] The present inventors have succeeded in translocating a protein to be functional in the central nervous system into the central nervous system by using a nucleic acid molecule containing a base sequence encoding a fusion protein comprising an anti-TfR antibody or its antigen-binding fragment and a protein to be functional in the central nervous system, and have demonstrated for the first time that the protein actually exerts a therapeutic effect in the central nervous system. It is also possible that recombinant cells, such as hematopoietic stem cells, into which the nucleic acid molecule of the present invention has been introduced may translocate to the central nervous system. Because both the recombinant cells and the fusion protein expressed and secreted by the cells (i.e., a fusion protein of a therapeutic protein such as IDS with an anti-TfR antibody) translocate to the central nervous system, it is difficult to predict the dynamics of the fusion protein, making it difficult to predict whether a therapeutic effect will actually be achieved. Furthermore, it is believed that the presence of an anti-TfR antibody causes a majority of the fusion protein to translocate to central tissues. Therefore, therapeutic effects in tissues other than the central nervous system cannot be expected, and therefore therapies that involve translocating therapeutic proteins to the central nervous system have been avoided. However, as demonstrated in the Examples, the fusion proteins of the present invention unexpectedly exert a certain therapeutic effect on tissues other than the central nervous system (e.g., the liver). Furthermore, because fusion proteins have a large molecular weight and are complex molecules, recombinant cells are unlikely to immediately produce such large and complex fusion proteins. This may result in a decrease in fusion protein productivity, making it even more difficult to predict therapeutic effects. Considering these factors, the effects of the present invention are unexpectedly significant.

[0014] In Example 1, the IDS activity in plasma from 1 month to 6 months after transplantation of gene-transduced hematopoietic stem cells is shown. In Example 1, the results of measuring IDS activity and GAG accumulation in the liver and spleen 6 months after transplantation of gene-transduced hematopoietic stem cells are shown. In Example 1, the results of measuring IDS activity and GAG accumulation in the kidney and heart 6 months after transplantation of gene-transduced hematopoietic stem cells are shown. In Example 1, the results of measuring IDS activity and GAG accumulation in the cerebrum and cerebellum 6 months after transplantation of gene-transduced hematopoietic stem cells are shown. In Example 2, the results of measuring IDS activity in plasma 4 weeks after transplantation of gene-transduced hematopoietic stem cells are shown. In Example 2, the results of measuring IDS activity and GAG accumulation in the liver and spleen 4 weeks after transplantation of gene-transduced hematopoietic stem cells are shown. In Example 2, the results of measuring IDS activity and GAG accumulation in the cerebrum and cerebellum 4 weeks after transplantation of gene-transduced hematopoietic stem cells are shown. In Example 3, the results of measuring IDS activity in plasma 4 weeks after transplantation of gene-transduced T cells are shown. Example 3 shows the results of measuring IDS activity and GAG accumulation in the cerebrum and cerebellum 4 weeks after transplantation of gene-transfected T cells. Example 4 shows the results of measuring GAA activity in gene-transfected HEK293T cells. Example 4 shows the results of measuring GAA activity in the serum, liver, and spleen 4 weeks after transplantation of gene-transfected hematopoietic stem cells. Example 4 shows the results of measuring GAA activity in the heart, quadriceps, diaphragm, and gastrocnemius 4 weeks after transplantation of gene-transfected hematopoietic stem cells. Example 4 shows the results of measuring glycogen accumulation in the heart, quadriceps, diaphragm, and gastrocnemius 4 weeks after transplantation of gene-transfected hematopoietic stem cells. Example 4 shows the results of measuring GAA activity and glycogen accumulation in the cerebrum and cerebellum 4 weeks after transplantation of gene-transfected hematopoietic stem cells. Example 5 shows the results of measuring GLB1 activity in the serum 4 weeks after transplantation of gene-transfected hematopoietic stem cells. In Example 5, the results of measuring GLB1 activity and GM1 accumulation in the cerebral cortex and cerebellum 4 weeks after transplantation of gene-transferred hematopoietic stem cells are shown. In Example 5, the results of measuring GLB1 activity and GM1 accumulation in the hippocampus 4 weeks after transplantation of gene-transferred hematopoietic stem cells are shown.

[0015] [Nucleic Acid Molecule] A nucleic acid molecule according to one embodiment of the present invention comprises a base sequence encoding a fusion protein of an anti-transferrin receptor (TfR) antibody or an antigen-binding fragment thereof with a protein to be functional in the central nervous system. Preferably, the anti-TfR antibody is an anti-human TfR antibody, and the IDS is a human IDS.

[0016] <Transferrin Receptor (TfR)> Transferrin receptor (TfR) is a transmembrane protein that takes up a complex of transferrin and iron (Fe) in the blood into cells, and is present on the surface of vascular endothelial cells, such as cerebrovascular endothelial cells. An anti-TfR antibody according to one embodiment of the present invention specifically binds to TfR and is thereby taken up into cells by TfR. At this time, an IDS that forms a fusion protein with the anti-TfR antibody is also taken up into the cells. Since the IDS, which cannot cross the blood-brain barrier (BBB) ​​by itself, is transported to brain tissue by TfR, it can exhibit its physiological activity in brain tissue. Therefore, a fusion protein of an anti-TfR antibody and an IDS can be used as a pharmaceutical agent that exerts its pharmacological effect in the brain.

[0017] In the present specification, the origin of the transferrin receptor (TfR) is not limited, and may be, for example, human-derived, or may be derived from a mouse, rat, rabbit, horse, or non-human primate, but is preferably human-derived. Human-derived TfR may be a wild-type TfR (protein: NP_001121620; gene: NM_001128148) or a mutant TfR. The mutant TfR is not particularly limited, and any antibody directed against it may be capable of binding to the wild-type TfR, and may be, for example, a mutant TfR with enhanced physiological activity or antigenicity.

[0018] When amino acids in the amino acid sequence of a protein having the physiological activity of wild-type TfR are substituted with other amino acids to form a mutant TfR, the number of amino acids to be substituted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When amino acids in the wild-type TfR are deleted, the number of amino acids to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Mutations that combine these amino acid substitutions and deletions can also be added. When amino acids are added to the wild-type TfR, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 amino acids are added within the amino acid sequence or to the N-terminus or C-terminus of the protein. Mutations that combine these amino acid additions, substitutions, and deletions can also be added. The amino acid sequence of the mutated protein preferably exhibits 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 amino acid sequence of wild-type TfR.

[0019] In the present invention, the location and type (deletion, substitution, addition) of each mutation compared to wild-type TfR 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 wild-type TfR and that of mutant TfR can be easily calculated using well-known homology calculation algorithms. Examples of such algorithms include BLAST (Altschul S. F., J. Mol. Biol. 215, 403-10, (1990)), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA. 85, 2444 (1988)), and the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2, 482-9 (1981)).

[0020] 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, in their side chains and chemical properties, and is predicted not to significantly alter the function of the protein (i.e., 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 affect 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.

[0021] <Anti-Transferrin Receptor (TfR) Antibody> As used herein, the term "antibody" refers to a protein immunoglobulin that specifically binds to an antigen. Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, IgE, and IgM. Generally, antibodies comprise at least two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), which comprises three constant domains, namely, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (VL) and a light chain constant region, which comprises one constant domain, namely, CL. The VH and VL regions each contain framework regions (FRs) and complementarity-determining regions (CDRs), each containing three CDRs and four FRs in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen.

[0022] As used herein, the term "antibody" includes monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, and tetrameric antibodies comprising two heavy chain molecules and two light chain molecules. In addition to common tetrameric antibodies, the term "antibody" also includes dimeric antibodies, single-chain antibodies, and single-domain antibodies, which will be described later, and these are also referred to as antibody fragments (antigen-binding fragments).

[0023] A "human antibody" refers to an antibody that is entirely encoded by a gene of human origin. However, antibodies encoded by genes in which mutations have been added to the original human gene for purposes such as increasing gene expression efficiency are also human antibodies. Furthermore, antibodies in which two or more genes encoding human antibodies are combined to replace a portion of one human antibody with a portion of another human antibody are also human antibodies. A human antibody has 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 referred to as CDR1, CDR2, and CDR3, starting from the N-terminus. The three CDRs in the immunoglobulin heavy chain are referred to as CDR1, CDR2, and CDR3, starting from the N-terminus. An antibody in which the antigen specificity, affinity, etc. of a human antibody have been modified by replacing the CDR of one human antibody with the CDR of another human antibody is also a human antibody.

[0024] As used herein, the term "human antibody" also refers to an antibody in which mutations such as substitutions, deletions, and additions have been added to the amino acid sequence of the original human antibody by modifying the gene of the original human antibody. When amino acids in the amino acid sequence of the original antibody are substituted with other amino acids, the number of substituted amino acids 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 antibody are deleted, the number of deleted amino acids is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3. Antibodies in which mutations have been added by combining these amino acid substitutions and deletions are also human 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 antibody. Antibodies in which mutations have been added by combining these amino acid additions, substitutions, and deletions are also human antibodies. The amino acid sequence of the mutated 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 antibody. In other words, in the present invention, the term "human-derived gene" includes not only the original human-derived gene, but also a gene obtained by modifying the original human-derived gene.

[0025] As used herein, the term "humanized antibody" refers to an antibody in which the amino acid sequence of a portion of the variable region (e.g., particularly all or part of the CDRs) is derived from a mammal other than human, and the remaining regions are derived from human. Examples of humanized antibodies include antibodies produced by replacing three complementarity-determining regions (CDRs) of an immunoglobulin light chain and three complementarity-determining regions (CDRs) of an immunoglobulin heavy chain that constitute a human antibody with CDRs from another mammal. The species of other mammal from which the CDRs to be grafted into appropriate positions in a human 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, and more preferably a mouse or rat, such as a mouse.

[0026] In the present specification, cases where the antibody is a human antibody or a humanized antibody will be described in detail below. The light chains of human antibodies and humanized antibodies include λ chains and κ chains. The light chains constituting the antibody may be either λ chains or κ chains. Furthermore, the heavy chains of human antibodies and humanized 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 the antibody may be any of γ chains, μ chains, α chains, σ chains, and ε chains, but are preferably γ chains. Furthermore, the γ chains of antibody heavy chains 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 antibody is a γ chain, the γ chain may be any of γ1 chains, γ2 chains, γ3 chains, and γ4 chains, but is preferably γ1 chains or γ4 chains. When the antibody is a humanized antibody or a human antibody and is an IgG, the light chain of the antibody may be either a λ chain or a κ chain, and the heavy chain of the antibody may be any of a γ1 chain, a γ2 chain, a γ3 chain, and a γ4 chain, but is preferably a γ1 chain or a γ4 chain. For example, one preferred embodiment of the antibody is one in which the light chain is a λ chain and the heavy chain is a γ1 chain.

[0027] As used herein, the term "chimeric antibody" refers to an antibody formed by linking fragments of two or more different antibodies derived from two or more different species. In one embodiment, the anti-TfR antibody is a humanized anti-TfR antibody.

[0028] A chimeric antibody between a human antibody and an antibody from another mammal is an antibody in which a portion of a human antibody is replaced with a portion of an antibody from a mammal other than human. The antibody consists of an Fc region, a Fab region, and a hinge region, as described below. A specific example of such a chimeric antibody is a chimeric antibody in which the Fc region is derived from a human antibody while the Fab region is derived from an antibody from another mammal. Conversely, a chimeric antibody in which the Fc region is derived from another mammal while the Fab region is derived from a human antibody is also a chimeric antibody. The hinge region may be derived from either a human antibody or an antibody from another mammal. The same applies to humanized anti-TfR antibodies.

[0029] Chimeric antibodies (e.g., humanized anti-TfR antibodies) can also be said to consist of a variable region and a constant region. Other specific examples of chimeric antibodies include those in which the heavy chain constant region (CH) and light chain constant region (CL) are derived from a human antibody, while the heavy chain variable region (VH) and light chain variable region (VL) are derived from an antibody of another mammal. Conversely, those in which the heavy chain constant region (CH) and light chain constant region (CL) are derived from an antibody of another mammal, while the heavy chain variable region (VH) and light chain variable region (VL) are derived from a human antibody. The other mammalian species 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, and more preferably a mouse.

[0030] Chimeric antibodies of a human antibody and a mouse antibody are particularly referred to as "human / mouse chimeric antibodies." Examples of human / mouse chimeric antibodies include chimeric antibodies in which the Fc region is derived from a human antibody and the Fab region is derived from a mouse antibody, and conversely, chimeric antibodies in which the Fc region is derived from a mouse antibody and the Fab region is derived from a human antibody. The hinge region is derived from either a human antibody or a mouse antibody. Other specific examples of human / mouse chimeric antibodies include those in which the heavy chain constant region (CH) and light chain constant region (CL) are derived from a human antibody and the heavy chain variable region (VH) and light chain variable region (VL) are derived from a mouse antibody, and conversely, those in which the heavy chain constant region (CH) and light chain constant region (CL) are derived from a mouse antibody and the heavy chain variable region (VH) and light chain variable region (VL) are derived from a human antibody.

[0031] An antibody originally has a basic structure consisting of a total of four polypeptide chains: two immunoglobulin light chains and two immunoglobulin heavy chains. However, in the present invention, the term "antibody" includes, in addition to tetrameric antibodies having this basic structure, the following: (1) dimeric antibodies consisting of two polypeptide chains, one immunoglobulin light chain and one immunoglobulin heavy chain; (2) single-chain antibodies comprising a linker at the C-terminus of an immunoglobulin light chain, and an immunoglobulin heavy chain attached to the C-terminus thereof; (3) single-chain antibodies comprising a linker at the C-terminus of an immunoglobulin heavy chain, and an immunoglobulin light chain attached to the C-terminus thereof; (4) single-chain antibodies (scFv) comprising a linker at the C-terminus of the variable region of an immunoglobulin heavy chain, and an immunoglobulin light chain attached to the C-terminus thereof; (5) single-chain antibodies (scFv) comprising a linker at the C-terminus of the variable region of an immunoglobulin light chain, and an immunoglobulin heavy chain attached to the C-terminus thereof; (6) antibodies consisting of an Fab region, which is the basic structure of the above-mentioned antibody with the Fc region deleted, and antibodies consisting of an Fab region and all or part of the hinge region (including Fab, F(ab'), and F(ab')2), and (7) single domain antibodies are also included in the term "antibody" herein. Furthermore, scFv, which is a single-chain antibody formed by linking the light chain variable region and the heavy chain variable region via a linker, is also included in the term "antibody" herein.

[0032] As used herein, 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 referred to as 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 and the C-terminus of another protein are linked 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.

[0033] In one embodiment of the present invention, Fab refers to a molecule in which one light chain containing a variable region and a CL region (light chain constant region) and one heavy chain containing a variable region and a CH1 region (part 1 of the heavy chain constant region) are bound by disulfide bonds between the cysteine ​​residues present in each. In Fab, the heavy chain may contain a portion of the hinge region in addition to the variable region and CH1 region (part 1 of the heavy chain constant region), but in this case, the hinge region lacks the cysteine ​​residues present in the hinge region that bind the heavy chains of the antibody. In Fab, the light chain and heavy chain are bound by disulfide bonds formed between cysteine ​​residues present in the light chain constant region (CL region) and cysteine ​​residues present in the heavy chain constant region (CH1 region) or hinge region. The heavy chains that form Fab are called Fab heavy chains. Fab lacks the cysteine ​​residues present in the hinge region that link the heavy chains of an antibody, and therefore consists of one light chain and one heavy chain. The light chain that constitutes Fab contains a variable region and a CL region. The heavy chain that constitutes Fab may consist of a variable region and a CH1 region, or may contain a portion of the hinge region in addition to the variable region and CH1 region. In this case, however, the hinge region is selected so as not to contain a cysteine ​​residue that links 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 contains, in addition to the variable region and CH1 region, all or part of the hinge region containing the cysteine ​​residue that links the heavy chains. F(ab')2 refers to a molecule in which two F(ab') are linked by disulfide bonds between the cysteine ​​residues present in the hinge regions. A 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 linking multiple antibodies directly or via a linker are also antibodies. Furthermore, without being limited to these, any substance that contains a portion of an antibody molecule and has the property of specifically binding to an antigen is included in the "antibody" referred to in the present invention. That is, the term "light chain" as used in the present invention includes a substance 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 a substance 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, for example, even one lacking the Fc region is considered a heavy chain.

[0034] Furthermore, Fc or Fc region herein refers to a region in an antibody molecule that includes a fragment consisting of the CH2 region (part 2 of the heavy chain constant region) and the CH3 region (part 3 of the heavy chain constant region).

[0035] 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 attaching a linker sequence to the C-terminus of the light chain and then attaching a heavy chain to that C-terminus, or by attaching a linker to the C-terminus of the heavy chain and then attaching a light chain to that C-terminus. Furthermore, antibodies of 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, a linker sequence may be attached to the C-terminus of a light chain variable region, and a heavy chain variable region may be attached to the C-terminus of that linker sequence. Alternatively, a linker sequence may be attached to the C-terminus of a heavy chain variable region, and a light chain variable region may be attached to the C-terminus of that linker sequence.

[0036] Furthermore, the term "antibody" as used herein includes not only full-length antibodies and those shown in (1) to (8) above, but also antigen-binding fragments (antibody fragments) in which a portion of a full-length antibody is deleted, which is a broader concept including (1) to (8). Antigen-binding fragments also include heavy chain antibodies, light chain antibodies, VHHs, VNARs, and those in which a portion of these is deleted.

[0037] The term "antigen-binding fragment" refers to an antibody fragment that retains at least a portion of its specific binding activity to an antigen, and may include the antigen complementarity-determining region (CDR). Examples of antigen-binding fragments include Fab, Fab', F(ab')2, variable region (Fv), single-chain antibody (scFv) in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked via an appropriate linker, diabodies, which are dimers of polypeptides containing a heavy chain variable region (VH) and a light chain variable region (VL), minibodies, which are dimers in which a portion of the constant region (CH3) is bound to the heavy chain (H chain) of an scFv, and other minibodies. However, the fragments are not limited to these molecules as long as they have the ability to bind to the antigen.

[0038] As used herein, 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 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 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, 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, a single-chain antibody can also be produced by deleting one or more CDRs, as long as the antigen-specific affinity of the antibody is maintained.

[0039] In a single-chain antibody, the linker disposed between the light and heavy chains of an immunoglobulin 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 antibody formed by linking both chains thereby retains affinity for the antigen, but is preferably composed of glycine alone or glycine and serine, and examples thereof include the amino acid sequence Gly-Ser, Gly-Gly-Ser, Gly-Gly-Gly, Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 9), Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 10), and Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 11), or sequences in which 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 heavy chain via a linker to the C-terminus of an amino acid sequence consisting of the entire variable region of an immunoglobulin light chain, a linker having a sequence in which the sequence of SEQ ID NO: 9 is repeated three times is preferably used.

[0040] 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 via 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.

[0041] In one embodiment of the present invention, specific examples of anti-TfR antibodies include those described in Patent Documents 2 to 4.

[0042] <Proteins that should function in the central nervous system> As used herein, a "protein that should function in the central nervous system" refers to a protein that has physiological activity in the central nervous system and that needs to function in the central nervous system. Here, proteins also include peptides, and may be proteins having, for example, 2 to 3,000, 10 to 1,500, or 20 to 1,000 amino acid residues. The protein to be made to function in the central nervous system is not particularly limited as long as it is a protein having physiological activity in the central nervous system, and examples thereof include lysosomal enzymes, neurotrophic factors, antibodies, hormones, somatomedins, insulin, glucagon, cytokines, lymphokines, blood coagulation factors, fusion proteins of antibodies with other proteins, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), macrophage-colony-stimulating factor (M-CSF), erythropoietin, darbepoetin, tissue plasminogen activator (t-PA), thrombomodulin, follicle-stimulating hormone (FSH), gonadotropin-releasing hormone (GnRH), gonadotropin, DNase I, thyroid-stimulating hormone (TSH), Examples of such antibodies include nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor α receptor (TNF-α receptor), an enzyme having the activity of degrading beta-amyloid, etanercept, pegvisomant, metreleptin, abatacept, asfotase, and GLP-1 receptor agonists.

[0043] Suitable examples of proteins that should function in the central nervous system as lysosomal enzymes include α-L-iduronidase, glucocerebrosidase, β-galactosidase (GLB1), GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, and acid sphingomyelinase. Examples of the lysosomal enzyme include lysosomal enzymes such as 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, aspartylglucosaminidase, palmitoyl protein thioesterase-1 (PPT-1), tripeptidyl peptidase-1 (TPP-1), hyaluronidase-1, CLN1, CLN2, and acid α-glucosidase (GAA). When the protein to be functional in the central nervous system is a lysosomal enzyme, it is more preferable that the lysosomal enzyme is β-galactosidase (GLB1) or acid α-glucosidase (GAA).

[0044] The protein to be functional in the central nervous system is preferably acid alpha-glucosidase (GAA), which is the enzyme responsible for the muscle disease known as Pompe disease, and a deficiency of GAA causes the accumulation of glycogen, a substrate for GAA, in tissues, resulting in various symptoms.

[0045] In the present specification, the origin of GAA is not limited, and may be, for example, human-derived, or may be derived from a mouse, rat, rabbit, horse, or non-human primate, but is preferably human-derived. Human-derived GAA may be wild-type GAA (e.g., protein: NP_000143 (Genbank); gene: NM_000152 (Genbank), etc.) or mutant GAA. The amino acid or nucleotide sequence of wild-type GAA may vary by several amino acids or several to several dozen bases depending on the database referenced, but is not particularly limited as long as it is recognizable as wild-type GAA by those skilled in the art. The mutant GAA is not particularly limited, and may be, for example, a mutant GAA with enhanced physiological activity. The same applies to mutant GAA as described for mutant TfR. Other proteins to function in the central nervous system may also be derived from a human, or may be derived from a mouse, rat, rabbit, horse, or non-human primate, but is preferably derived from a human. Furthermore, the protein may be a wild-type protein or a mutant protein.

[0046] The protein that should function in the central nervous system is preferably β-galactosidase (GLB1), which is the causative enzyme of a genetic disease known as GM1 gangliosidosis. A deficiency of GLB1 causes the accumulation of GM1 ganglioside, a substrate of GLB1, in tissues, resulting in various symptoms.

[0047] Herein, the origin of GLB1 is not limited and may be, for example, human-derived, or may be derived from mouse, rat, rabbit, horse, or non-human primate, but is preferably human-derived. Human-derived GLB1 may be wild-type GLB1 (e.g., protein: AAA51823 (GenBank), NP_000395 (GenBank), P16278 (Uniprot); gene: NM_000404 (GenBank), M34423 (GenBank), etc.), or may be mutant GLB1. The amino acid or nucleotide sequence of wild-type GLB1 may vary by several amino acids or several to several dozen nucleotides depending on the database referenced, but is not particularly limited as long as it is recognizable as wild-type GLB1 by those skilled in the art. The mutant GLB1 is not particularly limited and may be, for example, a mutant GLB1 with enhanced physiological activity. The description of mutant TfR also applies to mutant GLB1. Similarly, other proteins to be functional in the central nervous system may be derived from humans, mice, rats, rabbits, horses, or non-human primates, but are preferably derived from humans. Furthermore, they may be wild-type proteins or mutant proteins.

[0048] The protein that should function in the central nervous system is preferably iduronate-2-sulfate (IDS), an enzyme that causes mucopolysaccharidosis type II, also known as Hunter syndrome. Deficiency of IDS leads to the accumulation of glycosaminoglycans (GAGs), which are the substrates of IDS, in tissues, causing various symptoms.

[0049] In this specification, the origin of the IDS is not limited, and may be, for example, human-derived, or may be derived from a mouse, rat, rabbit, horse, or non-human primate, but is preferably human-derived. The human-derived IDS may be a wild-type IDS (protein: NP_000193; gene: NM_000202) or a mutant IDS. The mutant IDS is not particularly limited, and may be, for example, a mutant IDS with enhanced physiological activity. The description of the mutant TfR also applies to the mutant IDS. Other proteins that are to function in the central nervous system may also be human-derived, and may be derived from a mouse, rat, rabbit, horse, or non-human primate, but is preferably human-derived. Furthermore, the protein may be a wild-type protein or a mutant protein.

[0050] <Fusion Protein> The fusion protein of the present invention is a fusion protein of an anti-transferrin receptor (TfR) antibody or an antigen-binding fragment thereof with a protein to be functional in the central nervous system, which can diagnose, prevent, or treat central nervous system diseases. In the fusion protein, the anti-TfR antibody or its antigen-binding fragment and the protein to be functional in the central nervous system may be fused in this order from the N-terminus to the C-terminus, or in the reverse order, and it is preferable that the anti-TfR antibody or its antigen-binding fragment is present at the N-terminus. Furthermore, a peptide linker may be present between the anti-TfR antibody or its antigen-binding fragment and the protein to be functional in the central nervous system. Examples of peptide linkers are as described above.

[0051] The fusion protein of one embodiment of the present invention may contain a marker protein for detecting the fusion protein, and examples of the marker protein include GFP, EGFP, YFP, RFP, mCherry, luciferase, etc. The marker protein may be located at the N-terminus or C-terminus of the fusion protein, or between the anti-TfR antibody or antigen-binding fragment thereof and the protein to be functional in the central nervous system.

[0052] The fusion protein of one embodiment of the present invention may contain multiple proteins that function in the central nervous system. In this case, the multiple proteins that function in the central nervous system are functionally fused, preferably via a linker. The anti-TfR antibody or antigen-binding fragment thereof may be present at either the N-terminus or C-terminus, but is preferably present at the N-terminus.

[0053] <Nucleic Acid Molecule> As used herein, the term "nucleic acid molecule" refers 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.

[0054] 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.

[0055] 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 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. Modification of ribonucleotides is performed, for example, to suppress degradation of RNA by RNase and increase the stability of RNA in cells.

[0056] Among the nucleic acid molecules encoding the fusion protein, the nucleic acid molecule of the portion encoding the anti-TfR antibody or an antigen-binding fragment thereof is not particularly limited, and examples include the DNA sequences described in Patent Documents 2 to 4.

[0057] When the protein to function in the central nervous system is IDS, the nucleic acid molecule encoding the IDS portion of the nucleic acid molecule encoding the fusion protein may be the IDS gene (NM_000202) or cDNA. It may also be, for example, the codon-optimized DNA shown in SEQ ID NO: 3. When the protein to function in the central nervous system is GAA, the nucleic acid molecule encoding the GAA portion of the nucleic acid molecule encoding the fusion protein may be the GAA gene (e.g., NM_000152, etc.) or cDNA. It may also be, for example, the DNA shown in SEQ ID NO: 18. When the protein to function in the central nervous system is GLB1, the nucleic acid molecule encoding the GLB1 portion of the nucleic acid molecule encoding the fusion protein may be the GLB1 gene (e.g., NM_000404, M34423, etc.) or cDNA. It may also be, for example, the codon-optimized DNA shown in SEQ ID NO: 24.

[0058] When a peptide linker is present in the fusion protein between the anti-TfR antibody or antigen-binding fragment thereof and the protein to be functional in the central nervous system, the nucleic acid molecule encoding the fusion protein also contains a nucleic acid encoding the peptide linker.

[0059] <Vector> The vector of the present invention comprises the nucleic acid molecule of the present invention. The vector is preferably a vector capable of autonomous replication in host cells and / or a vector capable of integration into the chromosome of a host cell, a vector capable of transcribing the nucleic acid molecule, and a vector that stably expresses a fusion protein when the nucleic acid molecule is introduced into a host cell using the vector. Specific examples include lentiviral vectors, adenovirus-associated vectors, retroviral vectors, DNA vectors, RNA vectors, adenoviral vectors, baculoviral vectors, Epstein-Barr virus vectors, papovavirus vectors, vaccinia virus vectors, and herpes simplex virus vectors. Of these, lentiviral vectors and retroviral vectors are preferred from the viewpoint of stable expression in host cells.

[0060] Lentiviruses are viruses belonging to the genus Lentivirus in the Orthoretropenaeus subfamily, a subfamily of the Retroviridae family, and have a single-stranded (+) strand RNA genome (ssRNA). The lentivirus genome contains essential genes, such as gag (a region encoding structural proteins including capsid protein), pol (a region encoding a group of enzymes including reverse transcriptase), and env (a region encoding envelope proteins required for binding to host cells), which are sandwiched between two LTRs (5'LTR and 3'LTR). In addition to these, the lentivirus genome also contains auxiliary genes, such as Rev (a region encoding a protein that binds to an RRE (rev responsive element) present in the viral RNA and transports the viral RNA from the nucleus to the cytoplasm), tat (a region encoding a protein that binds to TAR in the 5'LTR and increases the promoter activity of the LTR), and vif, vpr, vpu, and nef.

[0061] 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 is developed based on the genome of HIV-1, a type of lentivirus, but is not limited to this.

[0062] First-generation lentiviral vectors 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 LTRs is packaged into the capsid protein. The packaging plasmid of the first-generation lentiviral vector 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 a ligand and a physiologically active protein. The promoter controlling the gene encoding the desired protein is preferably a promoter other than the CMV promoter, such as the MND promoter, the phosphoglycerate kinase (PGK) promoter, the CD11b promoter, the SV40 early promoter, the human elongation factor-1α (EF-1α) promoter, the human ubiquitin C promoter, the CAG promoter (a hybrid promoter of a cytomegalovirus enhancer, an chicken β-actin promoter, and a rabbit β-globin polyA), the retroviral Rous sarcoma virus LTR promoter, the dihydrofolate reductase promoter, the β-actin promoter, the mouse albumin promoter, the human albumin promoter, and the human α-1 antitrypsin promoter. Preferably, the MND promoter is as set forth in SEQ ID NO: 1.

[0063] Like the first-generation lentiviral vector, the second-generation lentiviral vector also consists of three plasmids: a packaging plasmid, an Env plasmid (envelope plasmid), and a transfer plasmid, except that the non-essential auxiliary genes vif, vpr, vpu, and nef have been deleted from the packaging plasmid.

[0064] In third-generation lentiviral vectors (i.e., lentiviral vector systems), the Rev gene contained in the packaging plasmid in the second generation is separated into an independent Rev plasmid. Tat is also deleted from the packaging plasmid. Furthermore, the U3 region in the 5' LTR of the transfer plasmid is replaced with a CMV promoter.

[0065] The packaging plasmid encodes viral genes other than the envelope gene and supplies proteins in trans for producing viral particles. Because the modifier gene products are not necessary for infection of non-dividing cells, the modifier genes (vif, fpr, vpu, nef) are deleted. While the modifier gene products are not essential for HIV-1 replication, they are important and deeply involved in HIV-1 pathogenesis, so deleting them increases safety. Furthermore, tat is essential for HIV-1 replication, so deleting it from the packaging plasmid further increases safety. Furthermore, because the packaging plasmid does not have a packaging signal (Ψ), RNA transcribed from this plasmid is not incorporated into viral particles.

[0066] Rev acts on transcribed RNA and selectively exports unspliced ​​mRNA out of the nucleus, resulting in the translation of structural proteins. In the third generation, the Rev plasmid is a separate plasmid from the packaging plasmid.

[0067] Since the HIV-1 envelope can only infect CD4-positive cells, the envelope plasmid uses VSV-G (vesicular stomatis virus G gylcoprotein) to broaden the host range. The receptor for VSV-G is thought to be a phospholipid, and by using VSV-G as the envelope, membrane fusion between the virus particle and the cell occurs independently of the cell surface receptor. Therefore, it is basically possible to infect any animal species or cell type. Furthermore, VSV-G is physically strong, and virus particles can be easily concentrated by ultracentrifugation.

[0068] The vector contains a gene of interest integrated between LTRs (transcriptional expression-regulating repeats) at both ends, each containing a packaging signal (Ψ). It also contains a primer binding site essential for reverse transcription, and RNA transcribed from this plasmid is incorporated into viral particles. Because HIV-1 LTR promoter activity is very weak in the absence of tat, an internal promoter is used to express the integrated foreign gene. In one embodiment, the internal promoter may be the PGK, CD11b, or MND promoter.

[0069] The RRE (rev-responsive element) is required for Rev to bind and for full-length viral genome RNA to be efficiently transported from the nucleus to the cytoplasm. During reverse transcription in retroviruses, plus-strand synthesis begins from the PPT (polypurine tract) sequence located immediately upstream of the 3'LTR, but lentiviruses have another identical sequence called the cPPT (central polypurine tract) in the center of the genome, and synthesis also occurs from here, resulting in the formation of a triple-stranded structure of approximately 100 base pairs in the center called a DNA flap in the final double-stranded cDNA. It has been suggested that the cPPT sequence affects the efficiency of reverse transcription, and incorporating the cPPT sequence into a vector increases gene transfer efficiency. The WPRE (woodchuk hepatitis virus posttranscriptional regulatory element) is incorporated to increase expression efficiency. Specifically, WPRE is believed to play a role in the active transport of mRNA from the nucleus to the cytoplasm and in increasing mRNA stability in the cytoplasm. Incorporation of this sequence into the vector increases the expression efficiency of titer and transgenes. Furthermore, deletion of the enhancer / promoter region in U3 of the 3'LTR results in both the 3' and 5'LTRs losing promoter activity in the proviral state, preventing transcription of the entire genome from the 5'R region. This makes it a lentiviral vector that corresponds to a strain defective in HIV-1 replication, etc., and satisfies the requirement of the position paper, "3. The provirus lacks LTR promoter activity, and the entire HIV genome is not transcribed." It is a self-inactivating (SIN) vector. Furthermore, by replacing U3 of the 5'LTR with a CMV promoter, tat dependency is eliminated, making it possible to delete tat from the packaging plasmid.

[0070] As an example, the self-inactivating lentivirus pLVSIN-CMV Neo vector (manufactured by Takara Bio Inc.) can be used. Furthermore, as described in the Examples, for example, pJLV1 is preferably used, which is a hybrid LTR obtained by substituting the U3 promoter region of the 5' LTR of the pLVSIN-CMV Neo vector with a CMV promoter, deleting the viral sequence downstream of the packaging signal, deleting the internal promoter PCMVIE through the neomycin resistance gene, and inserting the MND promoter. The vector of the present invention can be obtained by functionally inserting a nucleic acid molecule encoding a fusion protein downstream of the vector.

[0071] Because recombinant 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 nucleic acid molecule encoding a fusion protein. In host cells or the like into which the nucleic acid molecule has been introduced, the fusion protein is expressed from this nucleic acid molecule.

[0072] 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 necessary 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.

[0073] Retroviral vectors were primarily developed based on murine leukemia viruses. To eliminate pathogenicity and enhance safety, the viral genome is segmented to eliminate self-replication while maintaining infectivity. First-generation retroviral vectors consist of a packaging plasmid (the viral genome excluding the packaging signal) and a transfer plasmid (a packaging signal, a portion of gag, and a foreign gene flanked by 5' LTR and 3' LTR). Therefore, when homologous recombination occurs at the gag sequence shared by the packaging plasmid and the transfer plasmid, a self-replicating retrovirus, i.e., a replication-competent (RC) virus, emerges. In second-generation retroviral vectors, the 3' LTR of the first-generation packaging plasmid is replaced with a poly(A) addition signal. Therefore, for RC virus to emerge, simultaneous homologous recombination must occur at two sites, the gag sequence and upstream of the poly(A) addition signal, and the probability of this occurring is extremely low, enhancing safety. The third generation is composed of three plasmids, with the second generation packaging plasmid further split into a plasmid encoding gag / pol and a plasmid encoding env. This means that for RC virus to be generated, simultaneous homologous recombination must occur at three sites, which makes the probability of this occurring extremely low, further increasing safety.

[0074] Generally, to produce recombinant retroviral virions, the packaging plasmid and transfer plasmid 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 nucleic acid molecule encoding a fusion protein. In host cells, etc., into which the nucleic acid molecule has been introduced, the fusion protein is expressed from this nucleic acid molecule.

[0075] A method for constructing a vector according to the present invention will be described. The vector is constructed by a cloning procedure in which the desired insert DNA fragments (a nucleic acid molecule encoding an anti-TfR antibody or its antigen-binding fragment, and a nucleic acid molecule encoding a protein to be functionalized in the central nervous system) are placed into the desired vector (pJLV1 plasmid). In this case, the nucleic acid molecule encoding the anti-TfR antibody or its antigen-binding fragment and the nucleic acid molecule encoding the protein to be functionalized in the central nervous system may be prepared in a functionally linked state, or they may be prepared separately. When preparing them separately, restriction enzyme sites or the like are set in advance so that the two are functionally linked when cleaved and linked.

[0076] In preparing insert DNA, the restriction enzyme cleavage sites of the target DNA fragment are first identified, and then the restriction enzymes to be used for excision are selected according to the cloning site of the vector to be used. The restriction enzyme sites are not particularly limited, but may be, for example, 5'-NotI GCGGCCGC / 3'-XhoI CTCGAG.

[0077] Next, in preparing the vector, the cloning site of the target vector (pJLV1 plasmid) is cleaved with a restriction enzyme to linearize the vector, and then the insert DNA is ligated with the linearized plasmid to prepare the vector according to the present invention.

[0078] The plasmid system, which includes a packaging plasmid, an Env plasmid, a Rev plasmid, and a transfer plasmid (vector), is also known as a third-generation lentiviral vector. The entire system deletes more than one-third of the HIV-1 genome, making it highly unlikely that wild-type HIV-1 will be produced. The homologous regions between the plasmids are also minimized, making it highly unlikely that a virus capable of autonomous replication will be produced by homologous recombination.

[0079] Next, we will describe how lentiviral vectors are used. The constructed vectors are co-transfected with a third-generation packaging plasmid, a Rev plasmid, and a VSV-G envelope plasmid into large-scale cultures of, for example, 293T cells, and the supernatant culture medium is collected three days later. At this point, the culture medium contains viral particles, which are purified by ultracentrifugation and stored in aliquots at -80°C.

[0080] 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, primarily composed of phospholipids, particularly phosphatidylcholine. However, liposomes are not limited to this, and as long as they form a lipid bilayer, other lipids, such as egg yolk phosphatidylethanolamine, may also be added. 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 about 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. The 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 from the gene in cells, etc. into which the gene has been introduced. As used herein, 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).

[0081] In the present invention, the behavior of nucleic acid molecules introduced into cells, tissues, or living organisms in the form of a vector, encapsulated in a recombinant viral virion, or encapsulated in a liposome, lipid nanoparticle, or the like is exemplified below in (1) to (9). However, the behavior of nucleic acid molecules is not limited to these examples. (1) In one embodiment, the nucleic acid molecule is single-stranded (+) strand RNA. When introduced into a cell, a gene encoding a fusion protein of a ligand contained in the nucleic acid molecule and a physiologically active protein is translated, resulting in the expression of the fusion protein. (2) In one embodiment, 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) In one embodiment, 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) In one embodiment, the nucleic acid molecule is single-stranded (+) or (-) strand 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) In one embodiment, the nucleic acid molecule is single-stranded (+) strand DNA, and when introduced into a cell, the nucleic acid molecule is transcribed and translated to express the fusion protein. (6) In one embodiment, the nucleic acid molecule is single-stranded (-) strand DNA, and when introduced into a cell, double-stranded DNA is synthesized from the nucleic acid molecule, which is then transcribed and translated to express the fusion protein. (7) In one embodiment, the nucleic acid molecule is single-stranded (+) or (-) strand DNA, and when introduced into a cell, double-stranded DNA is synthesized from the nucleic acid molecule, and this DNA then undergoes random or homologous recombination with the genome of the host cell to be integrated into the genome, and the integrated DNA is transcribed and translated to express the fusion protein. (8) In one embodiment, 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.(9) In one embodiment, the nucleic acid molecule is double-stranded DNA, and this DNA undergoes random recombination or homologous recombination with the genome of a host cell to be integrated into the genome, and the integrated DNA is transcribed and translated to express the fusion protein.

[0082] Nucleic acid molecules in the form of vectors, encapsulated in recombinant viral virions, or encapsulated in liposomes, lipid nanoparticles, etc. can be introduced into cells, tissues, or living organisms.

[0083] 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, in the form of a vector, encapsulated in a recombinant viral virion, or encapsulated in a liposome, lipid nanoparticle, or the like.

[0084] 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 pharmaceuticals.

[0085] <Recombinant Cells> The recombinant cells of the present invention are obtained by introducing the vector of the present invention into host cells. The type of host cell is not particularly limited, and examples include stem cells such as hematopoietic stem cells, mesenchymal stem cells, dental pulp-derived 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, as well as immune cells such as T cells, B cells, and NK cells.

[0086] Recombinant cells into which the nucleic acid molecule has been introduced express the fusion protein of the invention and can then be implanted into a patient for therapeutic purposes.

[0087] [Drug] The drug of the present invention is a drug for diagnosing, preventing, or treating central nervous system diseases, comprising the recombinant cell of the present invention. Examples of central nervous system diseases include neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease; mental disorders such as schizophrenia and depression; multiple sclerosis, amyotrophic lateral sclerosis, central nervous system tumors including brain tumors, lysosomal diseases associated with brain disorders, glycogen storage diseases, muscular dystrophy, cerebral ischemia, cerebral inflammatory diseases, prion diseases, and traumatic central nervous system disorders. In addition, diseases caused by IDS deficiency, Gaucher disease, GM1-gangliosidosis types 1 to 3, GM2-gangliosidosis AB variant, Sandhoff disease and Tysachs disease, Sandhoff disease, I-cell disease, α-mannosidosis, β-mannosidosis, Krabbe disease, Gaucher-like storage disease, metachromatic leukodystrophy, fucosidosis, aspartylglucosaminuria, Schindler disease, Kawasaki disease, Examples of central nervous system disorders include IDS deficiency, Niemann-Pick disease, Fabry disease, Sly syndrome, Sanfilippo syndrome, Hurler syndrome, Farber disease, Cori disease (Forbes-Coli disease), sialidase deficiency, neuronal ceroid lipofuscinosis, Santavuori-Haltia disease, neuronal ceroid lipofuscinosis, Jansky-Bielschowsky disease, hyaluronidase deficiency, Pompe disease, and Batten disease. Among these, diseases caused by IDS deficiency, GM1-gangliosidosis types 1 to 3, and Pompe disease are preferred. Examples of diseases caused by IDS deficiency include mucopolysaccharidosis type II and Hunter syndrome, with mucopolysaccharidosis type II being particularly preferred.

[0088] In one embodiment, the pharmaceutical agent contains an effective amount of the recombinant cells. The effective amount may vary depending on the administration route, administration interval, body weight, and age. For example, ４ pieces ~ 10 １０ cells, preferably 10 ６ pieces ~ 10 ９ cells, more preferably 10 ７ pieces ~ 10 ８ Preferably, the number of cells is 1.

[0089] In one embodiment, the pharmaceutical agent may contain, in addition to the recombinant cells, a pharmaceutically acceptable carrier capable of preserving the cells. Such a carrier may be a physiological aqueous solvent (e.g., physiological saline, buffer solution, serum-free medium, etc.). If necessary, the pharmaceutical agent may contain commonly used preservatives, stabilizers, reducing agents, isotonicity agents, etc.

[0090] The method of the present invention is a method for diagnosing, preventing, or treating a central nervous system disease, which comprises transplanting the recombinant cell of the present invention into a subject in need thereof. The subject may be a patient with a central nervous system disease, particularly a patient with a disease caused by IDS deficiency, GM1-gangliosidosis types 1 to 3, or Pompe disease.

[0091] The transplantation method includes intravenously administering a cell solution containing an effective amount of the recombinant cells to the subject.

[0092] When the recombinant cells are hematopoietic stem cells, they engraft in the bone marrow 14 to 28 days after transplantation, and the engrafted recombinant cells self-proliferate, differentiate into various blood cells as well as microglia-like cells, and can engraft in the central nervous system. In this way, the recombinant cells can continuously express proteins that should function in the central nervous system in various tissues, including the central nervous system tissue.

[0093] [Use] A use of the present invention is the use of a recombinant cell of the present invention for diagnosing, preventing, or treating a central nervous system disease. A use of the present invention is also the use of a recombinant cell of the present invention in the manufacture of a medicament for diagnosing, preventing, or treating a central nervous system disease. The present invention also provides a recombinant cell of the present invention for use in diagnosing, preventing, or treating a central nervous system disease.

[0094] Example 1 Hematopoietic Stem Cell Gene Therapy Using Anti-Mouse TfR Antibody-Human IDS Mouse Female C57BL / 6 mice heterozygous for a mouse IDS gene deficiency (Garcia AR, et al. J Inherit Metab Dis. 2007;30(6):924-34.) were mated with normal male C57BL / 6 mice, and from the resulting males, mice hemizygous for the IDS deficiency were selected by PCR analysis. Male mice hemizygous for the IDS deficiency were used as MPS II mice, and male mice with wild-type IDS were used as normal controls.

[0095] <Construction of Lentiviral Vector> pJLV1-IDS (Patent Document 1: CMV hybrid 5'LTR-Ψ-RRE-cPPT-MND-IDS cDNA-WPREmut9-3'LTR (SEQ ID NO: 2)) was constructed, which contains human IDS (GenBank: NG_011900) directly downstream of the MND promoter (DNA: SEQ ID NO: 1) of the self-inactivating lentiviral vector pJLV1 plasmid (SEQ ID NO: 12), and pJLV1-mTfR-IDS was constructed which contains anti-mouse TfR antibody (mTfR)-IDS similarly directly downstream of the MND promoter.

[0096] (1) Construction of pJLV1-IDS The lentiviral vector pJLV1-IDS was constructed based on the pLVSIN-CMV Neo vector (Takara Bio). Specifically, the U3 promoter region of the 5'LTR was replaced with a CMV promoter to create a hybrid LTR. The virus-derived sequence downstream of the packaging signal was deleted, and the region from the internal promoter PCMVIE to the neomycin resistance gene was removed, followed by insertion of the MND promoter. A codon-optimized DNA fragment (SEQ ID NO: 3) encoding human IDS (SEQ ID NO: 4) was placed downstream of the MND promoter, and restriction enzyme sites, NotI and XhoI, were inserted upstream and downstream of the DNA fragment. Furthermore, downstream of the XhoI site, a modified WPRE sequence (one adenine base added at position 417) and a 3' SIN LTR in which the cHS4 core insulator sequence was inserted into the 3' LTR lacking the U3 region were placed, thereby obtaining pJLV1-IDS having the base sequence of SEQ ID NO: 2.

[0097] (2) Construction of pJLV1-mTfR-IDS Vector pJLV1-mTfR-IDS (SEQ ID NO: 13) was constructed by cloning a codon-optimized DNA fragment (SEQ ID NO: 7) encoding a fusion protein (mTfR-IDS, SEQ ID NO: 8) in which human IDS (SEQ ID NO: 4) was functionally fused to the 3' end of an anti-mouse TfR antibody (anti-mouse TfR_scFV, SEQ ID NO: 6; codon-optimized DNA: SEQ ID NO: 5) via a linker into vector pJLV1.

[0098] Specifically, DNA encoding mTfR-IDS was artificially synthesized with an EcoRI site at the 5' end and a HindIII site at the 3' end, followed by restriction enzyme digestion. Next, pCMV-Script (Agilent Technologies) was cleaved with EcoRI and HindIII, and pCMV-Script-mTfR-IDS was created using a Quick ligation kit (New England Biolab). Thereafter, pCMV-Script-mTfR-IDS and pJLV1-IDS were cleaved with restriction enzymes NotI and XhoI to excise the mTfR-IDS and pJLV1 plasmids, which were then ligated using a Quick ligation kit to prepare pJLV1-mTfR-IDS (SEQ ID NO: 13).

[0099] (4) Virus packaging Lentivirus packaging was carried out using a partially modified standard protocol (e.g., the protocol described in Patent Document 1) using the self-inactivating (SIN) lentivirus vector (pJLV1-IDS or pJLV1-mTfR-IDS) prepared in (1) or (2) above, as well as the packaging plasmid pCAG-HIVgp and the VSV-G / Rev plasmid pCMV-VSV-G-RSV-Rev.

[0100] Specifically, HEK293T cells were cultured to 70-80% confluence on a 245 mm x 245 mm dish treated with Poly-L-lysine, and the following plasmid DNAs were added to the cells in 2.5 M CaCl solution: 30 μg of packaging plasmid (pCAG-HIVgp), 30 μg of VSV-G, Rev plasmid (pCMV-VSV-G-RSV-Rev), and 60 μg of SIN lentiviral vector. ２ and incubated at 37°C, 5% CO ２ The cells were cultured in an incubator for 16 to 20 hours.

[0101] The culture supernatant was removed by suction, washed with PBS, and replaced with 60 ml of DMEM medium. After that, the cells were incubated at 37°C, 5% CO ２ The cells were cultured in an incubator for approximately 48 hours, after which the culture supernatant was collected, passed through a 0.45 μm filter, and then concentrated using a Centricon Plus-70 (100K).

[0102] The concentrated virus solution was made up to the upper limit of the centrifuge tube with PBS, and ultracentrifuged at 70,000 × g for 1.5 hours at 4°C. The supernatant was removed, and the pellet was suspended in 1 ml of PBS, then transferred to a 50 ml centrifuge tube and vortexed for 3 hours.

[0103] The Vortex suspension was centrifuged at 5000 rpm for 1 minute, and the supernatant was pipetted into screw-cap tubes and stored at -80°C.

[0104] The titers of the produced lentiviruses were measured using the Quick Titer HIV Lentivirus Quantitation Kit (Cell Biolabs, San Diego, CA), an ELISA for p24 protein. The titers were 1.6 × 10 for pJLV1-IDS and 1.6 × 10 for pJLV1-IDS. ９ IU / ml, pJLV1-mTfR-IDS: 1.9×10 ９ IU / ml.

[0105] <Preparation and transplantation of gene-transduced hematopoietic stem cells> Bone marrow cells were collected from the femur of 2-month-old MPS II mice, and mouse hematopoietic stem cells were isolated using a Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated mouse hematopoietic stem cells were transduced (infected) with the obtained lentivirus. Mouse hematopoietic stem cells were infected with the lentivirus according to a previously published method at a multiplicity of infection (MOI) of 50 (Non-Patent Document 2). Specifically, 2 × 10 mouse hematopoietic stem cells were transduced with the lentivirus. ６ 1 x 10 cells per recipient (number of cells for transplantation per recipient) ８ This was done with IU of lentivirus.

[0106] Transplantation of gene-transduced hematopoietic stem cells was also performed according to a previously reported literature (Non-Patent Document 2). The gene-transduced hematopoietic stem cells were administered via the tail vein to 2-month-old MPS II mice that had been lethally irradiated with 9 Gy using an X-ray irradiator MBR-1520-R (Hitachi Power Solutions). Plasma was collected every month after administration, and the liver, spleen, kidney, heart, cerebrum, and cerebellum were collected 6 months later. Non-transplanted wild-type (WT) C57BL / 6 mice and MPS II mice (MPS II) were used as control mice.

[0107] <Measurement of IDS activity and glycosaminoglycan (GAG)> The above tissues were extracted and IDS activity and GAG measurements were performed according to a previously published literature (Non-Patent Document 2). Protein quantification was performed using a DC protein assay kit (Bio-Rad), IDS activity measurement was performed using the fluorescent substrate 4-methylumbelliferone α-L-idopyranosiduronic acid 2-sulfate (Carbosynth, Berkshire, UK), and GAG measurement was performed using a triple quadrupole high-performance liquid chromatograph mass spectrometer LCMS-8040 (Shimadzu Corporation).

[0108] Figure 1 shows the IDS activity in plasma from 1 month to 6 months after transplantation. The IDS activity in Figure 1 is relative to the IDS activity in wild-type mice (WT), where the IDS activity in Figure 1 is set to 1. Figure 1 shows that MPS II mice (IDS in Figure 1) transfected with a lentivirus containing pJLV1-IDS maintained activity approximately 25 times higher than that of WT for 6 months after transplantation. Furthermore, IDS activity in MPS II mice (mTfR-IDS in Figure 1) transfected with a lentivirus containing pJLV1-mTfR-IDS was found to be equal to or greater than that of WT, and was comparable to that of WT even at 6 months. On the other hand, almost no IDS activity was observed in MPS II mice. Furthermore, the IDS activity in the plasma of mTfR-IDS mice was approximately 1 / 30 of that of IDS mice, suggesting that the amount of IDS secreted per cell in mTfR-IDS mice was lower than that in IDS mice.

[0109] The results of measuring IDS activity and GAG accumulation in the liver and spleen 6 months after transplantation are shown in Figure 2. IDS activity is the relative activity (%) when the IDS activity in wild-type mice (WT) is set to 100, and GAG accumulation is the relative amount (%) when the amount of GAG accumulation in MPS II mice (MPS II) is set to 100. As can be seen from Figure 2, the IDS activity in the liver and spleen of both IDS mice and mTfR-IDS mice exceeded that of the wild type, and GAG accumulation was also significantly reduced in both mice compared to MPS II.

[0110] The results of measuring IDS activity and GAG accumulation in the kidney and heart 6 months after transplantation are shown in Figure 3. As shown in Figure 3, both IDS mice and mTfR-IDS mice showed cardiac IDS activity equal to or greater than that of wild-type (WT) mice, but kidney IDS activity was higher in only IDS mice than in wild-type mice. However, GAG accumulation in the heart and kidney was significantly reduced in both IDS mice and mTfR-IDS mice.

[0111] Figure 4 shows the results of measuring IDS activity and GAG accumulation in the cerebrum and cerebellum 6 months after transplantation. As can be seen from Figure 4, although IDS activity was low in the mTfR-IDS mice, the reduction in GAG accumulation was significant. Furthermore, the IDS activity in the cerebrum of the mTfR-IDS mice was approximately one-sixth that of the IDS mice, and approximately five times higher than the activity ratio in plasma. This suggests that in the mTfR-IDS mice, not only is IDS secreted within the cerebrum, but IDS in the blood also migrates to the cerebrum. Furthermore, the reduction rate of GAG accumulation in the cerebrum of the mTfR-IDS mice was higher than that of the IDS mice. These findings suggest that IDS supply to the central nervous tissue of the mTfR-IDS mice may be due to the migration of cells derived from the transplanted hematopoietic stem cells to the central nervous system and local secretion, as well as the widespread distribution of IDS migrated from the blood throughout the tissue.

[0112] These results suggest that the fusion of anti-mouse TfR antibodies improved the tissue distribution of IDS, resulting in superior efficacy compared to conventional IDS. It has been believed that treatment of central nervous system lesions in MPS II requires a blood IDS activity significantly higher than that of the wild-type (WT). However, it has become clear that mTfR-IDS mice can achieve superior therapeutic effects (reduction of GAG accumulation in the cerebrum and cerebellum) compared to IDS mice with blood IDS activity comparable to that of the wild-type (WT) (see Figures 1 and 4).

[0113] Example 2: Hematopoietic stem cell gene therapy using anti-human TfR antibody-human IDS Mouse Female C57BL / 6 mice heterozygous for a deletion of the mouse IDS gene (Higuchi T et al., Mol. Genet. Metab. 2012;107:122-128) were mated with male knock-in mice heterozygous for the human transferrin receptor (Sonoda H, et al., Mol. Ther. 2018 May 2;26(5):1366-1374). From the resulting males, mice hemizygous for the deleted IDS and heterozygous for hTfR (human transferrin receptor-KI / MPS II mice) were selected by PCR analysis. C57BL / 6 male mice served as normal controls.

[0114] <Construction of Lentiviral Vector> pJLV1-hTfR-IDS was constructed by carrying a Fab-type anti-human TfR antibody-IDS (hTfR-IDS) directly downstream of the MND promoter (DNA: SEQ ID NO: 1) of pJLV1 plasmid (SEQ ID NO: 12).

[0115] (1) Construction of pJLV1-hTfR-IDS Vector pJLV1-hTfR-IDS (SEQ ID NO: 16) was constructed by cloning vector pJLV1 with a DNA fragment (SEQ ID NO: 15) encoding a fusion protein (hTfR-IDS, SEQ ID NO: 14) in which the N-terminus of human IDS was functionally fused to the C-terminus of the heavy chain of a Fab-type anti-human TfR antibody via a linker, and further the N-terminus of the light chain of the Fab-type anti-human TfR antibody was functionally fused to the C-terminus of the human IDS via the self-cleaving peptide p2A.

[0116] Specifically, DNA encoding MND-hTfR-IDS was artificially synthesized with an MluI site at the 5' end and an XhoI site at the 3' end, followed by restriction enzyme treatment. Next, pJLV1 plasmid was also cleaved with MluI and XhoI, and pJLV1-hTfR-IDS (SEQ ID NO: 16) was prepared using a Quick ligation kit.

[0117] Furthermore, the lentiviral vector pJLV1-IDS prepared in "(1) Construction of pJLV1-IDS" in Example 1 was also used in Example 2.

[0118] (2) Virus Packaging Lentivirus packaging was carried out in the same manner as in Example 1, using the self-inactivating (SIN) lentivirus vector (pJLV1-IDS or pJLV1-hTfR-IDS) prepared above, as well as the packaging plasmid pCAG-HIVgp and the VSV-G / Rev plasmid pCMV-VSV-G-RSV-Rev, with some modifications to a standard protocol (e.g., the protocol described in Patent Document 1).

[0119] The titers of the produced lentiviruses were measured using the Quick Titer HIV Lentivirus Quantitation Kit (Cell Biolabs, San Diego, CA), an ELISA for p24 protein. The titers were 1.6 × 10 for pJLV1-IDS and 1.6 × 10 for pJLV1-IDS. ９ IU / ml, pJLV1-hTfR-IDS: 1.7×10 ９ IU / ml.

[0120] <Preparation and transplantation of gene-transduced hematopoietic stem cells> Bone marrow cells were collected from the femur of a 5-month-old human transferrin receptor-KI / MPS II mouse, and mouse hematopoietic stem cells were isolated using a Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated mouse hematopoietic stem cells were transduced (infected) with the obtained lentivirus carrying IDS or hTfR-IDS. Mouse hematopoietic stem cells were infected with the lentivirus according to a previously published method (Non-Patent Document 2) at a multiplicity of infection (MOI) of 50. Specifically, 2 x 10 mouse hematopoietic stem cells were transduced with the lentivirus. ６ 1 x 10 cells per recipient (number of cells for transplantation per recipient) ８ This was done with IU of lentivirus.

[0121] Transplantation of gene-transduced hematopoietic stem cells was also performed according to a previously reported literature (Non-Patent Document 2). The gene-transduced hematopoietic stem cells were administered via the tail vein to 5-month-old hTfR-KI / MPS II mice that had been lethally irradiated with 9 Gy using an X-ray irradiator MBR-1520-R (Hitachi Power Solutions). Plasma, liver, spleen, cerebrum, and cerebellum were collected 4 weeks after administration. Non-transplanted wild-type (WT) C57BL / 6 mice and human transferrin receptor-KI / MPS II mice (MPS II) were used as control mice.

[0122] <IDS Activity Measurement and Glycosaminoglycan (GAG) Measurement> IDS activity and GAG measurement were carried out in the same manner as in Example 1.

[0123] The IDS activity in plasma 4 weeks after transplantation is shown in Figure 5. The IDS activity in Figure 5 is the relative activity (%) when the IDS activity in wild-type mice (WT) is set at 100. As shown in Figure 5, the IDS activity in plasma of both IDS mice and hTfR-IDS mice was higher than that of wild-type mice, being 9.36-fold higher in IDS mice and 1.85-fold higher in hTfR-IDS mice than that of wild-type mice.

[0124] The results of measuring IDS activity and GAG accumulation in the liver and spleen 4 weeks after transplantation are shown in Figure 6. IDS activity is the relative activity (%) when the IDS activity in wild-type mice (WT) is set to 100, and GAG accumulation is the relative amount (%) when the amount of GAG accumulation in human transferrin receptor-KI / MPS II mice (MPS II) is set to 100. As shown in Figure 6, the IDS activity in the liver of hTfR-IDS mice was 32.6% of that in wild-type mice, and the IDS activity in the spleen was 147% of that in wild-type mice, and a significant decrease in GAG accumulation was observed in both organs.

[0125] The results of measuring IDS activity and GAG accumulation in the cerebrum and cerebellum 4 weeks after transplantation are shown in Figure 7. IDS activity is the relative activity (%) when the IDS activity in wild-type mice (WT) is set to 100, and GAG accumulation is the relative amount (%) when the amount of GAG accumulation in human transferrin receptor-KI / MPS II mice (MPS II) is set to 100. As can be seen from Figure 7, the hTfR-IDS mice had low IDS activity, as with the hTfR-IDS mice of Example 1, but showed an excellent reduction in GAG accumulation.

[0126] Example 3: T cell gene therapy using anti-human TfR antibody-human IDS Mice The same mice as those obtained in Example 2 were used.

[0127] <Construction of Lentiviral Vector> Lentivirus packaging was performed using a partially modified standard protocol (for example, the protocol described in Patent Document 1) using the self-inactivating (SIN) lentiviral vector (pJLV1-IDS or pJLV1-hTfR-IDS) prepared in Example 2 above, as well as the packaging plasmid pCAG-HIVgp and the VSV-G / Rev plasmid pCMV-VSV-G-RSV-Rev.

[0128] <Preparation and transplantation of gene-transduced T cells> Mouse T cells were isolated from the spleens of 5-month-old human transferrin receptor-KI / MPS II mice using Pan T Cell Isolation Kit II (Miltenyi Biotec). The isolated mouse T cells were transduced (infected) with the lentivirus obtained above, carrying IDS or hTfR-IDS. Mouse T cells were infected with the lentivirus at a multiplicity of infection (MOI) of 50, after 24 hours of stimulation according to the protocol for Dynabeads Mouse T-Activator CD3 / CD28 (Thermo Fisher). Specifically, 1 x 10 mouse T cells were transduced with the lentivirus. ６ 5 x 10 cells per recipient (number of cells for transplantation per recipient) ７ This was done with IU of lentivirus.

[0129] Regarding the transplantation of transgenic T cells, the transgenic mouse T cells were administered via the tail vein to 5-month-old human transferrin receptor-KI / MPS II mice. Plasma, cerebrum, and cerebellum were collected 4 weeks after administration. Non-transplanted wild-type (WT) C57BL / 6 mice and human transferrin receptor-KI / MPS II mice (MPS II) were used as control mice.

[0130] <IDS Activity Measurement and Glycosaminoglycan (GAG) Measurement> IDS activity and GAG measurement were carried out in the same manner as in Example 1.

[0131] The results of measuring IDS activity in plasma, cerebrum, and cerebellum, and GAG accumulation in the cerebrum and cerebellum 4 weeks after transplantation are shown in Figures 8 and 9. IDS activity is the relative activity (%) when the IDS activity in wild-type mice (WT) is set to 100, and GAG accumulation is the relative amount (%) when the amount of GAG accumulation in human transferrin receptor-KI / MPS II mice (MPS II) is set to 100. As shown in Figure 8, the IDS activity in the plasma of both IDS mice and hTfR-IDS mice was higher than that of human transferrin receptor-KI / MPS II mice. As shown in Figure 9, even when mouse T cells were used, reduced GAG accumulation was observed in the cerebrum and cerebellum of hTfR-IDS mice.

[0132] Example 4: Hematopoietic stem cell gene therapy using anti-mouse TfR antibody-human GAA Mouse: Mice homozygously deficient in the acid α-glucosidase (GAA) gene were crossbred to generate GAA-KO mice. C57BL / 6 mice served as normal controls.

[0133] <Construction of Lentiviral Vector> pJLV1-GAA, which carries human GAA (wild-type human GAA, SEQ ID NO: 17) directly downstream of the MND promoter (DNA: SEQ ID NO: 1) of the self-inactivating lentiviral vector pJLV1 plasmid (SEQ ID NO: 12), and pJLV1-mTfR-GAA, which also carries anti-mouse TfR antibody-GAA (mTfR-GAA) directly downstream of the MND promoter, were constructed.

[0134] (1) Construction of pJLV1-GAA DNA (SEQ ID NO: 18) encoding human GAA (SEQ ID NO: 17) was artificially synthesized with a NotI site at the 5' end and an XhoI site at the 3' end, and then subjected to restriction enzyme treatment. Next, pJLV1 plasmid (SEQ ID NO: 12) was also cleaved with NotI and XhoI, and pJLV1-GAA (SEQ ID NO: 19) was prepared using a Quick ligation kit.

[0135] (2) Construction of pJLV1-mTfR-GAA DNA (SEQ ID NO: 21) encoding a fusion protein (mTfR-GAA, SEQ ID NO: 20) in which human GAA (SEQ ID NO: 17) was functionally fused to the 3' end of an anti-mouse TfR antibody (anti-mouse TfR_scFV, SEQ ID NO: 6; codon-optimized DNA: SEQ ID NO: 5) via a linker was artificially synthesized with a NotI site at the 5' end and an XhoI site at the 3' end, and then subjected to restriction enzyme treatment. Next, pJLV1 plasmid (SEQ ID NO: 12) was also cleaved with NotI and XhoI, and pJLV1-mTfR-GAA (SEQ ID NO: 22) was prepared using a Quick ligation kit.

[0136] (3) Virus Packaging Lentivirus packaging was carried out in the same manner as in Example 1, using the self-inactivating (SIN) lentivirus vector (pJLV1-GAA or pJLV1-mTfR-GAA) prepared in (1) or (2) above, as well as the packaging plasmid pCAG-HIVgp and the VSV-G / Rev plasmid pCMV-VSV-G-RSV-Rev, with some modifications to a standard protocol (for example, the protocol described in Patent Document 1).

[0137] The titers of the produced lentiviruses were measured using the Quick Titer HIV Lentivirus Quantitation Kit (Cell Biolabs, San Diego, CA), an ELISA for p24 protein. The titers were 1.7 × 10 for pJLV1-GAA and 1.7 × 10 for pJLV1-GAA. ９ IU / ml, pJLV1-mTfR-GAA: 1.2×10 ９ IU / ml.

[0138] <Evaluation of GAA activity of mTfR-GAA loaded on lentiviral vector> 1 x 10 ５HEK293T cells were infected with lentivirus carrying GAA or mTfR-GAA at an MOI of 10, 1, or 0.1 and cultured for 24 hours. The medium was then replaced with lentivirus-free medium, and after further culture for 48 hours, the medium and cells were collected. GAA activity was measured for the cell extract and medium extracted with distilled water. GAA activity was measured as follows.

[0139] <GAA activity measurement> Protein quantification was performed using a DC protein assay kit (Bio-Rad), and GAA activity was measured using the fluorescent substrate 4-methylumbelliferyl α-D-glucopyranoside (Sigma-Aldrich) according to a previously published literature (Non-Patent Document 8).

[0140] The results of measuring GAA activity for HEK293T cell extracts and culture media are shown in Figure 10. As shown in Figure 10, HEK293T cells infected with mTfR-GAA-carrying lentivirus had lower intracellular activity than HEK293T cells infected with GAA-carrying lentivirus, while their extracellular activity (secretory enzyme activity) was significantly higher than that of HEK293T cells infected with GAA-carrying lentivirus. Surprisingly, these results demonstrated that fusing GAA with an anti-mouse TfR antibody promoted the extracellular secretion of GAA. In other words, fusing an anti-TfR antibody to a protein that should function in the central nervous system not only improved the blood-brain barrier permeability of the protein itself, but also increased the amount of protein secreted extracellularly. These effects are expected to dramatically enhance the efficacy of ex vivo gene therapy.

[0141] <Preparation and Transplantation of Gene-Transduced Hematopoietic Stem Cells> Gene-transduced hematopoietic stem cells were prepared in 3-month-old GAA-KO mice using the same procedure as in Example 1, and transplanted into 3-month-old GAA-KO mice. Transplantation of the gene-transduced hematopoietic stem cells was also performed in the same manner as in Example 1, except that serum, liver, spleen, heart, quadriceps, diaphragm, gastrocnemius, cerebrum, and cerebellum were collected 4 weeks after administration, and control mice included non-transplanted wild-type (WT) C57BL / 6 mice, GAA-KO mice, and GAA-KO mice (ERT) administered recombinant GAA protein (Avaloglucosidase alpha (Sanofi)) at 20 mg / kg via the tail vein once every two weeks.

[0142] <GAA Activity Measurement and Glycogen Measurement> Protein quantification was performed using a DC Protein Assay Kit (Bio-Rad), and GAA activity was measured as described above. Glycogen measurement was performed using a Glycogen Assay Kit (abcam) according to the following method. With the Glycogen Assay Kit (abcam), glycogen is first hydrolyzed to glucose, and then the oxidized intermediate is labeled with an OxiRed Probe. Glycogen is quantified by measuring fluorescence using a Synergy H1 (BioTek). First, 50 μL each of a calibration curve standard sample containing a known concentration of glycogen and tissue homogenate supernatant was added to a 96F Untreated Black microwell (Thermo Scientific). Next, 1 μL of Hydrolysis Enzyme Mix, a hydrolysis reaction reagent, was added to each well, mixed, and allowed to react at room temperature for 30 minutes (except for the wells used for measuring endogenous glucose). Next, 50 μL of Development Enzyme Mix containing OxiRed Probe was added to each well, mixed, and allowed to react at room temperature for 30 minutes in the dark. The results were measured using a Synergy H1 (BioTek) at an excitation wavelength of 535 nm and a detection wavelength of 587 nm.

[0143] The results of measuring GAA activity in serum, liver, and spleen 4 weeks after transplantation are shown in Figure 11. GAA activity is expressed as a relative activity (%) when GAA activity in wild-type mice (WT) is defined as 100. Figure 11 shows that when GAA activity in serum, liver, and spleen was compared between the treatment groups (ERT mice, GAA mice, mTfR-GAA mice), the mTfR-GAA mice had the highest activity in all cases.

[0144] The results of measuring GAA activity in the heart, quadriceps, diaphragm, and gastrocnemius four weeks after transplantation are shown in Figure 12. GAA activity is relative activity (%) when GAA activity in wild-type mice (WT) is set to 100. As shown in Figure 12, when GAA activity in muscles such as the heart, quadriceps, diaphragm, and gastrocnemius was compared between treatment groups, the mTfR-GAA mice had the highest activity in all muscles. Furthermore, GAA activity equivalent to or greater than that of wild-type mice was observed in the heart, quadriceps, and gastrocnemius. GAA mice also showed the second highest GAA activity after mTfR-GAA mice, and GAA activity equivalent to or greater than that of wild-type mice was observed in the quadriceps and gastrocnemius.

[0145] Figure 13 shows the results of measuring glycogen accumulation in the heart, quadriceps, diaphragm, and gastrocnemius muscles 4 weeks after transplantation. Glycogen accumulation is expressed as a relative amount (%), with the amount of glycogen accumulation in GAA-KO mice (NT) set at 100. Figure 13 shows that the mTfR-GAA group showed a dramatic improvement in glycogen accumulation in various muscle tissues, with normalization observed in the diaphragm. On the other hand, in GAA mice, despite GAA activity comparable to that of mTfR-GAA in the quadriceps and other muscles, the decrease in glycogen accumulation was the smallest in the treatment group. This suggests that the fusion of anti-TfR antibodies improves the distribution and localization of GAA.

[0146] It is known that in Pompe disease, an abnormality called autophagic buildup occurs in muscle tissue, causing abnormal localization of therapeutic enzymes (e.g., GAA), resulting in resistance. The reason for the low glycogen reduction effect in GAA mice, despite the relatively high GAA enzyme activity, is thought to be the influence of the above-mentioned GAA abnormal localization. Although mTfR-GAA is also expected to accumulate in the autophagic buildup after being taken up into cells, unexpectedly, glycogen accumulation was dramatically reduced in mTfR-GAA mice. This is a surprising result.

[0147] The results of measuring GAA activity and glycogen accumulation in the cerebrum and cerebellum 4 weeks after transplantation are shown in Figure 14. GAA activity is the relative activity (%) when GAA activity in wild-type mice (WT) is set to 100, and glycogen accumulation is the relative amount (%) when the amount of glycogen accumulation in GAA-KO mice (NT) is set to 100. As shown in Figure 14, an increase in GAA activity was observed only in mTfR-GAA mice, and glycogen accumulation was also significantly reduced.

[0148] <Evaluation of anti-GAA antibody titer in serum of treated mice> Anti-GAA antibody titer in serum of GAA-KO mice after gene therapy using transgenic hematopoietic stem cells was measured using a modified version of the ELISA described in a previously published literature (Non-Patent Document 10). The ELISA was performed as follows: 250 ng of recombinant GAA protein (Avaloglucosidase alpha (Sanofi)) was bound to a microplate (Maxisorp, Thermo Scientific) overnight at 4°C, followed by blocking with PBS containing 1% BSA for 2 hours. After washing each well with PBS containing 0.05% Tween 20 (PBS-T), serum from each mouse or an anti-human GAA antibody (Proteintech) for preparing a standard curve was added and allowed to react for 1 hour at room temperature. After washing each well with PBS-T, peroxidase-labeled anti-mouse IgG antibody (Histofine Simple Stain MAX-PO(M), Nichirei Biosciences) was added to the wells containing each of the mouse sera, and peroxidase-labeled anti-rabbit IgG antibody (Histofine Simple Stain MAX-PO(R), Nichirei Biosciences) was added to the wells used for preparing the calibration curve, and the reaction was allowed to proceed for 1 hour at room temperature. Subsequently, each well was washed with PBS-T, and tetramethylbenzidine (SeraCare) was added to allow color development for 10 minutes, after which the reaction was stopped by adding 1.2 N sulfuric acid. For measurement, absorbance was measured at 450 nm and 650 nm using a Synergy H1 (BioTek), and the difference was taken as the true absorbance. The results are shown in Table 1.

[0149]

[0150] ERT mice treated with ERT had the highest anti-GAA antibody titers in their serum after treatment. Furthermore, GAA mice treated with gene therapy using GAA-introduced hematopoietic stem cells had lower anti-GAA antibody titers in their serum after treatment compared to ERT mice. These results are consistent with the fact that anti-GAA antibodies are often produced during treatment with ERT, the current standard of care, which not only weakens the therapeutic effect but also makes it difficult to continue treatment due to allergic reactions in some cases, and that hematopoietic stem cell gene therapy for other diseases is difficult to produce antibodies. Unexpectedly, mTfR-GAA mice treated with gene therapy using mTfR-GAA-introduced hematopoietic stem cells had the lowest anti-GAA antibody titers, at the same level as untreated mice and wild-type mice.

[0151] Example 5: Hematopoietic stem cell gene therapy using anti-mouse TfR antibody-human GLB1 Mice: Mice heterozygously deficient in the β-galactosidase (GLB1) gene (Non-Patent Document 9) were crossbred to generate GLB1-KO mice. C57BL / 6 mice were used as normal controls.

[0152] <Construction of Lentiviral Vectors> pJLV1-GLB1, which carries human GLB1 (wild-type human GLB1, SEQ ID NO: 23) directly downstream of the MND promoter (DNA: SEQ ID NO: 1) of the self-inactivating lentiviral vector pJLV1 plasmid (SEQ ID NO: 12), and pJLV1-mTfR-GLB1, which carries anti-mouse TfR antibody-GLB1 (mTfR-GLB1) similarly directly downstream of the MND promoter, were constructed.

[0153] (1) Construction of pJLV1-GLB1 DNA (SEQ ID NO: 24) encoding human GLB1 (SEQ ID NO: 23) was artificially synthesized and placed downstream of the MND promoter. Specifically, MND-GLB1 was artificially synthesized with an MluI site at the 5' end and an XhoI site at the 3' end, and then treated with the respective restriction enzymes. Next, pJLV1 plasmid (SEQ ID NO: 12) was also cleaved with MluII and XhoI, and pJLV1-GLB1 (SEQ ID NO: 25) was prepared using a Quick ligation kit.

[0154] (2) Construction of pJLV1-mTfR-GLB1 DNA (SEQ ID NO: 27) encoding a fusion protein (mTfR-GLB1, SEQ ID NO: 26) in which human GLB1 (SEQ ID NO: 23) was functionally fused to the 3' end of an anti-mouse TfR antibody (anti-mouse TfR_scFV, SEQ ID NO: 6; codon-optimized DNA: SEQ ID NO: 5) via a linker was placed downstream of the MND promoter and artificially synthesized. Specifically, MND-mTfR-GLB1 was artificially synthesized with an MluI site at the 5' end and an XhoI site at the 3' end, and then treated with the respective restriction enzymes. Next, pJLV1 plasmid (SEQ ID NO: 12) was also cleaved with MluI and XhoI, and pJLV1-mTfR-GLB1 (SEQ ID NO: 28) was prepared using a Quick ligation kit.

[0155] (3) Virus packaging In the same manner as in Example 1, lentivirus packaging was carried out using the self-inactivating (SIN) lentivirus vector (pJLV1-GLB1 or pJLV1-mTfR-GLB1) prepared in (1) or (2) above, as well as the packaging plasmid pCAG-HIVgp and the VSV-G / Rev plasmid pCMV-VSV-G-RSV-Rev, using a partially modified standard protocol (for example, the protocol described in Patent Document 1).

[0156] The titers of the produced lentiviruses were measured using the Quick Titer HIV Lentivirus Quantitation Kit (Cell Biolabs, San Diego, CA), an ELISA for p24 protein. The titers were 1.8 × 10 for pJLV1-GLB1 and 1.8 × 10 for pJLV1-GLB1. ８ IU / ml, pJLV1-mTfR-GLB1: 4.3×10 ８ IU / ml.

[0157] <Preparation and Transplantation of Gene-Transduced Hematopoietic Stem Cells> Gene-transduced hematopoietic stem cells were prepared in 10-week-old GLB1-KO mice using the same procedure as in Example 1, and transplanted into the 10-week-old GLB1-KO mice.

[0158] <Measurement of GLB1 activity and GM1 ganglioside> Protein quantification was performed using a DC protein assay kit (Bio-Rad), GLB1 activity was measured using the fluorescent substrate 4-methylumbelliferyl-β-D-galactopyranoside (Sigma-Aldrich) according to a previously published literature (Non-Patent Document 7), and GM1 ganglioside was measured using a triple quadrupole high-performance liquid chromatograph mass spectrometer LCMS-8040 (Shimadzu Corporation) according to a previously published literature (Non-Patent Document 7).

[0159] The results of measuring GLB1 activity and GM1 ganglioside accumulation (GM1 accumulation) in the serum, cerebral cortex, cerebellum, and hippocampus 4 weeks after transplantation are shown in Figures 15 to 17. GLB1 activity is the relative activity (%) when GLB1 activity in wild-type mice (WT) is set to 100, and GM1 accumulation is the relative amount (%) when the amount of GM1 accumulation in GLB1-KO mice (NT) is set to 100. Figures 16 to 17 show that mTfR-GLB1 mice had high GLB1 activity in the cerebral cortex, cerebellum, and hippocampus, and GM1 accumulation was also significantly reduced.

[0160] Generally, it is thought that the activity of a fusion protein formed by fusion with an anti-mTfR antibody is lower than the activity of a protein that does not form a fusion protein. However, unexpectedly, the blood activity of the anti-mTfR antibody fusion protein for GLB1 was higher than that of the wild-type enzyme. It is thought that the increased extracellular secretion and stabilization of the enzyme in the blood, likely brought about by the fusion with the anti-mTfR antibody, may have outweighed the decrease in activity. Because GAA and GLB1 are inherently poorly secreted or unstable enzymes, the fact that their blood activity increased rather than decreased, perhaps due to stabilization by fusion with the anti-mTfR antibody, was a truly unexpected result.

Claims

1. Recombinant cells obtained by introducing a nucleic acid molecule into a host cell using a vector containing a nucleic acid molecule that comprises a base sequence encoding a fusion protein containing an anti-transferrin receptor antibody or an antigen-binding fragment thereof and a protein to be made to function in the central nervous system.

2. The recombinant cell according to claim 1, wherein the protein to be activated in the central nervous system is a lysosomal enzyme, a neurotrophic factor, or an antibody.

3. The recombinant cell according to claim 2, wherein the lysosomal enzyme is acid α-glucosidase or β-galactosidase.

4. The recombinant cell according to claim 1, wherein the protein to be activated in the central nervous system is iduronate-2-sulfatase.

5. The recombinant cell according to claim 1, wherein the vector is a vector for stable expression of the fusion protein.

6. The recombinant cell according to claim 1, wherein the vector is a lentiviral vector.

7. The recombinant cell according to claim 1, wherein the host cell is a hematopoietic stem cell, a T cell, or a B cell.

8. A drug for diagnosing, preventing, or treating central nervous system diseases, comprising recombinant cells according to any one of claims 1 to 7.