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Definitions

• the present invention relates to nucleic acid molecules, vectors, recombinant cells, and drugs for the treatment of central nervous system diseases.
• MPS II Mucopolysaccharidosis type II
• IDS iduronic acid-2-sulfatase
• ERT using central nervous system-transferring IDS and intracerebroventricular administration of enzymes was approved in Japan.
• ERT has a high therapeutic effect, it requires weekly infusion and is extremely expensive, so establishing a cheaper and more effective treatment method is an important issue.
• Non-patent Documents 4 and 5 report the effects of hematopoietic stem cell gene therapy on MPS II mice.
• the present inventors used a (B6/MPS II) virus promoter (MND promoter: Moloney murine leukemia virus LTR/myeloproliferative sarcoma virus enhancer) to express the IDS gene in hematopoietic stem cells in a model mouse, thereby achieving ERT or HSCT.
• MND promoter Moloney murine leukemia virus LTR/myeloproliferative sarcoma virus enhancer
• the present inventors 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 improved central nervous system lesions in a mouse GM1-gangliosidosis model (Non-Patent Document 7).
• IDS gene therapy targeting hematopoietic stem cells such as Non-Patent Document 6
• cells derived from transplanted IDS gene-introduced hematopoietic stem cells migrate to the central nervous system, and IDS is also secreted in the central nervous system, resulting in a certain level of treatment. It is recognized that it is effective.
• IDS secreted into the blood outside the central nervous system cannot pass through the blood-brain barrier, and its contribution to therapeutic effects in the central nervous system is limited.
• the present invention aims to provide nucleic acid molecules, vectors, recombinant cells, and drugs for the treatment of central nervous system diseases that tend to spread to the central nervous system.
• the present inventors have previously developed a lentiviral vector system for gene therapy of mucopolysaccharidosis type II (Patent Document 1), and a nucleic acid molecule capable of expressing a fusion protein between IDS and an antibody against transferrin receptor (TfR).
• Patent Document 1 a lentiviral vector system for gene therapy of mucopolysaccharidosis type II
• TfR transferrin receptor
• the present invention includes the following.
• a nucleic acid molecule comprising a base sequence encoding a fusion protein comprising an anti-transferrin receptor (TfR) antibody or antigen-binding fragment thereof and a protein to function in the central nervous system.
• TfR anti-transferrin receptor
• the nucleic acid molecule according to [1] wherein the protein to be caused to function in the central nervous system is a lysosomal enzyme.
• the lysosomal enzyme is acid ⁇ -glucosidase (GAA) or ⁇ -galactosidase (GLB1).
• nucleic acid molecule according to [1], wherein the protein to be made to function in the central nervous system is iduronic acid-2-sulfatase (IDS).
• IDMS iduronic acid-2-sulfatase
• the nucleic acid molecule according to [1], wherein the protein to be caused to function in the central nervous system is a neurotrophic factor.
• the nucleic acid molecule according to [1], wherein the protein to be caused to function in the central nervous system is an antibody.
• a vector comprising the nucleic acid molecule according to any one of [1] to [6].
• 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.
• the vector according to [8] which is a lentivirus vector.
• a method for diagnosing, preventing or treating a central nervous system disease which comprises 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 central nervous system diseases.
• the recombinant cell according to [10] for use in diagnosing, preventing or treating central nervous system diseases.
• [16] Use of the recombinant cell according to [10] in the manufacture of a drug for diagnosing, preventing or treating central nervous system diseases.
• the present invention provides nucleic acid molecules, vectors, recombinant cells, and drugs for the treatment of 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.
• I will provide a.
• host cells such as hematopoietic stem cells expressing a fusion protein of an anti-TfR antibody or an antigen-binding fragment thereof and a protein that should function in the central nervous system, such as IDS
• the translocation of proteins such as IDS is increased, thereby treating central nervous system diseases such as mucopolysaccharidosis type II.
• the present invention can also use the patient's own autologous hematopoietic stem cells, there is no need to search for a compatible donor unlike in conventional hematopoietic stem cell transplantation, which is an allogeneic transplant, and graft-versus-host disease (GVHD) associated with transplantation can be avoided.
• GVHD graft-versus-host disease
• the risk of engraftment failure due to rejection is also extremely low.
• lifelong medical costs are expected to be lower than with enzyme replacement therapy, and the treatment is also highly effective.
• nucleic acid molecule containing a nucleotide sequence encoding a fusion protein comprising an anti-TfR antibody or an antigen-binding fragment thereof and a protein to function in the central nervous system By using a nucleic acid molecule containing a nucleotide sequence encoding a fusion protein comprising an anti-TfR antibody or an antigen-binding fragment thereof and a protein to function in the central nervous system, The researchers succeeded in translocating the desired protein to the central nervous system, and demonstrated for the first time that the protein actually exerts a therapeutic effect in the central nervous system. It is also conceivable that recombinant cells, such as hematopoietic stem cells, into which the nucleic acid molecules of the present invention have been introduced, themselves migrate into the central nervous system.
• both the recombinant cell and the fusion protein expressed and secreted by the cell i.e., the fusion protein of a therapeutic protein such as IDS and an anti-TfR antibody
• the fusion protein Because it is difficult to predict the dynamics, it is difficult to predict whether a therapeutic effect will actually be obtained.
• anti-TfR antibodies causes most of the fusion protein to be transferred to central tissues. In this case, therapeutic effects in tissues other than the central nervous system cannot be expected, so treatments that transfer therapeutic proteins to the central nervous system have been avoided.
• the fusion protein of the present invention can also exert a certain therapeutic effect on tissues other than the central nervous system (eg, liver, etc.). Furthermore, since fusion proteins have large molecular weights and are complex, it is thought that recombinant cells do not immediately try to produce fusion proteins that have large molecular weights and are complex. Therefore, the productivity of the fusion protein is expected to decrease, making prediction of therapeutic effects even more difficult. Taking these things into consideration, the effects of the present invention are unexpectedly significant.
• Example 1 IDS activity in plasma from 1 month to 6 months after transplantation of gene-transfected hematopoietic stem cells is shown.
• Example 1 measurement results of IDS activity and GAG accumulation in the liver and spleen 6 months after transplantation of gene-transfected hematopoietic stem cells are shown.
• Example 1 measurement results of IDS activity and GAG accumulation in the kidney and heart 6 months after transplantation of gene-transfected hematopoietic stem cells are shown.
• Example 1 measurement results of IDS activity and GAG accumulation in the cerebrum and cerebellum 6 months after transplantation of gene-transfected hematopoietic stem cells are shown.
• Example 2 the results of measuring IDS activity in plasma 4 weeks after transplantation of gene-transfected hematopoietic stem cells are shown.
• Example 2 measurement results of IDS activity and GAG accumulation in the liver and spleen 4 weeks after transplantation of gene-transfected hematopoietic stem cells are shown.
• Example 2 measurement results of IDS activity and GAG accumulation in the cerebrum and cerebellum 4 weeks after transplantation of gene-transfected hematopoietic stem cells are shown.
• Example 3 the results of measuring IDS activity in plasma 4 weeks after transplantation of gene-transferred T cells are shown.
• Example 3 measurement results of IDS activity and GAG accumulation in the cerebrum and cerebellum 4 weeks after transplantation of gene-transferred T cells are shown.
• Example 4 the results of measuring GAA activity in gene-transfected HEK293T cells are shown.
• Example 4 measurement results of GAA activity in serum, liver, and spleen 4 weeks after transplantation of gene-transfected hematopoietic stem cells are shown.
• Example 4 the results of measuring GAA activity in the heart, quadriceps, diaphragm, and gastrocnemius muscle 4 weeks after transplantation of gene-transfected hematopoietic stem cells are shown.
• Example 4 measurement results of glycogen accumulation in the heart, quadriceps, diaphragm, and gastrocnemius muscle 4 weeks after transplantation of gene-transferred hematopoietic stem cells are shown.
• Example 4 measurement results of GAA activity and glycogen accumulation in the cerebrum and cerebellum 4 weeks after transplantation of gene-transfected hematopoietic stem cells are shown.
• Example 5 the results of measuring GLB1 activity in serum 4 weeks after transplantation of gene-transfected hematopoietic stem cells are shown.
• Example 5 measurement results of GLB1 activity and GM1 accumulation in the cerebral cortex and cerebellum 4 weeks after transplantation of gene-transfected hematopoietic stem cells are shown.
• Example 5 measurement results of GLB1 activity and GM1 accumulation in the hippocampus 4 weeks after transplantation of gene-transfected hematopoietic stem cells are shown.
• a nucleic acid molecule of one embodiment of the present invention includes a base sequence encoding a fusion protein of an anti-transferrin receptor (TfR) antibody or antigen-binding fragment thereof and a protein that is to function in the central nervous system.
• TfR anti-transferrin receptor
• the anti-TfR antibody is an anti-human TfR antibody
• the IDS is a human IDS.
• Transferrin receptor is a transmembrane protein that takes into cells a conjugate of transferrin and iron (Fe) in the blood, and is present on the surface of vascular endothelial cells such as brain vascular endothelial cells.
• An anti-TfR antibody of one embodiment of the invention is an antibody that specifically binds to TfR and is thereby taken up into cells by TfR.
• IDS that forms a fusion protein with anti-TfR antibodies is also taken into cells, so IDS, which cannot pass through the blood-brain barrier (BBB) by itself, is transported to the brain tissue by TfR, and the IDS is transported into the brain tissue by TfR.
• BBB blood-brain barrier
• the physiological activity of IDS can be shown. Therefore, a fusion protein of an anti-TfR antibody and IDS can be used as a drug that should exert its efficacy in the brain.
• the origin of the transferrin receptor is not limited, and may be derived from humans, mice, rats, rabbits, horses, or non-human primates, but Preferably, it is of human origin.
• the human-derived TfR may be a wild type TfR (protein: NP_001121620; gene: NM_001128148) or a mutant TfR.
• Mutant TfR is not particularly limited, and any antibody against it may be an antibody that can also bind to wild-type TfR; for example, it may be a mutant TfR with enhanced physiological activity or antigenicity.
• the number of amino acids to be replaced is preferably 1 to 20, more preferably 1 to 10, More preferably, the number is 1 to 3.
• the number of amino acids to be deleted is preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 3.
• mutations that combine substitution and deletion of these amino acids can also be added.
• amino acids to wild-type TfR preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus of the protein. be done.
• the amino acid sequence of the mutated protein preferably exhibits 80% or more identity, preferably 85% or more identity, and more preferably 90% or more identity with the amino acid sequence of wild-type TfR. , more preferably 95% or more identity, even more preferably 98% or more identity.
• the position of each mutation and its type (deletion, substitution, addition) when compared with wild-type TfR can be easily confirmed by alignment of the amino acid sequences of the wild-type and mutant proteins.
• the identity between the amino acid sequence of wild type TfR and the amino acid sequence of mutant TfR can be easily calculated using a well-known homology calculation algorithm.
• homology calculation algorithm include BLAST (Altschul SF. J Mol. Biol. 215. 403-10, (1990)), Pearson and Lipman's similarity search method (Proc. Natl. Acad. Sci. USA. 85. 2444 (1988)), Smith and Waterman's local homology algorithm (Adv. Appl. Math. 2. 482-9 (1981)), etc.
• substitutions of amino acids by other amino acids in the amino acid sequences of the proteins mentioned above occur, for example, within families of amino acids that are related in their side chains and chemical properties. Substitutions within such amino acid families are expected not to result in significant changes in protein function (ie, are conservative amino acid substitutions).
• Such amino acid families include, for example: (1) Aspartic acid and glutamic acid, which are acidic amino acids, (2) basic amino acids histidine, lysine, and arginine; (3) Aromatic amino acids phenylalanine, tyrosine, tryptophan, (4) Serine and threonine, which are amino acids with hydroxyl groups (hydroxyamino acids), (5) hydrophobic amino acids methionine, alanine, valine, leucine, and isoleucine; (6) neutral hydrophilic amino acids cysteine, serine, threonine, asparagine, and glutamine; (7) Glycine and proline, which are amino acids that affect the orientation of peptide chains; (8) Asparagine and glutamine, which are amide-type amino acids (polar amino acids), (9) aliphatic amino acids alanine, leucine, isoleucine, and valine; (10) Alanine, glycine, serine, and threonine, which are amino acids
• 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, secreted IgA, IgG, IgE, and IgM. Generally, antibodies include at least two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region (CH), and the heavy chain constant region includes three constant domains: CH1, CH2, and CH3.
• VH heavy chain variable region
• CH heavy chain constant region
• Each light chain includes a light chain variable region (VL) and a light chain constant region, and the light chain constant region includes one constant domain, CL.
• the VH region and the VL region include a framework region (FR) and a complementarity determining region (CDR), each containing three regions from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Contains CDR and 4 FRs.
• the variable regions of heavy and light chains contain binding domains that interact with antigen.
• antibodies include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, and synthetic antibodies. , tetrameric antibodies comprising two heavy chain molecules and two light chain molecules.
• antibodies include dimeric antibodies, single chain antibodies, single domain antibodies, etc., which will be described later, and these are also referred to as antibody fragments (antigen-binding fragments). Called.
• Human antibody refers to an antibody that is encoded entirely by human-derived genes. However, antibodies encoded by genes in which mutations have been added to original human genes for the purpose of increasing gene expression efficiency are also human antibodies. Furthermore, an antibody in which two or more genes encoding human antibodies are combined and part of one human antibody is replaced with part of another human antibody is also a human antibody. Human antibodies have three complementarity determining regions (CDRs) in the immunoglobulin light chain and three complementarity determining regions (CDRs) in the immunoglobulin heavy chain. The three CDRs of the immunoglobulin light chain are called CDR1, CDR2, and CDR3 in order from the N-terminal side.
• CDRs complementarity determining regions
• the three CDRs of the immunoglobulin heavy chain are called CDR1, CDR2, and CDR3 in order from the N-terminal side.
• An antibody in which the antigen specificity, affinity, etc. of a human antibody is modified by replacing the CDRs of one human antibody with the CDRs of another human antibody is also a human antibody.
• an antibody in which mutations such as substitutions, deletions, additions, etc. are added to the amino acid sequence of the original antibody by modifying the genes of the original human antibody is also referred to as a human antibody.
• the number of amino acids to be replaced is preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5. 1 to 3, and even more preferably 1 to 3.
• the number of amino acids to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. The number is even more preferably 1 to 3.
• antibodies with mutations that combine these amino acid substitutions and deletions are also human antibodies.
• amino acids preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, and even more preferably in the amino acid sequence of the original antibody or on the N-terminus or C-terminus.
• 1-3 amino acids are added.
• Antibodies with mutations that combine additions, substitutions, and deletions of these amino acids are also human antibodies.
• the amino acid sequence of the mutated antibody preferably shows 80% or more identity, more preferably 90% or more identity, and still more preferably 95% or more identity with the amino acid sequence of the original antibody. and even more preferably 98% or more identity. That is, in the present invention, the term "human-derived gene" includes not only the original human-derived gene, but also genes obtained by modifying the human-derived original gene.
• humanized antibody means that the amino acid sequence of part of the variable region (e.g., all or part of the CDRs in particular) is derived from a mammal other than humans, and the other regions are derived from humans. refers to an antibody that is For example, as a humanized antibody, the three complementarity determining regions (CDRs) of the immunoglobulin light chain and the three complementarity determining regions (CDRs) of the immunoglobulin heavy chain that constitute a human antibody are combined with those of other mammals. Examples include antibodies made by replacing CDRs.
• the species of other mammals from which the CDRs to be grafted into appropriate positions of human antibodies are derived is not particularly limited as long as it is a non-human mammal, but is preferably mouse, rat, rabbit, horse, or Primates other than humans, more preferably mice and rats, such as mice.
• the light chains of human antibodies and humanized antibodies include ⁇ chains and ⁇ chains.
• the light chain constituting the antibody may be either a ⁇ chain or a ⁇ chain.
• the heavy chains of human antibodies and humanized antibodies include ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain, which correspond to IgG, IgM, IgA, IgD, and IgE, respectively.
• the heavy chain constituting the antibody may be any of the ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain, but preferably the ⁇ chain.
• the ⁇ chains of antibody heavy chains include ⁇ 1 chain, ⁇ 2 chain, ⁇ 3 chain, and ⁇ 4 chain, which correspond to IgG1, IgG2, IgG3, and IgG4, respectively.
• the heavy chain constituting the antibody is a ⁇ chain
• the ⁇ chain may be any of the ⁇ 1 chain, ⁇ 2 chain, ⁇ 3 chain, and ⁇ 4 chain, but is preferably the ⁇ 1 chain or the ⁇ 4 chain.
• the light chain of the antibody may be either a ⁇ chain or a ⁇ chain
• the heavy chain of the antibody may be a ⁇ 1 chain, a ⁇ 2 chain, or a ⁇ 2 chain.
• the antibody may be either a ⁇ 3 chain or a ⁇ 4 chain, but preferably a ⁇ 1 chain or a ⁇ 4 chain.
• one preferred embodiment of the antibody is one in which the light chain is a ⁇ chain and the heavy chain is a ⁇ 1 chain.
• chimeric antibody refers to an antibody formed by linking two or more different antibody fragments derived from two or more different species.
• the anti-TfR antibody is a humanized anti-TfR antibody.
• a chimeric antibody of a human antibody and another mammalian antibody is an antibody in which a part of the human antibody is replaced with a part of a non-human mammalian antibody.
• the antibody consists of an Fc region, a Fab region, and a hinge region, which will be explained below.
• Specific examples of such chimeric antibodies include chimeric antibodies in which the Fc region is derived from a human antibody while the Fab region is derived from another mammalian antibody.
• antibodies in which the Fc region is derived from another mammal while the Fab region is derived from a human antibody are also chimeric antibodies.
• the hinge region may be derived from either human or other mammalian antibodies. The same can be said for humanized anti-TfR antibodies.
• a chimeric antibody for example, a humanized anti-TfR antibody
• a chimeric antibody consists of a variable region and a constant region.
• the heavy chain constant region (CH) and the light chain constant region (CL) are derived from a human antibody
• the heavy chain variable region (VH) and the light chain variable region ( VL) is derived from antibodies of other mammals
• heavy chain constant regions (CH) and light chain constant regions (CL) are derived from antibodies of other mammals
• Examples include those in which the variable region (VH) and the variable region (VL) of the light chain are derived from human antibodies.
• the biological species of the other mammal 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. It is.
• a chimeric antibody of a human antibody and a mouse antibody is particularly referred to as a "human/mouse chimeric antibody.”
• 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, or conversely, the Fc region is derived from a mouse antibody while the Fab region is derived from a human antibody. Examples include chimeric antibodies.
• the hinge region is derived from either human or mouse antibodies.
• a human/mouse chimeric antibody is that 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 constant region (CL) are derived from a human antibody.
• the variable region (VL) is derived from a mouse antibody; conversely, the constant region (CH) of the heavy chain and the constant region (CL) of the light chain are derived from a mouse antibody, while the variable region (VH) of the heavy chain and those in which the variable region (VL) of the light chain is derived from a human antibody.
• Antibodies originally have a basic structure consisting of a total of four polypeptide chains: two immunoglobulin light chains and two immunoglobulin heavy chains.
• antibody in addition to the tetrameric antibody having this basic structure, (1) A dimeric antibody consisting of a total of two polypeptide chains, one immunoglobulin light chain and one immunoglobulin heavy chain, (2) a single chain antibody that is formed by linking a linker to the C-terminal side of an immunoglobulin light chain and further bonding an immunoglobulin heavy chain to the C-terminus; (3) a single chain antibody that is formed by linking a linker to the C-terminal side of an immunoglobulin heavy chain and further bonding an immunoglobulin light chain to the C-terminus; (4) a single chain antibody (scFv), which is formed by linking a linker to the C-terminal side of the variable region of an immunoglobulin heavy chain and further bonding the variable region of an immunoglobulin light chain to
• linker refers to, for example, a peptide chain in which multiple amino acids are linked via peptide bonds.
• a linker consisting of such a peptide chain can also be referred to as a "peptide linker.”
• Linker can also be referred to as "linker sequence” in the context of this specification. The N-terminus of this linker and the C-terminus of another protein are bonded via a peptide bond, and the N-terminus of another protein is further bonded to the C-terminus of the linker, thereby forming a conjugate between the two proteins via the linker. do.
• a Fab includes one light chain comprising a variable region and a CL region (constant region of a light chain), and one comprising a variable region and a CH1 region (portion 1 of a constant region of a heavy chain).
• the heavy chain may further include a part of the hinge region in addition to the variable region and the CH1 region (part 1 of the constant region of the heavy chain); It lacks the cysteine residue that binds the heavy chains of antibodies together.
• the light chain and heavy chain have a bond between cysteine residues present in the constant region (CL region) of the light chain and cysteine residues present in the constant region (CH1 region) or hinge region of the heavy chain. They are bound by disulfide bonds formed.
• the heavy chains that form Fab are called Fab heavy chains.
• Fab lacks the cysteine residue that exists in the hinge region and binds the heavy chains of antibodies, so it consists of one light chain and one heavy chain.
• the light chain that constitutes Fab includes a variable region and a CL region.
• the heavy chain constituting Fab may consist of a variable region and a CH1 region, or may include a part of a hinge region in addition to the variable region and CH1 region.
• the hinge region is selected so as not to contain a cysteine residue that binds between the heavy chains so that a disulfide bond is not formed between the two heavy chains at the hinge region.
• the heavy chain includes, in addition to the variable region and the CH1 region, all or part of a hinge region containing cysteine residues that bind the heavy chains together.
• F(ab')2 refers to a molecule in which two F(ab') molecules are bonded to each other by disulfide bonds between cysteine residues present in each other's hinge regions.
• a heavy chain forming F(ab') or F(ab')2 is called a Fab' heavy chain.
• polymers such as dimers and trimers formed by bonding multiple antibodies directly or via a linker are also antibodies.
• antibody as used in the present invention includes, but is not limited to, any antibody that contains a part of an antibody molecule and has the property of specifically binding to an antigen. That is, in the present invention, the term "light chain” includes those derived from a light chain and having the amino acid sequence of all or part of the variable region thereof. Moreover, when referring to a heavy chain, a chain derived from a heavy chain and having the amino acid sequence of all or part of the variable region thereof is included. Therefore, as long as it has the amino acid sequence of all or part of the variable region, for example, a chain with the Fc region deleted is also a heavy chain.
• the 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 constant region of the heavy chain) and the CH3 region (part 3 of the constant region of the heavy chain). means.
• the antibody in one embodiment of the invention is (8)
• the light chain and heavy chain constituting Fab, F(ab') or F(ab')2 shown in (6) above were linked via a linker sequence to form each single-chain antibody.
• scFab, scF(ab'), and scF(ab')2 may have a linker sequence attached to the C-terminal side of the light chain, and a heavy chain further attached to the C-terminal side.
• a linker may be attached to the C-terminus of the heavy chain, and a light chain may be further attached to the C-terminus.
• the antibodies of the present invention also include scFv, which is a single chain antibody obtained by linking a light chain variable region and a heavy chain variable region via a linker.
• a linker sequence may be attached to the C-terminus of the light chain variable region, and a heavy chain variable region may be further attached to the C-terminus of the linker sequence. It may be one in which a linker sequence is attached to the terminal side and a light chain variable region is further attached to the C-terminal side.
• antibody as used herein includes full-length antibodies, a broader concept that includes (1) to (8) in addition to those shown in (1) to (8) above, and full-length antibodies. Any form of antigen-binding fragment (antibody fragment) that is a partially deleted antibody is also included. Antigen-binding fragments include heavy chain antibodies, light chain antibodies, VHH, VNAR, and fragments lacking some of these.
• antigen-binding fragment refers to a fragment of an antibody that retains at least a portion of its specific binding activity with an antigen, and may include an antigen complementarity determining region (CDR).
• antigen-binding fragments include Fab, Fab', F(ab')2, variable region (Fv), heavy chain variable region (VH) and light chain variable region (VL) linked with an appropriate linker.
• Single-chain antibodies (scFv) diabodies that are polypeptide dimers containing a heavy chain variable region (VH) and a light chain variable region (VL), and a part of the constant region in the heavy chain (H chain) of the scFv. This includes minibodies that are dimers of (CH3) bound antibodies, other low-molecular-weight antibodies, and the like.
• the molecules are not limited to these molecules as long as they have the ability to bind to the antigen.
• single chain antibody refers to a linker attached to the C-terminus of an amino acid sequence that includes all or part of the variable region of an immunoglobulin light chain, and an immunoglobulin heavy chain antibody attached to the C-terminus.
• a linker is attached to the C-terminal side of an amino acid sequence that includes all or part of the variable region of an immunoglobulin heavy chain, and an amino acid sequence that further includes all or a part of the variable region of an immunoglobulin light chain at the C-terminus.
• a protein that is formed by binding to a specific antigen and is capable of specifically binding to a specific antigen is also a "single chain antibody" in the present invention.
• single chain antibody in which an immunoglobulin light chain is bound to the C-terminal side of an immunoglobulin heavy chain via a linker, the Fc region of the immunoglobulin heavy chain is usually deleted.
• the variable region of an immunoglobulin light chain has three complementarity determining regions (CDRs) that are involved in the antigen specificity of antibodies.
• CDRs complementarity determining regions
• the variable region of an immunoglobulin heavy chain also has three CDRs. These CDRs are the main regions that determine the antigen specificity of antibodies.
• a single chain antibody preferably includes all three CDRs of an immunoglobulin heavy chain and all three CDRs of an immunoglobulin light chain.
• a single-chain antibody with one or more CDRs deleted can also be used.
• the number of linkers disposed between the light chain and the heavy chain of the immunoglobulin is preferably 2 to 50, more preferably 8 to 50, still more preferably 10 to 30, and even more preferably It is a peptide chain composed of 12-18 or 15-25, 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 maintains affinity for the antigen, but it is preferably composed of only glycine or glycine and serine.
• amino acid sequence Gly-Ser amino acid sequence Gly-Ser, amino acid sequence Gly-Gly-Ser, amino acid sequence Gly-Gly-Gly, Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 9), Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 10), Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 11), or a sequence in which these amino acid sequences are repeated 2 to 10 times, or 2 to 5 times.
• the sequence of SEQ ID NO: 9 is repeated three times.
• a linker having a sequence is preferably used.
• a single domain antibody refers to an antibody that has the property of specifically binding to an antigen with a single variable region.
• Single domain antibodies include antibodies whose variable regions consist only of heavy chain variable regions (heavy chain single domain antibodies), and antibodies whose variable regions consist only of light chain variable regions (light chain single domain antibodies). It will be done.
• VHH and VNAR are a type of single domain antibody.
• anti-TfR antibodies include those described in Patent Documents 2 to 4.
• a protein to be made to function in the central nervous system is a protein that has physiological activity in the central nervous system and is required to be made to function in the central nervous system.
• the protein also includes a peptide, and may be a protein having, for example, 2 to 3000, 10 to 1500, or 20 to 1000 amino acid residues.
• Proteins that should function in the central nervous system are not particularly limited as long as they have physiological activity in the central nervous system, but examples include lysosomal enzymes, neurotrophic factors, antibodies, hormones, somatomedins, insulin, glucagon, cytokines, Lymphokines, blood coagulation factors, fusion proteins of antibodies and other proteins, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin , darbepoetin, tissue plasminogen activator (t-PA), thrombomodulin, follicle-stimulating hormone (FSH), gonadotropin-releasing hormone (GnRH), gonadotropin, DNasel, thyroid-stimulating hormone (TSH), nerve growth factor (NGF) , ciliary neurotrophic factor (CNTF), glial cell line neurotrophic
• Suitable examples when the proteins to be made to function in the central nervous system are lysosomal enzymes include ⁇ -L-iduronidase, glucocerebrosidase, ⁇ -galactosidase (GLB1), GM2 activation protein, ⁇ -hexosaminidase A, ⁇ -Hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, ⁇ -mannosidase, ⁇ -mannosidase, galactosylceramidase, saposin C, arylsulfatase A, ⁇ -L-fucosidase, aspartylglucosaminidase, ⁇ -N- Acetylgalactosaminidase, acid sphingomyelinase, ⁇ -galactosidase A, ⁇ -glucuronidase, heparan N-sulfatase, ⁇ -N-
• the protein to function in the central nervous system is a lysosomal enzyme
• the lysosomal enzyme is ⁇ -galactosidase (GLB1) or acid ⁇ -glucosidase (GAA).
• the protein that should function in the central nervous system is acid alpha-glucosidase (GAA).
• GAA is the enzyme responsible for the muscle disease known as Pompe disease, and when GAA is deficient, tissue , accumulation of glycogen, a substrate of GAA, causes various symptoms.
• the origin of GAA is not limited; for example, it may be derived from a human, a mouse, a rat, a rabbit, a horse, or a non-human primate, but it must be derived from a human. is preferred.
• the human-derived GAA may be a wild-type GAA (eg, protein: NP_000143 (Genbank); gene: NM_000152 (Genbank), etc.) or a mutant GAA.
• the amino acid or base sequence of wild-type GAA may vary by several amino acids or several bases to several dozen bases depending on the reference database, but it is not particularly limited as long as a person skilled in the art can recognize it as wild-type GAA.
• mutant GAA is not particularly limited, and may be, for example, a mutant GAA with enhanced physiological activity. Note that the description regarding mutant TfR is also the same for mutant GAA.
• other proteins that are to function in the central nervous system may be derived from humans, mice, rats, rabbits, horses, or non-human primates, but they must be derived from humans. is preferred.
• the protein may be a wild type protein or a mutant protein.
• the protein that should function in the central nervous system is ⁇ -galactosidase (GLB1)
• GLB1 is the enzyme responsible for the genetic disease known as GM1 gangliosidosis, and when GLB1 is deficient, Accumulation of GM1 ganglioside, a substrate of GLB1, in tissues causes various symptoms.
• the origin of GLB1 is not limited, and for example, it may be derived from a human, mouse, rat, rabbit, horse, or non-human primate, but it must be derived from a human. is preferred.
• the 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.), It may also be a mutant GLB1.
• the amino acid or base sequence of wild-type GLB1 may vary by several amino acids or several bases to several tens of bases depending on the reference database, but it is not particularly limited as long as a person skilled in the art can recognize it as wild-type GLB1.
• the mutant GLB1 is not particularly limited, and may be, for example, a mutant GLB1 with enhanced physiological activity. Note that the description regarding mutant TfR is also the same for mutant GLB1.
• other proteins that are to function in the central nervous system may be derived from humans, mice, rats, rabbits, horses, or non-human primates, but they must be derived from humans. is preferred.
• the protein may be a wild type protein or a mutant protein.
• the protein to be made to function in the central nervous system is preferably iduronate-2-sulfatase (IDS), and IDS is the causative enzyme of mucopolysaccharidosis type II known as Hunter syndrome, A deficiency of IDS causes various symptoms due to the accumulation of glycosaminoglycans (GAGs), which are substrates of IDS, in tissues.
• IDS iduronate-2-sulfatase
• GAGs glycosaminoglycans
• the origin of the IDS is not limited, and for example, it may be derived from a human, a mouse, a rat, a rabbit, a horse, or a non-human primate, but it must be derived from a human.
• 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. Note that the description for mutant TfR is also the same for mutant IDS.
• proteins that are to function in the central nervous system may be derived from humans, mice, rats, rabbits, horses, or non-human primates, but they must be derived from humans. is preferred.
• the protein may be a wild type protein or a mutant 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 and a protein that is to function in the central nervous system, and is capable of diagnosing, preventing or treating central nervous system diseases.
• TfR anti-transferrin receptor
• the anti-TfR antibody or antigen-binding fragment thereof and the protein to function in the central nervous system may be fused in this order or in the reverse order from the N-terminus to the C-terminus.
• the antigen-binding fragment is present at the N-terminus.
• a peptide linker may be present between the anti-TfR antibody or its antigen-binding fragment and the protein that is to function in the central nervous system. The peptide linker is as described above.
• the fusion protein of one embodiment of the present invention may include a marker protein for detecting the fusion protein, and examples of the marker protein include GFP, EGFP, YFP, RFP, mCherry, and luciferase.
• the marker protein may be located at the N-terminus, C-terminus of the fusion protein, or between the anti-TfR antibody or antigen-binding fragment thereof and the protein that is to function in the central nervous system.
• the fusion protein of one embodiment of the present invention may include proteins that are to function in multiple central nervous systems.
• the proteins to function in multiple central nervous systems 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 the C-terminus, but is preferably present at the N-terminus.
• nucleic acid molecule refers to either DNA, which is obtained by polymerizing deoxyribonucleotides through phosphodiester bonds, or RNA, which is obtained by polymerizing ribonucleotides through phosphodiester bonds.
• the DNA may be single-stranded (single-stranded) or double-stranded with complementary strands.
• the DNA may be a (+) strand or a (-) strand.
• the individual deoxyribonucleotides that make up DNA are naturally occurring types as long as the protein-encoding gene contained in the DNA can be translated into mRNA in mammalian (especially human) cells. It may be a natural type or a modified version of the natural type.
• individual deoxyribonucleotides constituting DNA are obtained by translating a gene encoding a protein contained in the DNA into mRNA in mammalian (particularly human) cells, and converting all or As long as a portion can be replicated, it may be a naturally occurring type or a modified natural type.
• the RNA 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.
• the individual ribonucleotides constituting the RNA are naturally occurring as long as the protein-encoding gene contained in the RNA can be reverse transcribed into DNA in mammalian (especially human) cells. It may be of the type that exists in , or it may be of a modified type.
• each ribonucleotide constituting the RNA can be used as long as the gene encoding the protein contained in the RNA can be translated into a protein in mammalian (particularly human) cells. It may be a naturally occurring type or a modified type. Modification of ribonucleotides is performed, for example, to suppress degradation of RNA by RNase and increase the stability of RNA within cells.
• nucleic acid molecules encoding the fusion protein is not particularly limited, and includes, for example, the DNA sequences described in Patent Documents 2 to 4.
• the part of the nucleic acid molecule encoding IDS among the nucleic acid molecules encoding the fusion protein may be the IDS gene (NM_000202) or cDNA. . Alternatively, for example, it may be a codon-optimized DNA shown in SEQ ID NO: 3.
• the part of the nucleic acid molecule encoding GAA in the nucleic acid molecule encoding the fusion protein may be a GAA gene (for example, NM_000152, etc.) or a cDNA. You can. Alternatively, for example, the DNA shown in SEQ ID NO: 18 may be used.
• the nucleic acid molecule encoding GLB1 may be a cDNA, even if it is the GLB1 gene (for example, NM_000404, M34423, etc.). It may be.
• the codon-optimized DNA shown in SEQ ID NO: 24 may be used.
• nucleic acid molecule encoding the fusion protein also contains the nucleic acid encoding the peptide linker.
• Vectors of the invention contain nucleic acid molecules of the invention.
• the vector is a vector that can autonomously replicate in a host cell and/or a vector that can be integrated into the chromosome of the host cell, and is capable of transcribing the above-mentioned nucleic acid molecule. It is preferable that the vector stably expresses the fusion protein when introduced.
• Lentivirus is a virus that belongs to the genus Lentivirus within the subfamily Orthoretrovirinae, which is a subfamily of the family Retroviridae, and has a single-stranded (+) strand RNA genome (ssRNA).
• the lentivirus genome contains essential genes such as gag (a region encoding structural proteins including capsid proteins), pol (a region encoding enzymes including reverse transcriptase), and env (envelope proteins necessary for binding to host cells). coding region), which are sandwiched between two LTRs (5'LTR and 3'LTR).
• the lentivirus genome contains a region that encodes a protein that has the function of binding to Rev (RRE (rev responsive element) present in viral RNA) and transporting viral RNA from the nucleus to the cytoplasm as an auxiliary gene.
• Rev rev responsive element
• tat a region encoding a protein that has the function of binding to TAR in the 5'LTR and increasing LTR promoter activity
• Lentivirus is an enveloped virus, and infects cells by fusing the envelope with the cell membrane. Furthermore, lentivirus is an RNA virus, and reverse transcriptase is present in the virion. After lentivirus infection, single-stranded plus-strand DNA is replicated from the (+)-strand RNA genome by reverse transcriptase, and double-stranded DNA is further synthesized. Proteins, which are constituents of virions, are expressed from this double-stranded DNA, and the (+) strand RNA genome is packaged into this, thereby causing virions to proliferate.
• lentiviral vectors include, but are not limited to, those developed based on the genome of HIV-1, a type of lentivirus.
• the first generation lentiviral vector consists of three types of plasmids: a packaging plasmid, an Env plasmid, and an introduction plasmid.
• the packaging plasmid has gag and pol genes under the control of the CMV promoter and the like.
• the Env plasmid has the env gene under the control of the CMV promoter.
• the introduced plasmid has a 5'LTR, an RRE, a gene encoding the desired protein under the control of the CMV promoter, and a 3'LTR.
• first generation lentiviral vectors also contain the virally derived rev, tat, vif, vpr, vpu, and nef accessory genes.
• the desired protein in the present invention is a fusion protein of a ligand and a physiologically active protein.
• the promoter that controls the gene encoding the desired protein may be a promoter other than the CMV promoter, such as the MND promoter, phosphoglycerate kinase (PGK) promoter, CD11b promoter, SV40 early promoter, or human elongation factor-1 ⁇ (EF-1 ⁇ ).
• PGK phosphoglycerate kinase
• CD11b CD11b promoter
• SV40 early promoter SV40 early promoter
• human elongation factor-1 ⁇ EF-1 ⁇
• Promoter human ubiquitin C promoter, CAG promoter (cytomegalovirus enhancer, avian ⁇ -actin promoter, rabbit ⁇ -globin polyA hybrid promoter) retrovirus Rous sarcoma virus LTR promoter, dihydrofolate reductase promoter, ⁇ -actin promoter, mouse albumin promoter , human albumin promoter, and human ⁇ -1 antitrypsin promoter are preferred.
• it is the MND promoter set forth in SEQ ID NO: 1.
• the second generation lentiviral vector also consists of three types of plasmids: a packaging plasmid, an Env plasmid (envelope plasmid), and an introduction plasmid.
• a packaging plasmid an Env plasmid (envelope plasmid)
• an introduction plasmid an auxiliary gene vif, vpr, vpu, and nef, which are not essential genes, have been deleted from the packaging plasmid.
• the Rev contained in the packaging plasmid in the second generation is separated to create an independent Rev plasmid.
• tat has also been deleted from the packaging plasmid.
• the U3 region within the 5'LTR of the introduced plasmid has been replaced with the CMV promoter.
• the packaging plasmid encodes viral genes other than the envelope and supplies proteins for making virus particles in trans.
• the modified genes (vif, fpr, vpu, nef) are deleted since the modified gene products are not required for infection of non-dividing cells.
• modified gene products are not essential for HIV-1 replication, they are important and are deeply involved in the pathogenesis of HIV-1, so deletion increases safety.
• tat is essential for HIV-1 replication, safety is further increased by deleting tat from the packaging plasmid. Note that since the packaging plasmid does not have a packaging signal ( ⁇ ), RNA transcribed from this plasmid is not incorporated into virus particles.
• Rev acts on RNA after transcription, and structural proteins are translated by selectively transporting unspliced mRNA out of the nucleus.
• a plasmid separate from the packaging plasmid is used.
• VSV-G vesicular stomatitis virus G gylcoprotein
• the receptor for VSV-G is thought to be a phospholipid, and by using VSV-G as an envelope, membrane fusion between the virus particle and the cell occurs independent of cell surface receptors. Therefore, it becomes possible to basically infect any animal species or cell tumor.
• VSV-G is physically strong, and virus particles can be easily concentrated by ultracentrifugation.
• the target gene is inserted between LTRs (repetitive sequences that control transcriptional expression) at both ends of the vector, which contain packaging signals ( ⁇ ). Furthermore, it has a primer binding site essential for reverse transcription, and RNA transcribed from this plasmid is incorporated into virus particles. Since the LTR promoter activity of HIV-1 is very weak in the absence of tat, an internal promoter is used to express the incorporated foreign gene. In one embodiment, the internal promoter may be the PGK, CD11b or MND promoter.
• RRE rev responsive element
• WPRE woodchuk hepatitis virus posttranscriptional regulatory element
• both 3' and 5' LTRs will not have promoter activity in the proviral state, and transcription of the entire genome from the 5'R region will be inhibited. It won't happen.
• This allows lentiviral vectors corresponding to HIV-1 replication-deficient strains to meet the requirements of the position paper, "3. A provirus that does not have LTR promoter activity and the entire HIV genome is not transcribed.” ) is considered a vector.
• U3 of the 5'LTR with the CMV promoter, tat dependence is eliminated, making it possible to delete tat from the packaging plasmid.
• the self-inactivating lentivirus pLVSIN-CMV Neo vector (manufactured by Takara Bio Inc.) can be used.
• pJLV1 was created by replacing the U3 promoter region with the CMV promoter, reducing the virus-derived sequences downstream of the packaging signal, removing the internal promoter PCMVIE up to 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.
• a foreign gene in the present invention is a nucleic acid molecule encoding a fusion protein.
• a fusion protein is expressed from this nucleic acid molecule in a host cell or the like into which the nucleic acid molecule has been introduced.
• Retroviruses have a single (+) strand RNA genome (ssRNA).
• the viral genome contains gag (encodes structural proteins including capsid proteins), pol (encodes enzymes including reverse transcriptase), env (encodes envelope proteins required for binding to host cells) and packaging signals ( ⁇ ), which are flanked by two long terminal repeats (LTR, 5'LTR and 3'LTR).
• LTR long terminal repeats
• 5'LTR and 3'LTR packaging signals
• Retroviral vectors are mainly developed based on the murine leukemia virus, and the viral genome has been segmented to maintain infectivity and lack self-replication ability, with the aim of losing pathogenicity and increasing safety.
• the first generation retroviral vector consists of a packaging plasmid (viral genome excluding packaging signal) and an introduction plasmid (packaging signal, part of gag, and foreign gene sandwiched between 5'LTR and 3'LTR). ). Therefore, when homologous recombination occurs in the gag sequence portion commonly present in the packaging plasmid and the introduction plasmid, a self-replicable retrovirus, ie, an RC (replication competent) virus, appears.
• the 3' LTR of the first generation packaging plasmid is replaced with a polyA addition signal.
• the third generation consists of three plasmids, and the packaging plasmid of the second generation is further divided into a plasmid encoding gag/pol and a plasmid encoding env.
• homologous recombination must occur simultaneously at three locations, and the probability of this occurrence is extremely low, further increasing safety.
• these packaging plasmids and introduction plasmids are first introduced into host cells by a common transfection technique. Then, the 5'LTR, 3'LTR, and the region containing the gene encoding the desired protein placed between these two LTRs are replicated in the host cell, and the resulting single-stranded (+) strand RNA is retro-transformed. It is packaged into the viral capsid protein to form recombinant retroviral virions. Since this recombinant retrovirus virion has infectivity, it can be used to introduce foreign genes into cells, tissues, or living bodies.
• a foreign gene in the present invention is a nucleic acid molecule encoding a fusion protein. A fusion protein is expressed from this nucleic acid molecule in a host cell or the like into which the nucleic acid molecule has been introduced.
• the vector is a cloning process in which the desired insert DNA fragment (a nucleic acid molecule encoding an anti-TfR antibody or its antigen-binding fragment, and a nucleic acid molecule encoding a protein that should function in the central nervous system) is placed into the desired vector (pJLV1 plasmid). It is done by manipulation.
• a nucleic acid molecule encoding an anti-TfR antibody or its antigen-binding fragment and a nucleic acid molecule encoding a protein to function in the central nervous system may be prepared in a functionally linked state, or they may be prepared separately. You may. When preparing them separately, the restriction enzyme sites should be set in advance so that they will be functionally linked when they are cut and linked.
• the restriction enzyme site is not particularly limited, but may be, for example, 5'-NotI GCGGCCGC/3'-XhoI CTCGAG.
• the cloning site of the target vector (pJLV1 plasmid) is cut with a restriction enzyme to linearize the vector. Then, the insert DNA and the linearized plasmid are ligated to produce a vector according to the present invention.
• the plasmid system which includes a packaging plasmid, Env plasmid, Rev plasmid, and introduction plasmid (vector), is also called a third generation lentiviral vector, and the entire system deletes more than 1/3 of the HIV-1 genome. Therefore, there is little possibility that wild-type HIV-1 will be produced. Since the homologous regions between each plasmid are also kept to a minimum, there is almost no possibility that a virus capable of autonomous replication will be produced by homologous recombination.
• the constructed vectors are co-transfected with the third generation packaging plasmid, Rev plasmid, and VSV-G envelope plasmid into, for example, 293T cells that have been cultured in large quantities, and the supernatant culture solution is collected 3 days later.
• the culture solution contains virus particles, which are purified by ultracentrifugation and stored in aliquots at -80°C.
• the nucleic acid molecule can also be in the form of being encapsulated in a liposome, a lipid nanoparticle (LNP), or the like.
• Liposomes are spherical vesicles with a lipid bilayer and are mainly composed of phospholipids, especially phosphatidylcholine.
• the present invention is not limited to this, and the liposome may contain other lipids such as egg yolk phosphatidylethanolamine, as long as a lipid bilayer is formed. Since cell membranes are mainly composed of phospholipid bilayer membranes, liposomes have the advantage of excellent biocompatibility.
• Lipid nanoparticles are lipid-based particles with a diameter of 10 nm to 1000 nm, typically less than about 200 nm, which can encapsulate hydrophobic (lipophilic) molecules, such as triglycerides, diglycerides, monoglycerides, and fatty acids.
• the main constituents are biocompatible lipids such as steroids. It is thought that when a gene encapsulated in liposomes or lipid nanoparticles is administered into a living body, it is taken into the cell by direct fusion with the cell membrane of the cell or by endocytosis, and then transferred to the nucleus and the gene is introduced into the cell.
• Liposomes, lipid nanoparticles, etc. encapsulating the nucleic acid molecules of the present invention can be used to introduce a gene for a fusion protein of a ligand and a physiologically active protein into cells, tissues, or living bodies. In cells etc. into which the gene has been introduced, a fusion protein comes to be expressed from this gene.
• liposomes, lipid nanoparticles, etc. refers to, in addition to the above-mentioned liposomes and lipid nanoparticles, polymer nanoparticles, micelles, emulsions, nanoemulsions, microspheres, nanospheres, microcapsules, nanocapsules, dendrimers, It includes nanogels, metal nanoparticles, and any other nano/microparticles that can be used as drug delivery systems (DDS).
• DDS drug delivery systems
• nucleic acid molecules introduced into cells, tissues, or living bodies in the form of vectors, encapsulated in recombinant virus virions, or encapsulated in liposomes, lipid nanoparticles, etc. is described below. Examples are shown in (1) to (9). However, the behavior of nucleic acid molecules is not limited to these.
• the nucleic acid molecule is a single (+) strand RNA, and 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 generated. is translated and the fusion protein is expressed.
• the nucleic acid molecule is a single (+) strand RNA, and when introduced into a cell, the nucleic acid molecule is reverse transcribed to become a single (+) strand DNA; This DNA is then transcribed and translated to express the fusion protein.
• the nucleic acid molecule is a single-stranded (+) strand RNA or (-) strand RNA, and when introduced into a cell, the nucleic acid molecule is reverse transcribed and then converted into a double-stranded DNA. This DNA is then transcribed and translated to express the fusion protein.
• the nucleic acid molecule is a single-stranded (+) strand or (-) strand RNA, and when introduced into a cell, the nucleic acid molecule becomes double-stranded DNA after being reverse transcribed. Then, this DNA undergoes random recombination or homologous recombination with the genome of the host cell and is integrated into the genome, and the integrated DNA is transcribed and translated to express the fusion protein.
• the nucleic acid molecule is a single (+) strand DNA, and when introduced into a cell, the nucleic acid molecule is transcribed and translated to express the fusion protein.
• the nucleic acid molecule is a single (-) stranded DNA, and when introduced into a cell, double-stranded DNA is synthesized from the nucleic acid molecule, and then this double-stranded DNA is The fusion protein is expressed through transcription and translation.
• the nucleic acid molecule is a single-stranded (+) stranded DNA or (-) stranded DNA, and when introduced into a cell, a double-stranded DNA is synthesized from the nucleic acid molecule, and then a double-stranded DNA is synthesized from the nucleic acid molecule.
• the nucleic acid molecule is a double-stranded DNA, and when introduced into a cell, the nucleic acid molecule is transcribed and translated to express the fusion protein.
• the nucleic acid molecule is double-stranded DNA, and this DNA undergoes random recombination or homologous recombination with the genome of the host cell and is integrated into the genome, and the integrated DNA is transcribed and translated. and the fusion protein is expressed.
• Nucleic acid molecules in the form of vectors, encapsulated in recombinant virus virions, or encapsulated in liposomes, lipid nanoparticles, etc. can be introduced into cells, tissues, or living bodies.
• the nucleic acid molecule When the nucleic acid molecule is introduced into a living body, the nucleic acid molecule can be in the form of a vector, encapsulated in a recombinant virus virion, or encapsulated in a liposome, lipid nanoparticle, etc., and can be injected subcutaneously or intramuscularly. , administered by parenteral means such as intravenous injection.
• Nucleic acid molecules in the form of plasmids, encapsulated in recombinant virus virions, or encapsulated in liposomes, lipid nanoparticles, etc. can be used as various medicines.
• the recombinant cell of the present invention is a recombinant cell obtained by introducing the vector of the present invention into a host cell.
• host cells There are no particular limitations on the type of host cells, but examples include 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.
• stem cells such as , and immune cells such as T cells, B cells, and NK cells.
• the recombinant cells into which the nucleic acid molecule has been introduced will express the fusion protein of the present invention, and thus can be transplanted into patients for therapeutic purposes.
• the drug of the present invention is a drug for diagnosing, preventing or treating central nervous system diseases, which contains the recombinant cell of the present invention.
• Central nervous system diseases include neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease; psychiatric disorders such as schizophrenia and depression; central nervous system diseases including multiple sclerosis, amyotrophic lateral sclerosis, and brain tumors. lysosomal diseases accompanied by brain damage, glycogen storage diseases, muscular dystrophy, cerebral ischemia, cerebral inflammatory diseases, prion diseases, and traumatic central nervous system disorders.
• diseases caused by IDS deficiency include IDS deficiency, Gaucher disease, GM1-gangliosidosis types 1 to 3, GM2-gangliosidosis AB variant, Sandhoff disease and Tisachs disease, Sandhoff disease, I-cell disease, ⁇ -mannosidosis , ⁇ -mannosidosis, Krabbe disease, Gaucher-like storage disease, metachromatic leukodystrophy, fucosidosis, aspartylglucosaminuria, Schindler disease, Kawasaki disease, Niemann-Pick disease, Fabry disease, Sly syndrome, Sanfilippo syndrome, Hurler syndrome, Farber disease, Coli disease (Forbes-Coli disease), sialidase deficiency, neuroceroid lipofuscinosis, Santavuori-Haltia disease, neuroceroid lipofuscinosis, Jansky-Bielschowsky disease, Examples include central nervous system disorders such as hyaluronidase deficiency, Pompe disease, and Batten disease.
• diseases caused by IDS deficiency GM1-gangliosidosis types 1 to 3, or Pompe disease are preferred.
• Diseases caused by IDS deficiency include mucopolysaccharidosis type II and Hunter syndrome, with mucopolysaccharidosis type II being particularly preferred.
• the drug of one embodiment contains an effective amount of the above-mentioned recombinant cells, and the effective amount may vary depending on the administration route, administration interval, body weight and age, but for example, 10 4 to 10 10 cells, preferably 10 6 cells.
• the number of cells is preferably 10 to 10 9 cells, more preferably 10 7 to 10 8 cells.
• the drug may contain, in addition to recombinant cells, a pharmaceutically acceptable carrier that can preserve the cells.
• a pharmaceutically acceptable carrier e.g., a physiological aqueous solvent (physiological saline, buffer solution, serum-free medium, etc.) can be used.
• the drug may contain commonly used preservatives, stabilizers, reducing agents, tonicity agents, and the like.
• 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, in particular a disease caused by IDS deficiency, GM1-gangliosidosis types 1-3, or Pompe disease.
• transplantation method examples include a method in which an effective amount of the cell fluid containing the above recombinant cells is administered intravenously to the subject.
• the recombinant cells are hematopoietic stem cells, they will engraft in the bone marrow for 14 to 28 days after transplantation, and the engrafted recombinant cells will self-proliferate and differentiate into microglia-like cells in addition to various blood cells. and can engraft in the central nervous system. In this way, recombinant cells can continuously express proteins to function in the central nervous system in various tissues, including the central nervous system.
• the use of the invention is the use of the recombinant cells of the invention for diagnosing, preventing or treating central nervous system diseases.
• a use of the invention is also the use of recombinant cells of the invention in the manufacture of a medicament for diagnosing, preventing or treating diseases of the central nervous system.
• the invention also provides recombinant cells of the invention for use in diagnosing, preventing or treating central nervous system diseases.
• Example 1 Anti-mouse TfR antibody-hematopoietic stem cell gene therapy using human IDS
• Female C57BL/6 mice heterozygous for mouse IDS gene deletion (GarciaAR, et al. J Inherit Metab Dis.2007;30(6):924-34.) were crossed with normal male C57BL/6 mice.
• Mice having hemizygous deletion IDS were selected from the generated males by PCR analysis.
• Male mice hemizygous for IDS deficiency were used as MPS II mice, and male mice with wild-type IDS were used as normal controls.
• Patent Document 1 CMV hybrid 5
• pJLV1-IDS pJLV1-IDS
• SEQ ID NO: 12 pJLV1 plasmid
• SEQ ID NO: 12 a self-inactivating lentiviral vector 'LTR- ⁇ -RRE-cPPT-MND-IDS cDNA-WPREmut9-3'LTR
• pJLV1-mTfR-IDS which also carries an anti-mouse TfR antibody (mTfR)-IDS directly under the MND promoter.
• pJLV1-IDS The lentivirus vector pJLV1-IDS was constructed based on the pLVSIN-CMV Neo vector (Takara Bio). Specifically, we created a hybrid LTR by replacing the U3 promoter region of the 5'LTR with the CMV promoter, reducing the virus-derived sequences downstream of the packaging signal, removing the internal promoter PCMVIE to the neomycin resistance gene, and replacing the MND promoter with the CMV promoter. Inserted.
• a codon-optimized DNA fragment (SEQ ID NO: 3) encoding human IDS (SEQ ID NO: 4) is placed downstream of the MND promoter, and NotI and XhoI sites are placed upstream and downstream of the DNA fragment as restriction enzyme sites. inserted. Furthermore, downstream of the XhoI site, a modified WPRE sequence (adding one base of adenine at position 417) and a 3' SIN LTR with a cHS4 core insulator sequence inserted into the 3' LTR lacking the U3 region were placed. pJLV1-IDS having the base sequence of SEQ ID NO: 2 was obtained.
• DNA encoding mTfR-IDS was artificially synthesized with an EcoRI site added to the 5' side and a HindIII site added to the 3' side, and treated with restriction enzymes.
• pCMV-Script (Agilent Technologies) was cut with EcoRI and HindIII, and pCMV-Script-mTfR-I was cut using Quick ligation kit (New England Biolab). I created a DS.
• pCMV-Script-mTfR-IDS and pJLV1-IDS are cut with restriction enzymes NotI and XhoI, mTfR-IDS and pJLV1 plasmid are excised, and pJLV1 -mTfR-IDS (SEQ ID NO: 13) was produced.
• lentivirus packaging in addition to the self-inactivating (SIN) lentivirus vector (pJLV1-IDS or pJLV1-mTfR-IDS) prepared in (1) or (2) above, The packaging plasmid pCAG-HIVgp and the VSV-G/Rev plasmid pCMV-VSV-G-RSV-Rev were used, and the standard protocol was partially modified (for example, as described in Patent Document 1). (protocol described).
• HEK293T cells cultured at 70-80% confluence on a 245 mm x 245 mm dish treated with Poly-L-Lysine Packaging plasmid (pCAG-HIVgp) 30 ⁇ g VSV-G, Rev plasmid (pCMV-VSV-G-RSV-Rev) 30 ⁇ g SIN lentiviral vector 60 ⁇ g
• pCAG-HIVgp 30 ⁇ g VSV-G
• Rev plasmid pCMV-VSV-G-RSV-Rev
• the culture supernatant was removed by suction, washed with PBS, and replaced with 60 ml of DMEM medium, followed by culturing in a 5% CO 2 incubator at 37° C. for about 48 hours. Thereafter, the culture supernatant was collected, passed through a 0.45 ⁇ m filter, and concentrated using Centricon Plus-70 (100K).
• the concentrated virus solution was diluted to the upper limit of the centrifuge tube with PBS, and ultracentrifuged at 70,000 x g and 4°C for 1.5 hours. After removing the centrifugation supernatant and suspending the pellet in 1 ml of PBS, it was transferred to a 50 ml centrifuge tube and vortexed for 3 hours.
• Vortex suspension was centrifuged at 5000 rpm for 1 minute, and the supernatant was pipetted into tubes with screw caps and stored at -80°C.
• the generated lentivirus titer was measured using the Quick Titer HIV Lentivirus Quantitation Kit (Cell Biolabs, San Diego, CA), which is an ELISA against p24 protein.
• the titers were pJLV1-IDS: 1.6 ⁇ 10 9 IU/ml and pJLV1-mTfR-IDS: 1.9 ⁇ 10 9 IU/ml, respectively.
• Non-Patent Document 2 The gene-transfected hematopoietic stem cells were administered through the tail vein to 2-month-old MPS II mice that had been irradiated with a lethal dose of 9 Gy using an X-ray irradiation device MBR-1520-R (Hitachi Power Solutions). Plasma was collected every month after administration, and after 6 months, the liver, spleen, kidney, heart, cerebrum, and cerebellum were collected. As control mice, non-transplanted wild type (WT) C57BL/6 mice and MPS II mice (MPS II) were used.
• WT wild type C57BL/6 mice
• MPS II mice MPS II mice
• ⁇ IDS activity measurement and glycosaminoglycan (GAG) measurement The above tissue was extracted and IDS activity and GAG measurements were performed according to a previously reported document (Non-Patent Document 2).
• GA was used for protein quantification using the DC protein assay kit (Bio-Rad), and for IDS activity measurement using the fluorescent substrate 4-methylumbelliferone ⁇ -L-idopyranosiduronic acid 2-sulfate (Carbosynth, Berkshire, UK).
• G The measurement was performed using a triple quadrupole high performance liquid chromatograph mass spectrometer LCMS-8040 (Shimadzu Corporation).
• FIG. 1 shows the IDS activity in plasma from 1 month to 6 months after transplantation.
• the IDS activity in FIG. 1 is the relative activity when the IDS activity in wild type mouse (WT) is set to 1. From FIG. 1, it was found that the MPS II mouse (IDS in FIG. 1) into which the lentivirus containing pJLV1-IDS was introduced maintained an activity approximately 25 times higher than that of WT for 6 months after transplantation. In addition, the IDS activity in MPS II mice (mTfR-IDS in Figure 1) transfected with lentivirus containing pJLV1-mTfR-IDS was found to be equivalent to or higher than that of WT, and was still at the same level as WT at 6 months.
• mTfR-IDS mice have approximately 1/30th the IDS activity in plasma of IDS mice, which means that the amount of IDS secreted on a cell-by-cell basis in mTfR-IDS mice is lower than that in IDS mice. suggested.
• FIG. 2 shows the measurement results of IDS activity and GAG accumulation in the liver and spleen 6 months after transplantation.
• IDS activity is the relative activity (%) when the IDS activity in wild type mice (WT) is taken as 100
• GAG accumulation is the relative amount (%) when the amount of GAG accumulation in MPS II mice (MPS II) is taken as 100. %).
• IDS activity in the liver and spleen of IDS mice and mTfR-IDS mice both exceeded that of the wild type, and GAG accumulation was also significantly reduced compared to MPS II.
• FIG. 3 shows the measurement results of IDS activity and GAG accumulation in the kidney and heart 6 months after transplantation.
• both IDS mice and mTfR-IDS mice exhibited cardiac IDS activity equivalent to or higher than wild type (WT), but kidney IDS activity exceeded that of wild type only in IDS mice.
• cardiac and renal GAG accumulation was significantly reduced in both IDS and mTfR-IDS mice.
• FIG. 4 shows the measurement results of IDS activity and GAG accumulation in the cerebrum and cerebellum 6 months after transplantation.
• mTfR-IDS mice had low IDS activity but excellent reduction in GAG accumulation.
• the IDS activity in the cerebrum of mTfR-IDS mice was about one-sixth that of IDS mice, and about five times higher than the activity ratio in plasma. This suggested that in mTfR-IDS mice, IDS was not only secreted within the cerebrum, but also that IDS in the blood was transferred to the cerebrum.
• the reduction rate of GAG accumulation in the cerebrum of mTfR-IDS mice was higher than that of IDS mice.
• Example 2 Anti-human TfR antibody-hematopoietic stem cell gene therapy using human IDS
• ⁇ Mouse> Knock-in of female C57BL/6 mice heterozygous for mouse IDS gene deletion (Higuchi T et al., Mol. Genet. Metab. 2012;107:122-128) and male heterozygous for human transferrin receptor. Mice that are hemizygous for 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.
• pJLV1-hTfR-IDS Fab type anti-human TfR antibody-IDS directly under the MND promoter (DNA: SEQ ID NO: 1) of pJLV1 plasmid (SEQ ID NO: 12).
• DNA encoding MND-hTfR-IDS was artificially synthesized with an MluI site added to the 5' side and an XhoI site added to the 3' side, and treated with restriction enzymes.
• pJLV1 plasmid was also digested with MluI and XhoI, and pJLV1-hTfR-IDS (SEQ ID NO: 16) was created using a Quick ligation kit.
• Example 2 Furthermore, the lentivirus vector pJLV1-IDS prepared in "(1) Construction of pJLV1-IDS" in Example 1 was also used in Example 2.
• lentivirus packaging in addition to the self-inactivating (SIN) lentivirus vector (pJLV1-IDS or pJLV1-hTfR-IDS) prepared above, Using the packaging plasmid pCAG-HIVgp and the VSV-G/Rev plasmid pCMV-VSV-G-RSV-Rev, the standard protocol was partially modified (for example, Patent Document 1). (protocol described in ).
• the generated lentivirus titer was measured using the Quick Titer HIV Lentivirus Quantitation Kit (Cell Biolabs, San Diego, CA), which is an ELISA against p24 protein.
• the titers were pJLV1-IDS: 1.6 ⁇ 10 9 IU/ml and pJLV1-hTfR-IDS: 1.7 ⁇ 10 9 IU/ml, respectively.
• ⁇ Preparation and transplantation of gene-transferred 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 the Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated mouse hematopoietic stem cells were transfected (infected) with the obtained lentivirus carrying IDS or hTfR-IDS. Lentivirus infection of mouse hematopoietic stem cells was performed according to the method described in the previously reported literature (Non-Patent Document 2), and was performed at a multiplicity of infection (MOI) of 50. Specifically, 1 ⁇ 10 8 IU of lentivirus was used per 2 ⁇ 10 6 mouse hematopoietic stem cells (number of cells for transplantation per recipient).
• MOI multiplicity of infection
• Non-patent Document 2 The above gene-transfected hematopoietic stem cells were introduced into 5-month-old hTfR-KI/MPS II mice through the tail vein, which had been irradiated with a lethal dose of 9 Gy using an X-ray irradiation device MBR-1520-R (Hitachi Power Solutions). administered. Plasma, liver, spleen, cerebrum, and cerebellum were collected 4 weeks after administration. As control mice, non-transplanted wild type (WT) C57BL/6 mice and human transferrin receptor-KI/MPS II mice (MPS II) were used.
• WT wild type
• MPS II human transferrin receptor-KI/MPS II mice
• IDS activity and GAG measurements were performed in the same manner as in Example 1.
• FIG. 5 shows IDS activity in plasma 4 weeks after transplantation.
• the IDS activity in FIG. 5 is the relative activity (%) when the IDS activity in wild type mouse (WT) is set as 100. From Figure 5, IDS activity in the plasma of both IDS mice and hTfR-IDS mice exceeds that of wild-type mice, with IDS mice being 9.36 times that of wild-type mice, and hTfR-IDS mice being 1.85 times that of wild-type mice. It was double that.
• FIG. 6 shows the measurement results of IDS activity and GAG accumulation in the liver and spleen 4 weeks after transplantation.
• the IDS activity is the relative activity (%) when the IDS activity in wild type mice (WT) is set as 100
• the GAG accumulation is the relative activity (%) when the IDS activity in wild type mice (WT) is set as 100. This is the relative amount (%) when From Figure 6, the IDS activity in the liver of hTfR-IDS mice was 32.6% of that of wild-type mice, and the IDS activity in the spleen was 147% of that of wild-type mice, and a significant decrease in GAG accumulation was observed in both organs. Ta.
• FIG. 7 shows the measurement results of IDS activity and GAG accumulation in the cerebrum and cerebellum 4 weeks after transplantation.
• the IDS activity is the relative activity (%) when the IDS activity in wild type mice (WT) is set as 100
• the GAG accumulation is the relative activity (%) when the IDS activity in wild type mice (WT) is set as 100. This is the relative amount (%) when From FIG. 7, as with the hTfR-IDS mice of Example 1, although the IDS activity was low, the reduction in GAG accumulation was excellent in the hTfR-IDS mice.
• Example 3 Anti-human TfR antibody-T cell gene therapy using human IDS.
• Example 2 The same mouse as that obtained in Example 2 was used.
• lentivirus packaging in addition to the self-inactivating (SIN) lentivirus vector (pJLV1-IDS or pJLV1-hTfR-IDS) prepared in Example 2 above, the packaging plasmids pCAG-HIVgp, VSV -G/Rev plasmid pCMV-VSV-G-RSV-Rev was used, and the standard protocol was partially modified (for example, the protocol described in Patent Document 1).
• Mouse T cells were isolated from the spleen 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 transfected (infected) with the obtained lentivirus carrying IDS or hTfR-IDS. Lentivirus infection of mouse T cells was performed at a multiplicity of infection (MOI) of 50 by applying lentivirus infection after 24 hours of stimulation according to the protocol of Dynabeads Mouse T-Activator CD3/CD28 (Thermo Fisher). Ta. Specifically, 5 ⁇ 10 7 IU of lentivirus was used per 1 ⁇ 10 6 mouse T cells (number of cells for transplantation per recipient).
• MOI multiplicity of infection
• Thermo Fisher Dynabeads Mouse T-Activator CD3/CD28
• the gene-transfected mouse T cells were administered to 5-month-old human transferrin receptor-KI/MPS II mice via the tail vein. Plasma, cerebrum, and cerebellum were collected 4 weeks after administration. As control mice, non-transplanted wild type (WT) C57BL/6 mice and human transferrin receptor-KI/MPS II mice (MPS II) were used.
• WT wild type
• MPS II human transferrin receptor-KI/MPS II mice
• IDS activity and GAG measurements were performed in the same manner as in Example 1.
• IDS activity is the relative activity (%) when the IDS activity in wild type mice (WT) is set as 100
• GAG accumulation is the relative activity (%) when the IDS activity in wild type mice (WT) is set as 100.
• This is the relative amount (%) when From FIG. 8, IDS activity in the plasma of both IDS mice and hTfR-IDS mice exceeded that of human transferrin receptor-KI/MPS II mice. From FIG. 9, even when mouse T cells were used, a decrease in GAG accumulation was observed in the cerebrum and cerebellum of hTfR-IDS mice.
• Example 4 Anti-mouse TfR antibody-hematopoietic stem cell gene therapy using human GAA] ⁇ Mouse> A mouse homozygously deficient in the acid ⁇ -glucosidase (GAA) gene was created by crossing (GAA-KO mouse). C57BL/6 mice served as normal controls.
• GAA acid ⁇ -glucosidase
• pJLV1-GAA which carries human GAA (wild type human GAA, SEQ ID NO: 17) directly under the MND promoter (DNA: SEQ ID NO: 1) of pJLV1 plasmid (SEQ ID NO: 12), which is a self-inactivating lentiviral vector
• pJLV1-mTfR-GAA was constructed carrying an anti-mouse TfR antibody-GAA (mTfR-GAA) directly under the MND promoter.
• pJLV1-GAA DNA (SEQ ID NO: 18) encoding human GAA (SEQ ID NO: 17) was artificially synthesized with NotI site added to the 5' side and XhoI site added to the 3' side, and restriction enzymes were added. Processed. Next, pJLV1 plasmid (SEQ ID NO: 12) was also cleaved with NotI and XhoI, and pJLV1-GAA (SEQ ID NO: 19) was created using a Quick ligation kit.
• lentivirus packaging was performed using the self-inactivating (SIN) lentivirus vector (pJLV1-GAA or pJLV1) prepared in (1) or (2) above.
• pJLV1-GAA self-inactivating lentivirus vector
• pJLV1-GAA self-inactivating lentivirus vector
• pJLV1-GAA self-inactivating lentivirus vector
• pJLV1-G-RSV-Rev VSV-G/Rev plasmid pCMV-VSV-G-RSV-Rev
• the generated lentivirus titer was measured using the Quick Titer HIV Lentivirus Quantitation Kit (Cell Biolabs, San Diego, CA), which is an ELISA against p24 protein.
• the titers were pJLV1-GAA: 1.7 ⁇ 10 9 IU/ml and pJLV1-mTfR-GAA: 1.2 ⁇ 10 9 IU/ml, respectively.
• ⁇ GAA activity measurement> For protein quantification, a DC protein assay kit (Bio-Rad) was used, and for GAA activity measurement, the fluorescent substrate 4-methylumbelliferyl ⁇ -D-glucopyranoside (Sigma-Aldrich) was used according to a previously reported document (Non-patent Document 8). Measured using
• Figure 10 shows the measurement results of GAA activity for HEK293T cell extract and culture medium. From Figure 10, in HEK293T cells infected with mTfR-GAA-equipped lentivirus, the intracellular activity is lower than that of HEK293T cells infected with GAA-equipped lentivirus, while the extracellular activity (secretory enzyme activity) is lower than that of HEK293T cells infected with GAA-equipped lentivirus. was significantly higher than that of HEK293T cells infected with . From this result, surprisingly, extracellular secretion of GAA was promoted by fusing GAA with an anti-mouse TfR antibody.
• ⁇ Preparation and transplantation of gene-transferred hematopoietic stem cells were generated in 3-month-old GAA-KO mice using the same procedure as in Example 1, and transplanted into 3-month-old GAA-KO mice.
• serum, liver, spleen, heart, quadriceps muscle, diaphragm, gastrocnemius muscle, cerebrum, and cerebellum were collected 4 weeks after administration, and as control mice.
• GAA protein Avalglucosidase alpha (Sanofi)
• Hydrolysis Enzyme Mix which is a hydrolysis reaction reagent, is added to each well and mixed, and the mixture is allowed to react at room temperature for 30 minutes (however, it is not added to the wells for measuring endogenous glucose).
• 50 ⁇ L of Development Enzyme Mix containing OxiRed Probe was added to each well and mixed, reacted for 30 minutes at room temperature in the dark, and measured using Synergy H1 (BioTek) at an excitation wavelength of 535 nm and a detection wavelength of 587 nm.
• FIG. 11 shows the measurement results of GAA activity in serum, liver, and spleen 4 weeks after transplantation.
• GAA activity is relative activity (%) when GAA activity in wild type mouse (WT) is set as 100. From FIG. 11, when comparing the GAA activity in serum, liver, and spleen among the treatment groups (ERT mice, GAA mice, and mTfR-GAA mice), mTfR-GAA mice had the highest activity in all of them.
• FIG. 12 shows the measurement results of GAA activity in the heart, quadriceps, diaphragm, and gastrocnemius muscles 4 weeks after transplantation.
• GAA activity is relative activity (%) when GAA activity in wild type mouse (WT) is set as 100.
• WT wild type mouse
• Figure 13 shows the measurement results of glycogen accumulation in the heart, quadriceps, diaphragm, and gastrocnemius muscles 4 weeks after transplantation.
• Glycogen accumulation is the relative amount (%) when the amount of glycogen accumulation in GAA-KO mice (NT) is taken as 100. From FIG. 13, dramatic improvement in glycogen accumulation was obtained in the mTfR-GAA group in various muscle tissues, and normalization was observed in the diaphragm. On the other hand, in GAA mice, although GAA activity comparable to mTfR-GAA was observed in quadriceps muscles, etc., the decrease in glycogen accumulation was the least in the treated group. This suggested that the distribution and localization of GAA was improved by the fusion of the anti-TfR antibody.
• FIG. 14 shows the measurement results of GAA activity and glycogen accumulation in the cerebrum and cerebellum 4 weeks after transplantation.
• GAA activity is the relative activity (%) when the GAA activity in wild type mice (WT) is set as 100
• glycogen accumulation is the relative amount (%) when the amount of glycogen accumulation in GAA-KO mice (NT) is set as 100. %). From FIG. 14, an increase in GAA activity was observed only in mTfR-GAA mice, and glycogen accumulation was also significantly reduced.
• ELISA ELISA was performed according to the following procedure. 250 ng of recombinant GAA protein (Avalglucosidase alpha (Sanofi)) was bound to a microplate (Maxisorp, Thermo Scientific) overnight at 4°C, and then blocked for 2 hours with PBS containing 1% BSA.
• a peroxidase-labeled anti-rabbit IgG antibody (Histofine Simple Stain MAX-PO (R), Nichirei Biosciences) was added and allowed to react at room temperature for 1 hour. Thereafter, each well was washed with PBS-T, tetramethylbenzidine (SeraCare) was added to develop color for 10 minutes, and then 1.2N sulfuric acid was added to stop the reaction. Regarding the measurement, the absorbance was measured at 450 nm and 650 nm using Synergy H1 (BioTek), and the difference was defined as the true absorbance. The results are shown in Table 1.
• mice treated with gene therapy using mTfR-GAA-transduced hematopoietic stem cells had the lowest anti-GAA antibody titer, which was at the same level as untreated mice and wild-type mice.
• Example 5 Anti-mouse TfR antibody-hematopoietic stem cell gene therapy using human GLB1
• ⁇ Mouse> A mouse heterozygously deficient in the ⁇ -galactosidase (GLB1) gene (Non-Patent Document 9) was created by crossing (GLB1-KO mouse). C57BL/6 mice served as normal controls.
• pJLV1-GLB1 which carries human GLB1 (wild type human GLB1, SEQ ID NO: 23) directly under the MND promoter (DNA: SEQ ID NO: 1) of pJLV1 plasmid (SEQ ID NO: 12), which is a self-inactivating lentiviral vector
• pJLV1-mTfR-GLB1 carrying anti-mouse TfR antibody-GLB1 (mTfR-GLB1) directly under the MND promoter was constructed.
• pJLV1-GLB1 DNA (SEQ ID NO: 24) encoding human GLB1 (SEQ ID NO: 23) was placed downstream of the MND promoter and created by artificial synthesis. Specifically, MND-GLB1 with an MluI site added to the 5' side and an XhoI site added to the 3' side was artificially synthesized and treated with each restriction enzyme. Next, pJLV1 plasmid (SEQ ID NO: 12) was also cleaved with MluII and XhoI, and pJLV1-GLB1 (SEQ ID NO: 25) was created using a Quick ligation kit.
• lentivirus packaging was performed using the self-inactivating (SIN) lentivirus vector (pJLV1-GLB1 or pJLV1- In addition to mTfR-GLB1), the packaging plasmid pCAG-HIVgp and the VSV-G/Rev plasmid pCMV-VSV-G-RSV-Rev were used, and the standard protocol was performed with some modifications. (For example, the protocol described in Patent Document 1).
• SIN self-inactivating
• the generated lentivirus titer was measured using the Quick Titer HIV Lentivirus Quantitation Kit (Cell Biolabs, San Diego, CA), which is an ELISA against p24 protein.
• the titers were pJLV1-GLB1: 1.8 ⁇ 10 8 IU/ml and pJLV1-mTfR-GLB1: 4.3 ⁇ 10 8 IU/ml, respectively.
• ⁇ GLB1 activity measurement and GM1 ganglioside measurement> For protein quantification, a DC protein assay kit (Bio-Rad) was used, and for GLB1 activity measurement, the fluorescent substrate 4-methylumbelliferyl- ⁇ -D-galactopyranoside, (Sigma- GM1 ganglioside was also measured using a triple quadrupole high performance liquid chromatography mass spectrometer LCMS-8040 (Shimadzu Corporation) according to a previously published document (Non-Patent Document 7).
• FIGS. 15 to 17 show the measurement results of GLB1 activity and GM1 ganglioside accumulation (GM1 accumulation) in serum, cerebral cortex, cerebellum, and hippocampus 4 weeks after transplantation.
• GLB1 activity is the relative activity (%) when the GLB1 activity in wild type mice (WT) is set as 100
• GM1 accumulation is the relative amount (%) when the amount of GM1 accumulation in GLB1-KO mice (NT) is set as 100. %).
• WT wild type mice
• GM1 accumulation is the relative amount (%) when the amount of GM1 accumulation in GLB1-KO mice (NT) is set as 100. %).
• FIGS. 16 and 17 From FIGS. 16 and 17, in mTfR-GLB1 mice, GLB1 activity was high in the cerebral cortex, cerebellum, and hippocampus, and GM1 accumulation was significantly reduced.
• the activity of the fusion protein due to anti-mTfR antibody fusion is lower than that of the protein that does not form the fusion protein, but unexpectedly, for GLB1, the anti-mTfR antibody fusion protein has a lower activity than the activity of the protein that does not form the fusion protein.
• the activity in the blood was higher than that of the type enzyme. It is considered that the increase in extracellular secretion and the stabilizing effect of the enzyme in the blood, which may be brought about by the fusion of the anti-mTfR antibody, may outweigh the decrease in activity. Since GAA and GLB1 are enzymes that are difficult to secrete or are unstable, the fusion of the anti-mTfR antibody may have stabilized them.It was an unexpected result that their blood activity increased instead of decreasing. there were.

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