Polynucleotide binding agent, polynucleotide transport composition, virus production method, and cell preparation method

Abstract

This polynucleotide binding agent includes a first binding site and a second binding site, the first binding site includes an amino acid sequence capable of binding to a receptor-binding protein that binds to a receptor of a target cell, and the second binding site includes an amino acid sequence capable of binding to a polynucleotide. The molecular weight of the polynucleotide is preferably 350 kDa or more. The number of amino acid residues contained in the first binding site and the second binding site is preferably 5 to 100.

Description

Polynucleotide binder, polynucleotide transport composition, method for producing a virus, and method for producing cells

[0001] The present invention relates to a polynucleotide binder, a polynucleotide transport composition, a method for producing a virus, and a method for producing cells. This application claims priority to Japanese Patent Application No. 2025-048171, filed in Japan on March 24, 2025, and Japanese Patent Application No. 2025-184584, filed in Japan on October 31, 2025, the contents of which are incorporated herein by reference.

[0002] With the sequencing of the human and mouse genomes, there is an increasing need in fields such as medicine and pharmacy to introduce molecules such as polynucleotides (DNA or RNA, or their derivatives), proteins (signaling proteins or transcription factors, or their derivatives), small molecules of physiologically active substances, and drug candidates into target cells to test the function of genes or the physiological activity of physiologically active molecules within those target cells. This is necessary when conducting molecular biology-related research and development or drug development.

[0003] There are three main methods for introducing molecules into target cells: chemical, physical, and biological methods. Chemical methods involve using transfection reagents such as cationic polymers, cationic lipids, or calcium phosphate to introduce molecules into target cells via endocytosis. Physical methods involve directly introducing molecules into target cells through physical manipulations such as microinjection, sonoporation, laser irradiation, or electroporation. Biological methods involve using viral vectors such as retroviruses, lentiviruses, adeno-associated viruses (AAVs), or adenoviruses to transfer nucleic acids within the viral particles into target cells using the infectivity of the virus.

[0004] Non-patent document 1 describes that when introducing genes into hematopoietic stem cells using a retroviral vector, the addition of fibronectin improves the introduction efficiency.

[0005] Non-patent document 2 describes that when introducing genes into blood cells using a retroviral vector, the introduction efficiency is improved by adding a recombinant protein (Retronectin®, recombinant human fibronectin CH-296) containing three functional domains of human fibronectin: the cell adhesion domain (C-domain), the heparin-binding domain (H-domain), and the CS-1 site.

[0006] Patent Document 1 describes a method for introducing a foreign gene into target cells using a retroviral vector, which includes the steps of (a) adding a liquid containing a retroviral vector carrying a foreign gene to a culture vessel immobilized with a retrovirus-binding substance such as fibronectin or fibronectin fragment CH-296 [RetroNectin], and then incubating it at a temperature of less than 25°C for 4 hours or more to obtain a culture vessel to which the retroviral vector has been bound, and (b) adding target cells to the culture vessel obtained in step (a) and incubating it.

[0007] Patent Document 2 describes a method for producing a cell population into which a desired gene has been introduced, comprising the steps of (1) culturing a cell population containing T cells and / or T cell progenitor cells in a container containing fibronectin or a fragment thereof and a CD3 ligand, and (2) adding a vector containing the desired gene to the container of step (1).

[0008] In addition to the methods described above, cell-penetrating peptides (CPPs) are being studied as means of polynucleotide transport because they have the ability to introduce substances into cells across the cell membrane. For example, Patent Document 3 and Non-Patent Documents 3 and 4 report the intracellular introduction of nucleic acids or biomolecules using CPPs. However, the nucleic acids targeted in Patent Document 3 and Non-Patent Documents 3 and 4 are limited to relatively small nucleic acids, short oligonucleotides, or small gene sequences, making them unsuitable for the introduction of polynucleotides into cells, such as genes for AAV vector expression in cells (double-stranded DNA plasmid vectors 8-20 kbp) or chimeric antigen receptor (CAR) genes (e.g., mRNA 1400 bases or more). Furthermore, since the interaction between CPPs and nucleic acids mainly depends on electrostatic interactions, as the size of the nucleic acid increases, the complex with CPPs tends to aggregate, which can lead to nonspecific adsorption, increased cytotoxicity, and decreased delivery and expression efficiency (Non-Patent Document 5).

[0009] Given this technical background, it has been understood by those skilled in the art that increasing nucleic acid size is disadvantageous in the field of nucleic acid delivery using CPP, and that it is difficult to deliver large nucleic acids by CPP and establish expression. Therefore, CPP delivery targeting large nucleic acids has not been a subject that those skilled in the art have actively considered.

[0010] International Publication No. 2012 / 002452, International Publication No. 2009 / 119793, US007943581B2

[0011] Moritz, T., et al., “Fibronectin Improves Translation of Reconstructing Hematopoietic Stem Cells by Retroviral Vectors: Evidence of Direct Viral Binding to Chymogenic Carboxy-Terminal Fragments,” Blood, August 1, 1996, Vol. 88, No. 3, pp. 855-862. Hanenberg, H. Laurence Crombez, et al., “Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic translation of mammalian cells,” Nature Medicine, August 1996, Vol. 2, No. 8, pp. 876-882. , and 7 others, “A New Potent Secondary Amphipathic Cell-penetrating Peptide for siRNA Delivery Into Mammalian Cells”, Molecular Therapy, January 2009, Volume 17, Issue 1, p. 95-103Corina Ciobanasu. , and 2 others, “Cell-penetrating HIV1 TAT peptides can generate pores in model membranes”, Biophysical Journal, July 2010, Vol. 99, p. 153-162 Chanuk Jeong, et al., “A branched TAT cell-penetrating peptide as a novel delivery carrier for the efficient gene transfer”, Biomaterials Research, 2016, Vol. 20, Article No. 28.

[0012] Methods for transporting molecules to the cell membrane of target cells require further improvements in efficiency. Furthermore, conventional methods for transporting molecules to the cell membrane of target cells often require complex procedures; therefore, there is a need for the development of simpler, non-viral vector-based transport methods.

[0013] As described in Patent Document 3 and Non-Patent Documents 3 and 4, conventional nucleic acid delivery technologies using CPP have made it difficult to introduce large polynucleotides into cells and to express the genes encoded by those polynucleotides. Furthermore, the inhibition of cell proliferation when polynucleotides are introduced into cells makes it difficult to secure a sufficient number of cells.

[0014] The present invention aims to provide a polynucleotide binder and a polynucleotide transport composition that have high molecular transport efficiency to the cell membrane of target cells and are easy to handle.

[0015] The present invention encompasses the following embodiments: [1] A method for introducing polynucleotides, comprising: a complexing step of obtaining a complex formed by complexing a polynucleotide with a polynucleotide binder composed of at least a peptide; and a contact step of contacting the complex with the cell membrane of a target cell to introduce the polynucleotide from the cell membrane into the target cell, wherein the contact step is performed in the presence of at least one of receptors and cell adhesion factors contained in the cell membrane. [2] The method for introducing polynucleotides according to [1], wherein the molecular weight of the polynucleotide is 350 kDa or more. [3] The method for introducing polynucleotides according to [1] or [2], wherein the complex is formed by the non-covalent bonding of the polynucleotide and the polynucleotide binder. [4] The method for introducing polynucleotides according to any one of [1] to [3], wherein the amino acid sequence of the polynucleotide binder has 80% or more sequence identity with the amino acid sequence represented by SEQ ID NOs: 36 to 48, SEQ ID NOs: 86 to 117, or SEQ ID NOs: 132 to 140. [5] The polynucleotide introduction method according to any one of [1] to [4], wherein the content of cationic polyethyleneimine or cationic lipid in the complex is 0.1% by mass or less. [6] The polynucleotide introduction method according to any one of [1] to [5], wherein the introduction in the contact step is naturally introduced from the cell membrane into the target cell by the feeding action of the target cell. [7] The polynucleotide introduction method according to any one of [1] to [6], wherein the target cell is a cell expressing a caveolae-related gene. [8] The polynucleotide introduction method according to any one of [1] to [7], wherein the target cell is a cell in which at least one of caveolin 1, caveolin 2, and cabin 1 is overexpressed. A polynucleotide binder used in a polynucleotide introduction method according to any one of [9], [1] to [7], wherein the polynucleotide binder comprises a first binding site and a second binding site, the first binding site comprising an amino acid sequence capable of directly or indirectly binding to a receptor on a target cell, and the second binding site comprising an amino acid sequence capable of binding to a polynucleotide.

[10] The polynucleotide binder according to [9], wherein the sum of the number of amino acid residues contained in the first binding site and the second binding site is 5 or more and 100 or less.

[11] The polynucleotide binder according to [9] or

[10] , wherein the amino acid sequence of the first binding site has 80% or more sequence identity with the amino acid sequence represented by SEQ ID NOs: 1 to 17, SEQ ID NOs: 60 to 75, or SEQ ID NOs: 122 to 127.

[12] The polynucleotide binder according to any one of [9] to

[11] , wherein the second binding site contains a cationic amino acid.

[13] The polynucleotide binder according to any one of [9] to

[12] , wherein the polynucleotide is at least one of single-stranded or double-stranded DNA and RNA.

[14] The polynucleotide binder according to any one of [9] to

[13] , wherein the polynucleotide is at least one of circular DNA and linear DNA.

[15] The polynucleotide binder according to any one of [9] to

[14] , wherein the polynucleotide is a double-stranded polynucleotide forming a complementary chain, and the number of base pairs contained in the polynucleotide is 1 kbp or more.

[16] A polynucleotide transport composition comprising the polynucleotide binder according to any one of [9] to

[14] and a polynucleotide.

[17] The polynucleotide transport composition according to

[16] , wherein the content of cationic polyethyleneimine or cationic lipid is 0.1% by mass or less.

[18] The polynucleotide transport composition according to

[16] or

[17] , further comprising a receptor-binding protein.

[19] A polynucleotide transport composition comprising the polynucleotide binder according to any one of [9] to

[14] , a polynucleotide and a target cell.

[20] The polynucleotide transport composition according to

[19] , wherein the content of cationic polyethyleneimine or cationic lipid is 0.1% by mass or less.

[21] The polynucleotide transport composition according to

[19] or

[20] , further comprising a receptor-binding protein.

[0016] The following A1 to A14 are other aspects of the present invention. [A1] A polynucleotide binder comprising a first binding site and a second binding site, wherein the first binding site comprises an amino acid sequence capable of binding to a receptor-binding protein that binds to a receptor on a target cell, and the second binding site comprises an amino acid sequence capable of binding to a polynucleotide. [A2] The polynucleotide binder according to [A1], wherein the molecular weight of the polynucleotide is 350 kDa or more. [A3] The polynucleotide binder according to [A1] or [A2], wherein the number of amino acid residues contained in the first binding site and the second binding site is 5 or more and 100 or less. [A4] The polynucleotide binder according to any one of [A1] to [A3], wherein the amino acid sequence of the first binding site has 80% or more sequence identity with the amino acid sequence represented by SEQ ID NOs: 1 to 17, SEQ ID NOs: 60 to 75, or SEQ ID NOs: 122 to 127. [A5] The polynucleotide binder according to any one of [A1] to [A4], wherein the second binding site comprises a cationic amino acid. [A6] The polynucleotide binder according to any one of [A1] to [A5], wherein the polynucleotide is at least one of single-stranded or double-stranded DNA and RNA. [A7] The polynucleotide binder according to any one of [A1] to [A6], wherein the polynucleotide is at least one of circular DNA and linear DNA. [A8] The polynucleotide binder according to any one of [A1] to [A7], wherein the polynucleotide is a double-stranded polynucleotide forming a complementary strand, and the number of base pairs contained in the polynucleotide is 1 kbp or more. [A9] A polynucleotide transport composition comprising the polynucleotide binder according to any one of [A1] to [A8] and a polynucleotide. [A10] The polynucleotide transport composition according to [A9], wherein the content of cationic polyethyleneimine or cationic lipid is 0.1% by mass or less. [A11] The polynucleotide transport composition according to [A9] or [A10], further comprising a receptor-binding protein.A polynucleotide transport composition comprising the polynucleotide binder, polynucleotide, and target cell according to any one of [A12] [A1] to [A8]. [A13] The polynucleotide transport composition according to [A12], wherein the content of cationic polyethyleneimine or cationic lipid is 0.1% by mass or less. [A14] The polynucleotide transport composition according to [A13], further comprising a receptor-binding protein. A method for producing a virus, comprising the step of introducing at least a polynucleotide from the cell membrane into the cytoplasm by contacting the target cell with the polynucleotide binder according to any one of [A1] to [A8] or the polynucleotide transport composition according to any one of [A9] to [A14], and producing a virus in the target cell. [A16] The method for producing a virus according to [A15], wherein the virus is an adeno-associated virus. A method for producing cells, comprising the step of introducing at least a polynucleotide from the cell membrane into the cytoplasm by contacting a target cell with a polynucleotide binder according to any one of [A17], [A1] to [A8], or a polynucleotide transport composition according to any one of [A9] to [A14], thereby causing the target cell to express an exogenous membrane protein or overexpress an intrinsic membrane protein on its cell membrane. [A18] The method for producing cells according to [A17], wherein the exogenous membrane protein is a chimeric antigen receptor.

[0017] According to the above embodiment, it is possible to provide a polynucleotide binder and a polynucleotide transport composition that have high molecular transport efficiency to the cell membrane of target cells and are easy to handle.

[0018] Figure 1(A) is a schematic diagram of a complex containing a polynucleotide binder according to one aspect of the present invention. Figure 1(B) is a schematic diagram of a complex containing multiple polynucleotide binders according to one aspect of the present invention. Figure 2(A) is a schematic diagram of a complex containing a polynucleotide binder according to one aspect of the present invention. Figure 2(B) is a schematic diagram of a complex containing multiple polynucleotide binders according to one aspect of the present invention. Figure 3(A) is an agarose gel electrophoresis image confirming the binding of the polynucleotide binder and DNA according to one aspect of the present invention. Figure 3(B) is an agarose gel electrophoresis image confirming the binding of the polynucleotide binder and RNA according to one aspect of the present invention. Figure 4 is an agarose gel electrophoresis image confirming the formation of a complex containing a polynucleotide binder according to one aspect of the present invention. Figure 5(A) is a microscopic image when DNA is introduced into target cells using the polynucleotide binder according to one aspect of the present invention. Figure 5(B) is a graph showing the introduction efficiency when DNA is introduced into target cells using the polynucleotide binder according to one aspect of the present invention. Figure 6 is a graph showing the introduction efficiency when DNA is introduced into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 7 is a graph showing the introduction efficiency when plasmid vectors with different base sequences are introduced into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 8 is a graph showing the introduction efficiency when plasmid vectors with different base sequences are introduced into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 9 is a graph showing the number of viable cells after DNA is introduced into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 10 is a graph showing the number of GFP-positive cells after DNA is introduced into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 11 is a graph showing the introduction efficiency when DNA is introduced into HEK293 cells using polynucleotide binders with different amino acid sequences. Figure 12 is a graph showing the introduction efficiency when DNA is introduced into Jurkat cells using polynucleotide binders with different amino acid sequences.Figure 13 is a graph showing the introduction efficiency when DNA is introduced into Jurkat cells using polynucleotide binders with different amino acid sequences. Figures 14(A) and 14(B) are graphs showing the introduction efficiency when DNA is introduced into caveola 1 overexpressing target cells using a polynucleotide binder according to one aspect of the present invention. Figure 15 is a graph showing the introduction efficiency when polynucleotides are introduced into target cells overexpressing any of Cav1, Cav2, and Cavin1 using a polynucleotide binder according to one aspect of the present invention. Figure 16 is a graph showing the introduction efficiency when polynucleotides are introduced into target cells overexpressing any of Cav1, Cav2, and Cavin1 using a polynucleotide binder according to one aspect of the present invention. Figure 17 is a graph showing the introduction efficiency when polynucleotides are introduced into target cells overexpressing any of Cav1, Cav2, and Cavin1 using a polynucleotide binder according to one aspect of the present invention. Figure 18 is a graph showing the introduction efficiency when polynucleotides are introduced into target cells overexpressing any of Cav1, Cav2, or Cavin1 using a polynucleotide binder according to one aspect of the present invention. Figure 19 is a microscopic image of human peripheral blood single cells after DNA has been introduced using a polynucleotide binder according to one aspect of the present invention. Figure 20 is a microscopic image of human preadipocytes after DNA has been introduced using a polynucleotide binder according to one aspect of the present invention. Figure 21 is a microscopic image of human peripheral blood single cells after RNA has been introduced using a polynucleotide binder according to one aspect of the present invention. Figure 22 is a microscopic image of Jurkat cells after RNA has been introduced using a polynucleotide binder according to one aspect of the present invention. Figure 23 is a graph showing the results of confirming the storage stability of the polynucleotide binder according to one aspect of the present invention. Figure 24 is a graph showing the introduction efficiency when the polynucleotide binder according to one aspect of the present invention is stored under different storage conditions. Figure 25 is a graph showing the delivery efficiency after introducing DNA into target cells using a polynucleotide binder according to one embodiment of the present invention.Figure 26 is a graph showing the delivery efficiency after introducing DNA into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 27 is a graph showing the delivery efficiency after introducing DNA into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 28 is a graph showing the delivery efficiency after introducing DNA into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 29 is a graph showing the delivery efficiency and the number of viable cells after introducing DNA into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 30 is a graph showing the delivery efficiency after introducing DNA into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 31 is a graph showing the delivery efficiency after introducing DNA into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 32 is a graph showing the titer of the viral extract after introducing a viral vector expression plasmid vector into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 33 shows (A) a fluorescence microscopy image, (B) a bright-field microscopy image, and (C) a superimposed image of the fluorescence microscopy and bright-field microscopy images after introducing a viral vector prepared using a polynucleotide binder according to one aspect of the present invention into target cells. Figure 34 is a graph showing the number of virus-infected cells after introducing a viral vector prepared using a polynucleotide binder according to one aspect of the present invention into target cells. Figure 35 is a graph showing the flow cytometry analysis results after introducing a viral vector expression plasmid vector into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 36 is a graph showing the relative number of viable cells after introducing a viral vector expression plasmid vector into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 37(A) is a graph showing the flow cytometry analysis results after introducing mRNA encoding the CD19 chimeric antigen receptor (CD19-CAR) sequence into target cells using a polynucleotide binder according to one aspect of the present invention.Figure 38(A) is a graph showing the flow cytometry analysis results after introducing mRNA encoding the CD19 chimeric antigen receptor (CD19-CAR) sequence into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 39(A) is a graph showing the flow cytometry analysis results after introducing mRNA encoding the CD19 chimeric antigen receptor (CD19-CAR) sequence into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 40 is a graph showing the flow cytometry analysis results after introducing mRNA encoding the CD19 chimeric antigen receptor (CD19-CAR) sequence into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 41 is an agarose gel electrophoresis image confirming the size of the plasmid vector used in the example. Figure 42 is an agarose gel electrophoresis image confirming the size of the plasmid vector used in the example. Figure 43 is an agarose gel electrophoresis image showing the results of confirming the formation of the complex in the example and comparative example. Figure 44 is an agarose gel electrophoresis image showing the results of confirming the formation of the complex in the examples and comparative examples. Figure 45 is a graph showing the introduction efficiency after introducing DNA into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 46 is a graph showing the introduction efficiency relative to the number of cells when DNA is introduced into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 47 is a graph showing the introduction efficiency relative to the number of cells when DNA is introduced into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 48 is a graph showing the introduction efficiency after introducing DNA into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 49 is a graph showing the amount of virus produced after introducing a viral vector expression plasmid vector into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 50 is a graph showing the amount of virus produced after introducing a viral vector expression plasmid vector into target cells using a polynucleotide binder according to one aspect of the present invention.Figure 51 is a graph showing the titer of the viral extract after introducing a viral vector expression plasmid vector into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 52 is a graph showing the titer of the viral extract after introducing a viral vector expression plasmid vector into target cells using a polynucleotide binder according to one aspect of the present invention. Figure 53 is a graph showing the number of virus-infected cells after introducing a viral vector prepared using a polynucleotide binder according to one aspect of the present invention into target cells. Figure 54 is a microscopic image of mRNA introduced into induced pluripotent stem cells (iPS cells) using a polynucleotide binder according to one aspect of the present invention. Figure 55 is a graph showing the introduction efficiency relative to the number of cells when mRNA is introduced into iPS cells using a polynucleotide binder according to one aspect of the present invention. Figure 56 is a graph showing the introduction efficiency relative to the number of cells when DNA is introduced into iPS cells using a polynucleotide binder according to one aspect of the present invention. Figure 57 is a microscopic image of mRNA introduced into mesenchymal stem cells (BM-MSCs) using a polynucleotide binder according to one aspect of the present invention. Figure 58 is a graph showing the introduction efficiency relative to the number of cells when mRNA is introduced into BM-MSCs using a polynucleotide binder according to one aspect of the invention. Figure 59 is a graph showing the introduction efficiency relative to the number of cells when DNA is introduced into BM-MSC cells using a polynucleotide binder according to one aspect of the invention. Figure 60 is a microscopic image of adipose-derived stem cells (ADSCs) after mRNA has been introduced using a polynucleotide binder according to one aspect of the invention. Figure 61 is a graph showing the introduction efficiency relative to the number of cells when mRNA is introduced into ADSCs using a polynucleotide binder according to one aspect of the invention. Figure 62 is a graph showing the introduction efficiency relative to the number of cells when DNA is introduced into ADSCs using a polynucleotide binder according to one aspect of the invention. Figure 63 is a microscopic image of human fibroblasts (HDFs) after mRNA has been introduced using a polynucleotide binder according to one aspect of the invention.Figure 64 is a graph showing the number of GFP-positive cells after introducing DNA into HDF using the polynucleotide binding agent according to one embodiment of the present invention. Figure 65 shows the results of confirming the expression of Cav1, Cav2 and Cavin1 in the Cav1-overexpressing strain, Cav2-overexpressing strain and Cavin1-overexpressing strain prepared in Experimental Examples 8 and 9. Figure 66 is a graph showing the results of confirming the expression levels of Cav1, Cav2 and Cavin1 in the Cav1-overexpressing strain, Cav2-overexpressing strain and Cavin1-overexpressing strain prepared in Experimental Examples 8 and 9.

[0019] Hereinafter, embodiments of the present invention will be specifically described, but the present invention is not limited to the following embodiments, and can be implemented with various modifications within the scope of the gist thereof.

[0020] In the following specification, the term "polynucleotide" refers to a substance in which 10 or more nucleotide monomers are linked, and does not include substances bound to nucleoproteins such as ribosomes and nucleosomes.

[0021] In the following specification, the term "complex" refers to a complex containing at least the polynucleotide binding agent and the polynucleotide according to one embodiment of the present invention, which may or may not contain a receptor-binding protein.

[0022] When a numerical range is described as, for example, "1 to 10 µm", it means the range from 1 µm to 10 µm, including the lower limit value of 1 µm and the upper limit value of 10 µm. In addition, the upper limit and lower limit of a numerical range can be arbitrarily combined. Furthermore, the numerical ranges for each physical property, composition, measurement step and the like can be arbitrarily combined with each other.

[0023] <Polynucleotide Binding Agent> (First Embodiment) The polynucleotide binding agent of this embodiment is a polynucleotide binding agent for bringing a polynucleotide closer to the cell membrane of a target cell, and includes a first binding site and a second binding site. The first binding site preferably includes an amino acid sequence that can directly or indirectly bind to the receptor of the target cell. The first binding site preferably includes an amino acid sequence that can bind to a receptor-binding protein that binds to the receptor of the target cell. The second binding site includes an amino acid sequence that can bind to the polynucleotide. The second binding site preferably includes an amino acid sequence represented by at least one of the following sequences A and B.

[0024] (First Embodiment) The polynucleotide binder in the first embodiment of this embodiment will be described with reference to Figure 1(A). Figure 1(A) is a schematic diagram of the complex containing the polynucleotide binder in the first embodiment. The complex 1 in this embodiment includes a polynucleotide 10, a polynucleotide binder 20a, and a receptor-binding protein 30. The polynucleotide binder 20a has a first binding site 21 and a second binding site 23 that are directly bound. The first binding site 21 is bound to the receptor-binding protein 30, and the second binding site 23 is bound to the polynucleotide 10. The receptor-binding protein 30 contained in the complex 1 binds to the receptor 40 of the target cell, thereby enabling the transport of the polynucleotide 10 to the cell membrane of the target cell.

[0025] In the polynucleotide binding agent 20a, the first binding site 21 and the second binding site 23 are directly bonded. Here, the term "directly bonded" means that the main chain of the first binding site 21 and the main chain of the second binding site 23 directly form a chemical bond. There is no particular limitation on the type of chemical bond, but a peptide bond formed by dehydration condensation between a carboxyl group and an amino group contained in amino acids is preferred. The first binding site 21 may be located on the C-terminal side or the N-terminal side of the second binding site 23. The polynucleotide binding agent in this embodiment is preferably linear. As used herein, the term "linear" refers to a structure in which the first binding site, the second binding site, and a non-binding site described below are all linked by peptide bonds and connected in a linear sequence.

[0026] There is no particular limitation on the sum of the number of amino acid residues contained in the first binding site 21 and the second binding site 23. The sum of the number of said amino acid residues is preferably 5 to 100 residues, more preferably 10 to 60 residues, and even more preferably 15 to 40 residues. When the sum of the number of said amino acid residues is equal to or higher than the above lower limit, the function of transporting the polynucleotide 10 to the cell membrane of a target cell is more likely to be exhibited. When the sum of the number of said amino acid residues is equal to or lower than the above upper limit, the distance between the polynucleotide 10 and the receptor 40 of the target cell does not become unnecessarily long, so a decrease in the introduction efficiency of the polynucleotide 10 can be further suppressed. In addition, the cost for preparing the polynucleotide binding agent 20a can be further reduced.

[0027] [First Binding Site] The first binding site 21 of the present embodiment comprises a peptide having an amino acid sequence that binds to a receptor-binding protein 30. Since the first binding site 21 binds to the receptor 40 via the receptor-binding protein 30, it can be expressed that the first binding site 21 is capable of indirectly binding to the receptor 40.

[0028] There are no particular limitations on the number of amino acid residues in the first binding site 21, but it is preferably 2 to 30 residues, more preferably 5 to 25 residues, and even more preferably 7 to 20 residues. When the number of amino acid residues is above the lower limit, the function of transporting the polynucleotide 10 to the cell membrane of the target cell is more easily exerted. When the number of amino acid residues is below the upper limit, the distance between the polynucleotide 10 and the receptor 40 of the target cell does not become unnecessarily long, so the decrease in the efficiency of introducing the polynucleotide 10 can be further suppressed. In addition, the cost of preparing the polynucleotide binder 20a can be further reduced.

[0029] The amino acid sequences included in the first binding site 21 that binds to the receptor-binding protein 30 include the amino acid sequences shown in Tables 1A to 1B below as first binding site amino acid number 1 (SEQ ID NO: 1), first binding site amino acid number 5 (SEQ ID NO: 5), first binding site amino acid number 54 (SEQ ID NO: 63), and first binding site amino acid number 55 (SEQ ID NO: 64). Hereinafter, in this specification, the first binding site amino acid number may be simply referred to as the amino acid number. In Tables 1A to 1B below, the first binding site amino acid number indicates the number assigned to each first binding site, and the SEQ ID NO indicates the SEQ ID NO in the sequence listing. A "-" indicates that the first binding site can bind directly to the receptor on the target cell without the receptor-binding protein. The first binding site of this embodiment can bind to a receptor or a receptor-binding protein. For example, when a complex of the polynucleotide binder of this embodiment and an expression plasmid containing a target gene (e.g., the GFP gene) is brought into contact with a target cell, the complex is introduced into the target cell via the receptor or a receptor-binding protein. As a result, the target gene (e.g., GFP) is expressed in the target cells. In other words, whether or not the polynucleotide binder of this embodiment has a first binding site can be determined by whether or not the target gene (e.g., GFP) can be expressed in the target cells by the method described above.

[0030]

[0031]

[0032]

[0033] The first binding site 21 preferably has 80% or more sequence identity with the amino acid sequence indicated by amino acid numbers 1, 5, 54, or 55 (SEQ ID NOs: 1, 5, 63, or 64) in Tables 1A to 1B, more preferably 85% or more, and even more preferably 90% or more. The upper limit of the sequence identity is not particularly limited and may be 100%. If the sequence identity is equal to or greater than the lower limit, the first binding site 21 and the receptor-binding protein 30 will bind more easily.

[0034] In this specification, "sequence identity" of amino acids refers to the value (%) calculated by performing a global alignment using the Needleman-Wunsch method between a reference sequence and a comparison sequence, dividing the number of residues in which amino acids at corresponding positions match in the optimally aligned alignment by the alignment length (including gaps), and multiplying by 100. It is preferable to use BLOSUM62 as the substitution score matrix for the alignment and to perform gap generation penalties and gap extension penalties under conditions considered standard in the art. An example of a program for performing global alignment is EMBOSS needle. For example, conditions such as a gap generation penalty of 10 to 12 (preferably 11) and a gap extension penalty of 0.5 to 1 (preferably 1) are used.

[0035] [Second Binding Site] The second binding site 23 of this embodiment contains a peptide having an amino acid sequence that binds to the polynucleotide 10. Preferably, the second binding site 23 contains an amino acid sequence represented by at least one of the following sequences A and B.

[0036] The second binding site 23 of this embodiment includes an amino acid sequence that binds to the polynucleotide 10. The number of amino acid residues in the second binding site 23 is preferably, for example, 2 to 30 residues, more preferably 5 to 25 residues, and even more preferably 7 to 20 residues. Also, for example, it is preferably 2 to 25 residues, more preferably 3 to 22 residues, even more preferably 4 to 20 residues, particularly preferably 5 to 17 residues, and most preferably 5 to 15 residues. If the number of amino acid residues is above the lower limit, the function of transporting the polynucleotide 10 to the cell membrane of the target cell is more easily exerted. If the number of amino acid residues is below the upper limit, the distance between the polynucleotide 10 and the receptor 40 of the target cell does not become unnecessarily long, so the decrease in the efficiency of introducing the polynucleotide 10 can be further suppressed. Furthermore, after the polynucleotide 10 is introduced into the target cell, the second binding site 23 and the polynucleotide 10 become more easily dissociated, and the polynucleotide 10 is more easily transported into the nucleus of the target cell. Furthermore, the cost of preparing the polynucleotide binder 20a can be further reduced.

[0037] The second binding site 23 preferably contains a cationic amino acid. Here, a cationic amino acid is an amino acid having a positive charge, and examples include arginine (R: Arg), lysine (K: Lys), and histidine (H: His).

[0038] There are no particular limitations on the isoelectric point (pI) of the second binding site 23, but it is preferably 7.5 to 13, more preferably 7.8 to 12.8, and even more preferably 8 to 12.7. When the isoelectric point of the second binding site 23 is within the above preferred numerical range, the second binding site 23 becomes more likely to become positively charged and readily binds to the negatively charged polynucleotide 10 through electrostatic interaction. The isoelectric point can be determined based on the amino acid sequence using a database or calculation software. As an example, the isoelectric point can be calculated using online software provided by the Swiss Institute of Bioinformatics (SIB) (https: / / web.expasy.org / compute_pi / ).

[0039] (Sequence A) The number of amino acid residues included in the second binding site 23 represented by Sequence A is preferably 15 residues or less, preferably 2 to 15 residues, more preferably 4 to 15 residues, even more preferably 6 to 15 residues, and particularly preferably 8 to 14 residues. When the number of amino acid residues is above the lower limit, the function of transporting the polynucleotide 10 to the cell membrane of the target cell is more easily exhibited. When the number of amino acid residues is below the upper limit, the introduction efficiency of the polynucleotide 10 is further increased.

[0040] The number of cationic amino acid residues in the second binding site 23 represented by sequence A is preferably 4 residues or less, more preferably 1 to 4 residues, even more preferably 2 to 4 residues, and particularly preferably 4 residues. When the number of cationic amino acid residues is within the above numerical range, the binding strength between the second binding site 23 and the polynucleotide 10 becomes appropriate. As a result, after the polynucleotide 10 is introduced into the target cell, the second binding site 23 and the polynucleotide 10 become more easily dissociated, and the polynucleotide 10 is more easily transported into the nucleus of the target cell.

[0041] (Sequence B) The number of amino acid residues in the second binding site 23 represented by Sequence B is preferably 20 residues or less, more preferably 2 to 20 residues, more preferably 4 to 18 residues, more preferably 5 to 16 residues, more preferably 6 to 15 residues, and particularly preferably 8 to 14 residues. When the number of amino acid residues is above the lower limit, the function of transporting the polynucleotide 10 to the cell membrane of the target cell is more easily exerted. When the number of amino acid residues is below the upper limit, the introduction efficiency of the polynucleotide 10 is further increased.

[0042] The number of cationic amino acid residues in the second binding site 23 represented by sequence B is preferably 5 residues or less, more preferably 1 to 5 residues, even more preferably 2 to 5 residues, particularly preferably 3 to 5 residues, and most preferably 3 to 4 residues. When the number of cationic amino acid residues is within the above numerical range, the binding strength between the second binding site 23 and the polynucleotide 10 becomes appropriate. As a result, after the polynucleotide 10 is introduced into the target cell, the second binding site 23 and the polynucleotide 10 become more easily dissociated, and the polynucleotide 10 is more easily transported into the nucleus of the target cell.

[0043] The proportion of tryptophan (W:Trp) contained in the second binding site 23 represented by sequence B is preferably 20% or less, more preferably 0-20%, more preferably 3-20%, more preferably 5-20%, more preferably 8-20%, and even more preferably 10-20%. Also, for example, 0-20% is preferred, more preferably 0-17%, more preferably 0-15%, more preferably 0-12%, and even more preferably 0-10%. In this specification, the proportion of tryptophan contained in the second binding site refers to the ratio of the number of tryptophan residues to the number of amino acid residues contained in the second binding site. When the proportion of tryptophan is within the above range, the introduction efficiency of the polynucleotide 10 is increased.

[0044] The amino acid sequences included in the second binding site 23 that binds to the polynucleotide 10 are the amino acid sequences shown in Tables 2A to 2C below, with second binding site amino acid numbers 20 to 37 (SEQ ID NOs: 18 to 35), second binding site amino acid numbers 67 to 76 (SEQ ID NOs: 76 to 85), and second binding site amino acid numbers 115 to 118 (SEQ ID NOs: 128 to 131). Hereinafter in this specification, the second binding site amino acid number may be simply referred to as the amino acid number. In Tables 2A to 2C below, the second binding site amino acid number indicates the number assigned to each second binding site, and the SEQ ID NO number indicates the SEQ ID NO number in the sequence listing.

[0045]

[0046]

[0047]

[0048] The second binding site 23 preferably has 80% or more sequence identity with the amino acid sequence represented by amino acid numbers 20 to 37 (SEQ ID NOs: 18 to 35), amino acid numbers 67 to 76 (SEQ ID NOs: 76 to 85), or amino acid numbers 115 to 118 (SEQ ID NOs: 128 to 131) in Tables 2A to 2C, more preferably 85% or more, and even more preferably 90% or more. The upper limit of the sequence identity is not particularly limited and may be 100%. If the sequence identity is equal to or greater than the lower limit, the second binding site is more likely to become positively charged and more likely to bind to the negatively charged polynucleotide 10 through electrostatic interaction.

[0049] There are no particular limitations on the mode of binding between the second binding site 23 and the polynucleotide 10; for example, non-covalent bonding can be used. Examples of non-covalent bonding include binding by electrostatic interaction, sequence-specific binding, and binding by structural interaction. Of these binding modes, binding by electrostatic interaction is preferred. When the second binding site 23 and the polynucleotide 10 are bound by electrostatic interaction, it is thought that after the complex 1 containing the polynucleotide binder 20a and the polynucleotide 10 is introduced into the cytoplasm, the polynucleotide binder 20a and the polynucleotide 10 become more likely to dissociate, and the polynucleotide 10 is more likely to be transported into the nucleus of the target cell.

[0050] The second binding site 23 and the polynucleotide 10 may be reversibly bound or irreversibly bound, but reversibly bound is preferred. When the second binding site 23 and the polynucleotide 10 are reversibly bound, it is thought that after the complex 1 containing the polynucleotide binder 20a and the polynucleotide 10 is introduced into the cytoplasm, the polynucleotide binder 20a and the polynucleotide 10 become more easily dissociated, and the polynucleotide 10 is more easily transported into the nucleus of the target cell. The second binding site in this embodiment can be used to bind, for example, a plasmid (for example, the one used in the examples described later). The presence or absence of binding can be examined by a gel shift assay on an agarose gel.

[0051] [Polynucleotides] There are no particular limitations on the type of polynucleotide, but it is preferable that it be at least one selected from single-stranded or double-stranded DNA and RNA. Figures 1 and 2 illustrate the case where polynucleotide 10 is a circular plasmid, but the form of the polynucleotide may be circular or linear. Of these polynucleotides, circular DNA is preferred because it is less likely to be degraded by endonucleases in target cells.

[0052] The polynucleotide may consist of one, two, or three polynucleotides containing the target gene sequence, the adeno-associated virus gene, and the viral helper gene. These genes may be contained in different polynucleotides or in a single polynucleotide. Adeno-associated viruses are relatively safe because they do not exhibit pathogenicity, and they have various tissue-specific properties, making them suitable for use as gene therapy vectors.

[0053] The target gene sequence contained in the polynucleotide may include a base sequence encoding a desired gene to be contained within the adeno-associated virus produced. Examples of such polynucleotides include a polynucleotide in which, in the single-stranded DNA genome of a wild-type adeno-associated virus, the desired gene sequence is contained between the ITR sequences in the nucleic acid sequence, leaving the terminal inverted repeat (ITR) sequences at both ends and excluding the REP and CAP genes. The desired gene encoded by the desired gene sequence is expressed in cells infected with the produced adeno-associated virus.

[0054] Examples of desired gene sequences include nucleic acids that supply any foreign gene, such as polypeptides (enzymes, growth factors, cytokines, receptors, structural proteins, etc.), antisense RNA, ribozymes, decoys, RNA that causes RNA interference, etc. In addition, appropriate promoters, enhancers, terminators, and other transcriptional regulatory elements may be inserted into the nucleic acids to control the expression of the foreign gene. For example, the target gene sequence to be encapsulated in an adeno-associated virus may contain any foreign gene that is desired to be encapsulated in the adeno-associated virus between two ITR sequences, or it may contain any foreign gene that is desired to be encapsulated in the adeno-associated virus and one or more elements for controlling the expression of this foreign gene between two ITR sequences. Foreign membrane proteins expressed from foreign genes refer to proteins that are present or localized in the cell membrane after being introduced from outside, even though they are not originally present in the target cell. Examples of foreign membrane proteins include virus-derived membrane proteins and artificially designed membrane proteins. An example of an artificially designed membrane protein is a chimeric antigen receptor. Chimeric antigen receptors are exogenous membrane proteins expressed by gene transfer after artificially fusing multiple originating elements (e.g., antigen-binding domain, transmembrane region, and intracellular signaling region) that are not naturally present in the cell. The polynucleotide containing the target gene is preferably introduced into cells as a nucleic acid construct in the form of a plasmid equipped with an expression cassette. Plasmids can be constructed using, for example, commercially available rAAV vector plasmids such as pAAV-CMV Vector (manufactured by Takara Bio).

[0055] Adeno-associated virus genes are the genes used to prepare adeno-associated viruses. These genes include the REP gene and the CAP gene. These genes encode structural proteins (also called capsid proteins) and non-structural proteins necessary for the replication and packaging of adeno-associated viruses.

[0056] The adeno-associated virus gene preferably contains transcriptional regulatory sequences such as promoters and enhancers to enable gene expression within the introduced cell. The polynucleotide containing the adeno-associated virus gene is preferably introduced into cells as a nucleic acid construct in the form of a plasmid equipped with an expression cassette. As a plasmid, for example, the commercially available pRC vector plasmid pRC2-mi342 Vector (manufactured by Takara Bio Inc.) can be used.

[0057] A viral helper gene is a gene that codes for a helper protein derived from adenovirus. Viral helper genes express and supply essential auxiliary elements for the formation of viral particles produced. Examples of viral helper genes include E1A, E1B, E2A, E4, and VA genes. Of these, the gene not present in the cell into which the polynucleotide is introduced is introduced as the viral helper gene. For example, if the cell is a HEK293 cell, since HEK293 cells transform between E1A and E1B, the E2A, E4, and VA genes are introduced into the HEK293 cell as viral helper genes. It is preferable to introduce the polynucleotide containing the viral helper gene into the cell as a nucleic acid construct in the form of a plasmid equipped with an expression cassette. As a plasmid, for example, a commercially available pHelper vector plasmid (manufactured by Takara Bio Inc.) can be used.

[0058] There are no particular limitations on the number of base pairs in a polynucleotide when it is a double-stranded polynucleotide forming a complementary chain. The number of base pairs is preferably 1 to 22 kbp, more preferably 1 to 20 kbp, even more preferably 4 to 14 kbp, particularly preferably 5 to 14 kbp, and most preferably 6 to 14 kbp. If the number of base pairs is above the lower limit, the number of selectable gene sequences increases, allowing for selection from a wider range of options for proteins expressed in target cells and cell mutant lines to be produced. If the number of base pairs is below the upper limit, the adjustment cost is lower and the gene transfer efficiency is higher.

[0059] There are no particular limitations on the number of bases in a polynucleotide when it is a single-stranded polynucleotide that does not form a complementary strand. The number of bases is preferably 10 to 3000 nts, more preferably 100 to 3000 nts, and even more preferably 1000 to 3000 nts. If the number of bases is above the lower limit, the number of selectable gene sequences increases, allowing for selection from a wider range of options for proteins expressed in target cells and cell mutant lines to be produced. If the number of bases is below the upper limit, the adjustment cost is lower and the gene transfer efficiency is higher.

[0060] The molecular weight of the polynucleotide is preferably 340 kDa or more, more preferably 450 to 13,000 kDa, even more preferably 3,800 kDa or more, and particularly preferably 6,300 kDa or more. According to the polynucleotide binder of this embodiment, molecular transport efficiency can be increased not only when transporting polynucleotides with small molecular weights, but also when transporting polynucleotides with molecular weights within the above numerical range to target cells.

[0061] [Target Cells] There are no particular limitations on target cells; for example, eukaryotic cells are used, and animal cells are preferred. Examples of animal cells include vertebrate cells and invertebrate cells, with vertebrate cells being preferred. Mammalian cells are preferred among vertebrate cells. Insect cells are preferred among invertebrate cells.

[0062] Examples of mammalian cells include cells derived from mammals, including humans, and cells derived from non-human mammals. Examples of non-human mammals include mice, rats, Chinese hamsters, chimpanzees, gorillas, orangutans, monkeys, cows, pigs, horses, dogs, and cats.

[0063] Furthermore, the human-derived cells mentioned above may be cells intended for reintroduction into a human organism. Also, the human-derived cells mentioned above may be cells cultured from cells collected from a human organism. In addition, the non-human animal cells mentioned above may be cells present within an organism, cells present within a tissue, cells collected from an organism or tissue, or cells cultured from cells collected from an organism or tissue.

[0064] Specific examples of target cells include HEK293 cells; mesenchymal stem cells; adipose-derived stem cells; iPS cells; blood-derived cells such as macrophages, T cells (CD3-positive cells), B cells, NK cells, monocytes, dendritic cells, plasma cells, and lymphocytes; adipose-derived stem progenitor cells; Jurkat cells; HuT-78 cells; human peripheral blood stem cells (PBMCs); human preadipocytes (hPADs); human fibroblasts, and hematopoietic stem cells. It is preferable to use at least one cell selected from this group as the target cell.

[0065] The target cells preferably overexpress at least one gene selected from the group consisting of caveolin 1 (Cav1), caveolin 2 (Cav2), and cabin 1 (Cavin1), and more preferably overexpress at least the Cav1 gene. When the target cells overexpress the Cav1 gene 5 to 50 times compared to normal mRNA levels, the efficiency of introducing polynucleotides into the cytoplasm using caveolae pathway endocytosis can be further improved.

[0066] Cav1 is the main scaffolding protein of cell membrane caveolae in target cells and is sometimes referred to as caveolin 1. The amino acid sequence of Cav1 that can be used in this embodiment is preferably a sequence that has at least 80%, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more sequence identity with SEQ ID NO: 49, or contains a fragment in which a part of it has been deleted or removed. The Cav1 that can be used in this embodiment preferably functions as a component of cell membrane caveolae in target cells.

[0067] Cav2, like Cav1, is a major scaffolding protein for cell membrane caveolae in target cells and is known to interact with Cav1; it is sometimes referred to as caveolin 2. The amino acid sequence of Cav2 that can be used in this embodiment is preferably a sequence that has at least 80%, preferably 90% or more, more preferably 95% or more, sequence identity with SEQ ID NO: 50, or preferably includes a fragment in which a part of it has been deleted or removed. The Cav2 that can be used in this embodiment preferably functions as a component of cell membrane caveolae in target cells.

[0068] Cavin1 is a protein that plays a crucial role in the formation and function of cell membrane caveolae in target cells and is said to interact with caveolin. It is also sometimes referred to as PTRF (Polymerase I and Transcript Release Factor). The amino acid sequence of Cavin1 that can be used in this embodiment is preferably a sequence that has at least 80%, preferably 90% or more, more preferably 95% or more, sequence identity with SEQ ID NO: 51, or contains a fragment in which a part of it has been deleted or removed. The Cavin1 that can be used in this embodiment preferably has the function of interacting with cell membrane caveolin in target cells.

[0069] The target cells may artificially overexpress at least one gene selected from the group consisting of Cav1, Cav2, and Cavin1. In other words, the target cells may have a polynucleotide containing at least one protein encoding gene selected from the group consisting of Cav1, Cav2, and Cavin1 pre-introduced into them.

[0070] In this specification, gene overexpression means that the target protein encoded by the gene is expressed in significantly higher amounts compared to wild-type cells. Whether the target protein is significantly overexpressed can be confirmed by comparing the amount of messenger RNA in wild-type cells and gene-overexpressing cells using real-time PCR.

[0071] When molecules are introduced from the cell membrane surface into the cytoplasm via endocytosis, they are introduced while enclosed in vesicles called endosomes. Endocytosis involves two pathways: the clathrin-mediated pathway, in which molecules are introduced via clathrin coating, and the caveolae-mediated pathway, in which molecules are introduced via caveolae.

[0072] Caveolas are primarily formed when Cav1 and Cav2 bind to lipid rafts to form oligomers consisting of 14 to 16 molecules. Cavin1 is a major regulator of caveolae formation. When target cells overexpress at least one protein selected from the group of Cav1, Cav2, and Cavin1, endocytosis via the caveolae pathway is activated, and the efficiency of molecular delivery to target cells is further improved.

[0073] In target cells, specifically Jurkat cells and T cells, which are CD3-positive cells with low molecular transduction efficiency, overexpression of at least one protein selected from the group of Cav1, Cav2, and Cavin1 further activates endocytosis via the caveolae pathway, thereby improving molecular transduction efficiency.

[0074] [Receptor-binding protein] The receptor-binding protein 30 is not particularly limited as long as it is a protein that can bind to the receptor 40 of the target cell, and examples include insulin, transferrin, albumin, endothelin, and leptin.

[0075] In Table 1A above, the first binding site 21 indicated by amino acid number 1 (SEQ ID NO: 1) has an amino acid sequence that can bind to insulin. In Table 1A above, the first binding site 21 indicated by amino acid number 5 (SEQ ID NO: 5) has an amino acid sequence that can bind to transferrin. In Table 1B above, the first binding sites 21 indicated by amino acid number 54 (SEQ ID NO: 63) and amino acid number 55 (SEQ ID NO: 64) have amino acid sequences that can bind to albumin.

[0076] [Receptor] The receptor 40 expressed on the cell membrane surface of the target cell can be any receptor to which the receptor-binding protein 30 can bind, and is not particularly limited. Examples of receptors 40 to which the receptor-binding protein 30 can bind include insulin receptor, transferrin receptor, integrin, albumin receptor, endothelin receptor, leptin receptor, LDL receptor, cubilin, megalin, LRP2 (Low Density Lipoprotein Receptor-Related Protein 2 Receptor), EGF receptor (Epidermal Growth Factor Receptor), and cluster of difference (CD). Examples of CD receptors include CD3, CD4, CD7, CD8, CD28, and CD25. In this specification, megalin and LRP2 may be collectively referred to as "LRP2 / megalin". The receptor 40 may consist of one of these receptors or two or more.

[0077] The polynucleotide binder of this embodiment allows for the transport of polynucleotides to the cell membrane of target cells with simple operation and high efficiency.

[0078] (Second Embodiment) The polynucleotide binder in the second embodiment of this embodiment will be described with reference to Figure 2(A). Figure 2(A) is a schematic diagram of the complex containing the polynucleotide binder in the second embodiment. The complex 2 in this embodiment contains polynucleotide 10 and polynucleotide binder 20b. The polynucleotide binder 20b includes a first binding site 22 and a second binding site 24, the first binding site 22 and the second binding site 24 are directly bound. The first binding site 22 is directly bound to the receptor 40, and the second binding site 24 is bound to the polynucleotide 10. The first binding site 22 contained in the complex 2 directly binds to the receptor 40, thereby enabling the transport of polynucleotide 10 to the cell membrane of the target cell.

[0079] The descriptions and preferred specific examples of polynucleotides, target cells, and target cell receptors in this embodiment are as described in the polynucleotide binder of the first embodiment, so a detailed explanation is omitted.

[0080] The first binding site 22 contains a peptide having an amino acid sequence that directly binds to the receptor 40 of the target cell. There are no particular limitations on the number of amino acid residues in the first binding site 22, but it is preferably 2 to 30 residues, more preferably 5 to 25 residues, and even more preferably 7 to 20 residues. If the number of amino acid residues is above the lower limit, the function of transporting the polynucleotide 10 to the cell membrane of the target cell is more easily exerted. If the number of amino acid residues is below the upper limit, the distance between the polynucleotide 10 and the receptor 40 of the target cell does not become unnecessarily long, so the decrease in the efficiency of introducing the polynucleotide 10 can be further suppressed. In addition, the cost of preparing the polynucleotide binder 20b can be further reduced.

[0081] Examples of amino acid sequences that can directly bind to the receptor 40 and include the first binding site 22 are the amino acid sequences shown in Table 1A with amino acid numbers 2-4 (SEQ ID NOs: 2-4) and 6-19 (SEQ ID NOs: 6-17). Also, examples of amino acid sequences shown in Table 1B are the amino acid sequences shown with amino acid numbers 51-53 (SEQ ID NOs: 60-62) and 56-66 (SEQ ID NOs: 65-75). Furthermore, examples of amino acid sequences shown in Table 1C are the amino acid sequences shown with amino acid numbers 109-114 (SEQ ID NOs: 122-127).

[0082] The first binding site 22 preferably has 80% or more sequence identity with the amino acid sequence represented by any of the amino acid numbers 2-4 (SEQ ID NOs: 2-4), 6-19 (SEQ ID NOs: 6-17), 51-53 (SEQ ID NOs: 60-62), 56-66 (SEQ ID NOs: 65-75), or 109-114 (SEQ ID NOs: 122-127) in Tables 1A to 1C above, more preferably 85% or more, and even more preferably 90% or more. The upper limit of the sequence identity is not particularly limited and may be 100%. If the sequence identity is equal to or greater than the lower limit, the first binding site 22 and the receptor 40 will bind more easily.

[0083] In Tables 1A to 1C above, the first binding sites 22 indicated by amino acid numbers 2 to 4 (SEQ ID NOs: 2 to 4), amino acid numbers 51 to 52 (SEQ ID NOs: 60 to 61), and amino acid number 109 (SEQ ID NO: 122) have amino acid sequences that can directly bind to the insulin receptor. In Tables 1A to 1B above, the first binding sites 22 indicated by amino acid number 6 (SEQ ID NO: 6) and amino acid number 53 (SEQ ID NO: 62) have amino acid sequences that can directly bind to the transferrin receptor. In Tables 1A to 1B above, the first binding sites 22 indicated by amino acid numbers 7 to 15 (SEQ ID NOs: 7 to 13) have amino acid sequences that can directly bind to integrins. In Tables 1A to 1B above, the first binding sites 22 indicated by amino acid numbers 16 to 17 (SEQ ID NOs: 14 to 15) have amino acid sequences that can directly bind to the endothelin receptor. In Tables 1A and 1B above, the first binding site 22 indicated by amino acid numbers 18-19 (SEQ ID NOs. 16-17) and amino acid number 57 (SEQ ID NO. 66) has an amino acid sequence that can directly bind to the leptin receptor. In Table 1B above, the first binding site 22 indicated by amino acid numbers 54-56 (SEQ ID NOs. 63-65) has an amino acid sequence that can directly bind to the albumin receptor. In Table 1B above, the first binding site 22 indicated by amino acid numbers 58-59 (SEQ ID NOs. 67-68) has an amino acid sequence that can directly bind to the LDL receptor. In Table 1B above, the first binding site 22 indicated by amino acid number 60 (SEQ ID NO. 69) has an amino acid sequence that can directly bind to cubilin. In Tables 1B and 1C above, the first binding site 22 indicated by amino acid number 61 (SEQ ID NO: 70) and amino acid numbers 110-112 (SEQ ID NOs: 123-125) has an amino acid sequence that can directly bind to megalin and LRP2. In Table 1B above, the first binding site 22 indicated by amino acid numbers 62-63 (SEQ ID NOs: 71-72) has an amino acid sequence that can directly bind to CD4. In Table 1B above, the first binding site 22 indicated by amino acid numbers 64-65 (SEQ ID NOs: 73-74) has an amino acid sequence that can directly bind to CD28. In Table 1B above, the first binding site 22 indicated by amino acid number 66 (SEQ ID NO: 75) has an amino acid sequence that can directly bind to CD25.In Table 1C above, the first binding site 22, indicated by amino acid numbers 113-114 (sequence numbers 126-127), has an amino acid sequence that can directly bind to EGFR.

[0084] The second binding site 24 of this embodiment contains a peptide having an amino acid sequence that binds to the polynucleotide 10. Preferably, the second binding site 24 contains an amino acid sequence represented by at least one of sequence A and sequence B. The number of amino acid residues in the second binding site 24 is preferably 2 to 30 residues, more preferably 5 to 25 residues, and even more preferably 7 to 20 residues. Furthermore, the number of amino acid residues in the second binding site 24 is preferably 2 to 25 residues, more preferably 3 to 22 residues, even more preferably 4 to 20 residues, particularly preferably 5 to 17 residues, and most preferably 5 to 15 residues. If the number of amino acid residues is above the lower limit, the function of transporting the polynucleotide 10 to the cell membrane of the target cell is more easily exerted. If the number of amino acid residues is below the upper limit, the distance between the polynucleotide 10 and the receptor 40 of the target cell does not become unnecessarily long, so the decrease in the efficiency of introducing the polynucleotide 10 can be further suppressed. Furthermore, after the polynucleotide 10 is introduced into the target cell, the second binding site 23 and the polynucleotide 10 become more readily dissociable, facilitating the transport of the polynucleotide 10 into the nucleus of the target cell. In addition, the cost of preparing the polynucleotide binder 20b can be further reduced.

[0085] Examples of amino acid sequences that can directly bind to the first binding site 22 and bind to the polynucleotide 10 include the amino acid sequences shown in Tables 2A to 2C above, specifically amino acid numbers 20 to 37 (SEQ ID NOs: 18 to 35), amino acid numbers 67 to 76 (SEQ ID NOs: 76 to 85), and amino acid numbers 115 to 118 (SEQ ID NOs: 128 to 131).

[0086] The second binding site 24 preferably has 80% or more sequence identity with the amino acid sequence represented by amino acid numbers 20 to 37 (SEQ ID NOs: 18 to 35), amino acid numbers 67 to 76 (SEQ ID NOs: 76 to 85), or amino acid numbers 115 to 118 (SEQ ID NOs: 128 to 131) in Tables 2A to 2C, more preferably 85% or more, and even more preferably 90% or more. The upper limit of the sequence identity is not particularly limited and may be 100%. If the sequence identity is equal to or greater than the lower limit, the second binding site is more likely to become positively charged and more likely to bind to the negatively charged polynucleotide 10 through electrostatic interaction.

[0087] The polynucleotide binder of this embodiment allows for the transport of polynucleotides to the cell membrane of target cells with simple operation and high efficiency. Furthermore, since the first binding site contains a peptide having an amino acid sequence that directly binds to the receptor of the target cell, polynucleotides can be transported to the cell membrane of target cells without the need to add receptor-binding proteins such as insulin.

[0088] (Other Embodiments) Embodiments other than those described above are shown below. In the complex, the polynucleotide transporter and the polynucleotide are not limited to being bound in a 1:1 ratio. For example, as shown in Figures 1(B) and 2(B), a complex may be formed in which multiple polynucleotide binders are bound to one polynucleotide molecule and aggregated. In the complex shown in Figure 1(B), at least one of the receptor-binding proteins included in the complex binds to the receptor, thereby enabling the transport of polynucleotides to the cell membrane of the target cell. In the complex shown in Figure 2(B), at least one of the first binding sites included in the complex binds to the receptor, thereby enabling the transport of polynucleotides to the cell membrane of the target cell.

[0089] The polynucleotide binder may or may not contain amino acid sequences other than the first and second binding sites (hereinafter sometimes referred to as "non-binding sites"). There are no particular limitations on the number of amino acid residues included in the non-binding sites, but it is preferably 1 to 50 residues, more preferably 5 to 35 residues, and even more preferably 8 to 20 residues.

[0090] The amino acid sequence of the non-binding site is preferably glycine-rich and serine-rich, at least one of the following: glycine-rich and serine-rich. Here, glycine-rich means that the ratio of glycine residues to the total number of amino acid residues in the non-binding site is 30% or more. Serine-rich means that the ratio of glycine residues to the total number of amino acid residues in the non-binding site is 30% or more.

[0091] Specific examples of non-binding sites include the amino acid sequences of the non-binding sites contained in the polynucleotide binders shown in Tables 3A to 3C below, with binder amino acid numbers 39, 41, 43-45, 47-50 (SEQ ID NOs: 37, 39, 41-43, 45-48).

[0092] If the polynucleotide binder includes a non-binding site, the non-binding site may be located at the C-terminus or N-terminus of the amino acid sequence of the first binding site. The non-binding site may also be located at the C-terminus or N-terminus of the amino acid sequence of the second binding site.

[0093] In the first and second embodiments of the polynucleotide binder, the first binding site and the second binding site are directly bound, but the present invention is not limited thereto. The first binding site and the second binding site of the polynucleotide binder may be indirectly bound via a non-binding site. Here, the indirect binding of the first binding site and the second binding site via a non-binding site means that the first binding site, the non-binding site, and the second binding site may be linked in this order to form a single peptide chain. Alternatively, the first binding site and the second binding site may be bound via a linker portion described later.

[0094] The molecular structure of the polynucleotide binder may have one first binding site and two or more second binding sites. The binding order of the first and second binding sites in the molecular structure of the binder can be arbitrarily set. In particular, the order corresponding to the polynucleotide binder shown by binding amino acid number 50 (SEQ ID NO: 48) in Table 3C below is preferred because it provides higher molecular transport efficiency to the cell membrane of the target cell. That is, a polynucleotide binder bound in the order of first binding site, non-binding site, second binding site, non-binding site, and first binding site from the N-terminus is preferred.

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] In Tables 3A to 3F above, the binding amino acid number indicates the number assigned to each polynucleotide binding agent, and the sequence number indicates the sequence number in the sequence listing. The number listed under Amino Acid Number (N) indicates the amino acid number of the amino acid sequence located at the N-terminus of the polynucleotide binding agent. The number listed under Amino Acid Number (C) indicates the amino acid number of the amino acid sequence located at the C-terminus of the polynucleotide binding agent.

[0102] For example, IBP-NBP1, indicated by amino acid number 38 (SEQ ID NO: 36) in Table 3A above, contains a first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above, and a second binding site indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A above, in that order from the N-terminus. Furthermore, the C-terminus of the first binding site located on the N-terminus side of the polynucleotide binder is directly bound to the N-terminus of the second binding site located on the C-terminus side. IBP-NBP1 can indirectly bind to the insulin receptor via insulin, which is a receptor-binding protein.

[0103] [Linker portion] The linker portion is a structure that connects the first binding site and the second binding site, and can be formed by the reaction of a linker molecule between the first and second binding sites. The linker molecule is not particularly limited, and examples include a molecule in which reactive groups are introduced at both ends of a polyalkylene glycol. The reactive groups are not particularly limited as long as they react with the C-terminus or N-terminus of each peptide to form a covalent bond. The linker portion thus formed contains repeating units derived from the alkylene glycol unit.

[0104] Linker molecules containing repeating units derived from alkylene glycol units are sometimes called polyalkylene glycol linkers. Examples of alkylene glycol units include ethylene glycol units.

[0105] The number of repeating units (alkylene glycol units) derived from alkylene glycol contained in the linker portion is preferably 2 to 10, and more preferably 2 to 6.

[0106] The linker substance may be further modified by the addition of amino acids and other compounds.

[0107] According to the polynucleotide binder of this embodiment, the inclusion of a linker portion creates a space between the polynucleotide and the first binding site, making it less likely for the polynucleotide-containing complex to be hindered by the three-dimensional structure of the membrane protein when approaching the receptor on the target cell.

[0108] <Method for preparing polynucleotide binders> (Second aspect) As a method for preparing the polynucleotide binder according to the first aspect, for example, one method is to synthesize it as a single peptide chain in which the first binding site and the second binding site are linked in that order. Alternatively, one method is to synthesize it as a single peptide chain including a first binding site, a second binding site and an unbinding site. The method for synthesizing a single peptide chain is not particularly limited, and for example, known solid-phase synthesis methods can be applied. Specifically, one example is to synthesize a single polypeptide chain in which the C-terminus of the main chain of the second binding site is peptide-bonded to the N-terminus of the main chain of the main chain of the first binding site.

[0109] Another preparation method involves synthesizing the first and second binding sites separately and then chemically linking them later. In this case, a peptide bond may be directly formed between the first and second binding sites, or a linker molecule may be reacted to link them via the linker. The order in which the first and second binding sites are linked is not particularly limited; the second binding site may be attached to the N-terminus of the first binding site, or the first binding site may be attached to the N-terminus of the second binding site.

[0110] When using a polyalkylene glycol linker as the linker molecule, there are no particular limitations on the number of repeating units (alkylene glycol units) derived from alkylene glycol, but it is preferably 2 to 10, and more preferably 2 to 6.

[0111] The polynucleotide binder preferably has 80% or more sequence identity with respect to the amino acid sequences indicated by binder amino acid numbers 38-50 (SEQ ID NOs. 36-48), binder amino acid numbers 77-108 (SEQ ID NOs. 86-117), or binder amino acid numbers 119-127 (SEQ ID NOs. 132-140) in Tables 3A to 3F above, more preferably 85% or more, and even more preferably 90% or more. The upper limit of the sequence identity is not particularly limited and may be 100%. If the sequence identity is above the lower limit, the first binding site and the receptor-binding protein or receptor will bind more easily. If the sequence identity is above the lower limit, the second binding site will be more likely to become positively charged and will bind more easily to negatively charged polynucleotides through electrostatic interactions.

[0112] <Polynucleotide Transport Composition> (Third Embodiment) The polynucleotide transport composition in this embodiment comprises the polynucleotide binder of the first embodiment and a polynucleotide or a target cell. In other words, the polynucleotide transport composition may comprise the polynucleotide binder and polynucleotide of the first embodiment, or it may comprise the polynucleotide binder and target cell of the first embodiment. Specific examples and preferred embodiments of the polynucleotide binder, polynucleotide and target cell are as described in the description of the first embodiment, so a detailed explanation is omitted.

[0113] The polynucleotide binder may further contain a receptor-binding protein. In other words, the polynucleotide transport composition may contain the polynucleotide binder of the first embodiment, a polynucleotide, and a receptor-binding protein. Alternatively, the polynucleotide transport composition may contain the polynucleotide binder of the first embodiment, a target cell, and a receptor-binding protein. Specific examples and preferred embodiments of the receptor-binding protein are as described in the description of the first embodiment, so a detailed explanation is omitted.

[0114] The polynucleotide transport composition preferably does not contain cationic lipids. Here, cationic lipids are compounds having at least one fatty acid and a hydrocarbon chain, and are hydrophobic compounds. Note that the cationic lipids are lipids other than those that constitute the cell membrane of the target cell. The polynucleotide transport composition of this embodiment preferably does not contain cationic lipids, or the content of cationic lipids is 0.1% by mass or less of the total mass of the polynucleotide transport composition. Furthermore, the polynucleotide transport composition preferably does not contain cationic polyethyleneimine (PEI) or its derivatives, which are conventionally used as cell transfection reagents. Since PEI or its derivatives are known to be toxic to cells, the polynucleotide transport composition of this embodiment preferably does not contain PEI or its derivatives, or the content of PEI or its derivatives is 0.1% by mass or less of the total mass of the polynucleotide transport composition. Note that the molecular structure of linear PEI is CH ３ - (NH-CH ２ -CH ２ ) ｎ It is represented as -OH. Examples of PEI derivatives include linear PEI, deacetylated PEI, and chemically modified PEI. Examples of chemically modified PEI include PEI modified with polyethylene glycol (PEG).

[0115] The polynucleotide transport composition may be a solution. There are no particular limitations on the solvent when the polynucleotide transport composition is a solution; examples include purified water, buffer solutions, and cell culture media.

[0116] There are no particular limitations on the mixing ratio of the polynucleotide binder to the polynucleotide. For example, the mass ratio represented by polynucleotide binder:polynucleotide can be appropriately set to preferably 1:0.1 to 1:50, more preferably 1:0.5 to 1:25, and even more preferably 1:0.8 to 1:5.

[0117] There are no particular limitations on the mixing ratio of the polynucleotide binder to the target cells. For example, 1 × 10 ５The total mass of the polynucleotide binder relative to the cell can be appropriately set to preferably 0.1 to 10 μg, more preferably 0.15 to 5 μg, and even more preferably 0.2 to 1 μg.

[0118] There are no particular limitations on the mixing ratio of the polynucleotide binder to the receptor-binding protein. For example, the mass ratio of the polynucleotide binder to the receptor-binding protein can be appropriately set to preferably 1:0.1 to 1:50, more preferably 1:0.5 to 1:25, and even more preferably 1:0.8 to 1:10.

[0119] <Method for introducing polynucleotides> (Fourth embodiment) The method for introducing polynucleotides in this embodiment includes the steps of: (A) bringing the polynucleotide binder of the first embodiment into contact with the polynucleotide to form a complex containing the polynucleotide binder and the polynucleotide; and (B) bringing the complex into contact with the cell membrane of the target cell to introduce at least the polynucleotide from the cell membrane into the cytoplasm, wherein step (B) is performed after step (A).

[0120] Specific examples and preferred embodiments of the polynucleotide binder, first binding site, second binding site, polynucleotide, target cell, receptor-binding protein, and receptor in this embodiment are as described in the description of the first embodiment, so a detailed explanation is omitted.

[0121] In step (A), a polynucleotide binder and a polynucleotide are brought into contact to form a complex containing the polynucleotide binder and the polynucleotide. Methods for bringing the polynucleotide binder and the polynucleotide into contact include, for example, mixing a solution containing the polynucleotide binder with a solution containing the polynucleotide. Alternatively, a method of mixing the polynucleotide transport composition of the third embodiment with a solution containing the polynucleotide is also possible.

[0122] The solution containing the polynucleotide binder and the solvent used to prepare the solution containing the polynucleotide are as described in the description of the third embodiment, so a detailed explanation is omitted.

[0123] There are no particular limitations on the mixing ratio when mixing a solution containing a polynucleotide binder with a solution containing a polynucleotide. For example, the mass ratio of polynucleotide binder to polynucleotide can be appropriately set to preferably 1:0.1 to 1:50, more preferably 1:0.5 to 1:25, and even more preferably 1:0.8 to 1:5. If the mixing ratio is above the lower limit, the introduction efficiency will be high. If the mixing ratio is below the upper limit, the cost will be low.

[0124] A complex is formed when a polynucleotide binds to the second binding site of a polynucleotide binder. The mode of binding between the polynucleotide and the second binding site is as described in the description of the first embodiment, so a detailed explanation is omitted.

[0125] There are no particular limitations on the mixing ratio of the polynucleotide binder and the polynucleotide complex to the receptor-binding protein. For example, the mass ratio of the polynucleotide binder to the receptor-binding protein contained in the complex can be appropriately set to preferably 1:0.1 to 1:50, more preferably 1:0.5 to 1:25, and even more preferably 1:0.8 to 1:10.

[0126] Step (B) is preferably carried out in the presence of a receptor-binding protein. Here, carrying out the step in the presence of a receptor-binding protein means, for example, that step (B) may be carried out after a complex containing a polynucleotide binder, a polynucleotide, and a receptor-binding protein is formed in step (A). Alternatively, in step (B), the receptor-binding protein may be added when the complex containing the polynucleotide binder and polynucleotide is brought into contact with the target cells.

[0127] Step (B) is preferably carried out in the presence of at least one of the receptor-binding proteins insulin, transferrin, and albumin.

[0128] Step (B) is preferably carried out in the presence of at least one of the receptor and the cell adhesion molecule. The receptor is a receptor expressed on the cell membrane surface of the target cell. The cell adhesion molecule is a cell adhesion molecule expressed on the cell membrane surface or inside the cell of the target cell. The receptor is as described in the description of the first embodiment, so a detailed explanation is omitted.

[0129] Cell adhesion molecules are a general term for molecules that adhere cells to each other. Examples of cell adhesion molecules include integrins and cadherins. Polynucleotides can also be transported to the target cell surface by the binding of receptor-binding proteins or primary binding sites contained in the complex to the cell adhesion molecules of the target cell.

[0130] In step (B), the target cells are preferably cells that express endocytosis-related genes. Here, endocytosis-related genes are a general term for genes involved in endocytosis (phagocytosis) in cells. When target cells express endocytosis-related genes, the complex comes into contact with the target cells, and polynucleotides are spontaneously introduced into the cells from the target cell membrane through phagocytosis.

[0131] Examples of endocytosis-related genes include genes that form clathrin, which is involved in endocytosis via the clathrin-mediated pathway, and genes that form caveolae, which are involved in endocytosis via the caveolae-mediated pathway (hereinafter sometimes referred to as caveolae-related genes).

[0132] In clathrin-mediated endocytosis, endosomes are formed by multiple clathrins, and molecules encapsulated within these endosomes are introduced into the cytoplasm. In step (B), if clathrin-mediated endocytosis occurs, at least polynucleotides are encapsulated within the endosomes formed by the clathrins, and these polynucleotides are introduced into the cytoplasm.

[0133] In caveolae-mediated endocytosis, caveolae, which are endosomes, are formed, and molecules encapsulated within the caveolae are introduced into the cytoplasm. The formation of caveolae is as described in the description of the first embodiment, so a detailed explanation will be omitted. In step (B), if caveolae-mediated endocytosis occurs, at least polynucleotides are encapsulated within the caveolae, and the polynucleotides are introduced into the cytoplasm.

[0134] In step (B), if endocytosis occurs via the caveolae-mediated pathway, it is preferable that the target cells express caveolae-related genes. Examples of caveolae-related genes include caveolin (Cav) and cavin. Examples of caveolin include Cav1, Cav2, and Cav3. Examples of cavin include Cavin1, Cavin2, Cavin3, and Cavin4.

[0135] In step (B), when endocytosis occurs via the caveolae-mediated pathway, the target cells preferably overexpress at least one gene selected from the group consisting of Cav1, Cav2, and Cavin1 as caveolae-related genes, and more preferably overexpress at least the Cav1 gene. Since Cav1, Cav2, and Cavin1 are as described in the description of the first embodiment, a detailed explanation thereof will be omitted.

[0136] The contact time of the complex with the target cells in step (B) should be the time required for the complex to be taken up by the target cells, for example, 1 minute to 1 hour is a guideline.

[0137] According to the polynucleotide transport method of this embodiment, polynucleotides can be introduced into target cells with high introduction efficiency through simple operations.

[0138] <Virus Production Method> (Fifth Embodiment) The virus production method in this embodiment includes a step of introducing at least the polynucleotide from the cell membrane into the cytoplasm by contacting the polynucleotide with a target cell using a polynucleotide binder or a polynucleotide transport composition, thereby producing a virus in the target cell. An example of a method for contacting the polynucleotide with the target cell is the method described in the fourth embodiment. Furthermore, specific examples and preferred embodiments of the polynucleotide binder, first binding site, second binding site, target cell, receptor binding protein, and receptor in this embodiment are as described in the description of the first embodiment, so a detailed explanation is omitted. For example, the virus production method in this embodiment includes a step (A) of contacting the polynucleotide with the polynucleotide binder of the first embodiment to form a complex containing the polynucleotide binder and the polynucleotide, and a step (B) of introducing at least the polynucleotide from the cell membrane into the cytoplasm by contacting the complex with the cell membrane of the target cell, wherein step (B) is performed after step (A).

[0139] The polynucleotide in this embodiment includes a base sequence encoding a viral gene. Preferably, the polynucleotide includes a base sequence encoding an adeno-associated virus gene. The polynucleotide may not only encode a viral gene, but also, for example, a gene for expressing an arbitrary protein or a viral helper gene. An example of the arbitrary protein is the target gene in the first embodiment. These genes may be contained in different polynucleotides or in a single polynucleotide. The amount of virus produced in the target cell and the viral titer are measured by the method described in the examples below.

[0140] According to the virus production method of this embodiment, polynucleotides can be introduced into target cells with high introduction efficiency through simple operations, and viruses can be produced within the target cells.

[0141] <Method for Cell Production> (Sixth Embodiment) The cell production method in this embodiment includes a step of introducing at least polynucleotides from the cell membrane into the cytoplasm by contacting the polynucleotides with target cells using a polynucleotide binder or a polynucleotide transport composition, thereby causing the target cells to express foreign membrane proteins or overexpress intrinsic membrane proteins on their cell membranes. This makes it possible to produce cells in which foreign membrane proteins are expressed or intrinsic membrane proteins are overexpressed on their cell membranes. An example of a method for introducing polynucleotides into target cells using a polynucleotide binder or a polynucleotide transport composition is the method described in the fourth embodiment. Furthermore, specific examples and preferred embodiments of the polynucleotide binder, first binding site, second binding site, target cells, receptor-binding protein, and receptor in this embodiment are as described in the description of the first embodiment, so a detailed explanation is omitted. For example, the cell production method in this embodiment includes the steps of: (A) contacting a polynucleotide with a polynucleotide binder of the first embodiment or a polynucleotide transport composition of the third embodiment to form a complex containing the polynucleotide binder and the polynucleotide; and (B) contacting the complex with the cell membrane of the target cell to introduce at least the polynucleotide from the cell membrane into the cytoplasm, wherein step (B) is performed after step (A).

[0142] The polynucleotide in this embodiment is a polynucleotide that, when introduced into target cells, causes the cell membrane of the target cells to express foreign membrane proteins or overexpress intrinsic membrane proteins. The foreign membrane proteins are as described in the first embodiment, so a detailed explanation is omitted. Intrinsic membrane proteins include proteins that have a transmembrane region, membrane proteins with a hairpin structure having a hydrophobic region that is inserted into the cell membrane, and membrane proteins with a region that binds to other membrane proteins. Examples of intrinsic membrane proteins include Cav1, Cav2, and Cavin1.

[0143] According to the cell production method of this embodiment, polynucleotides can be introduced into target cells with high introduction efficiency through simple operations, and cells can be produced in which foreign membrane proteins are expressed or intrinsic membrane proteins are overexpressed on the cell membrane.

[0144] The present invention will be described more specifically below with reference to examples. However, the present invention is not limited to the examples described below. In the following examples, plasmids as polynucleotides may be referred to as "DNA," and polynucleotide binders may be referred to as "peptides." Table 7 shows the number of base pairs (bp) and molecular weight of the plasmids as polynucleotides used in these examples, as well as the number of base pairs (nt) and molecular weight of the mRNA.

[0145] [Experimental Example 1] The formation of a complex containing a polynucleotide and a polynucleotide binder was confirmed by the following procedure. A plasmid was used as the polynucleotide. The plasmid used was a plasmid vector (pCMV-eGFP, SEQ ID NO: 52) capable of expressing green fluorescent protein (GFP). Insulin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog number: 099-06473) was used as the receptor-binding protein.

[0146] As the polynucleotide binder, IBP-NBP1, indicated by binder amino acid number 38 (SEQ ID NO: 36) in Table 3A above, was used. The polynucleotide binder was prepared by the following procedure. In the following examples, the polynucleotide binder was also prepared by the same procedure. The polynucleotide binder was prepared by the Fmoc solid-phase synthesis method. The purification purity was >70% (HPLC purity). The dry powder was dissolved in pure water (>1 μg / μL) to prepare the solution. The polynucleotide binder was used as a solution containing the polynucleotide binder, and purified water was used as the solvent.

[0147] The results are shown in Figures 3(A) and 3(B). Figure 3(A) is an agarose gel electrophoresis image confirming the binding of the polynucleotide binder to DNA. Figure 3(B) is an agarose gel electrophoresis image confirming the binding of the polynucleotide binder to RNA.

[0148] In Figure 3(A), the darkest band in the lane with 0 μg of polynucleotide binder is considered to be the plasmid band. In all lanes where polynucleotide binder was added, a band appeared that was lighter than the plasmid band in the lane with 0 μg of polynucleotide binder, and was broadly shifted toward the high molecular weight side. This indicates that a complex containing the polynucleotide binder and the plasmid is formed by contacting the polynucleotide binder and plasmid of the present invention.

[0149] In Figure 3(B), the darkest band in the lane with 0 μg of polynucleotide binder is considered to be the RNA band. In all lanes where polynucleotide binder was added, a band appeared that was lighter than the RNA band in the lane with 0 μg of polynucleotide binder, and was broadly shifted toward the higher molecular weight side. This indicates that contact between the polynucleotide binder of the present invention and RNA results in the formation of a transport complex containing the polynucleotide binder and RNA.

[0150] [Experimental Example 2] The formation of a complex containing a polynucleotide, a polynucleotide binder, and a receptor-binding protein was confirmed by the following procedure. The same polynucleotides and polynucleotide binders as in Experimental Example 1 were used. Insulin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog number: 099-06473) was used as the receptor-binding protein.

[0151] (Example 1) A DNA-peptide complex was prepared by mixing 10 μL of plasmid (1 μg / μL) with 10 μL of polynucleotide binder (5 μg / μL) and reacting at room temperature for 5 minutes. A sample solution containing the complex from Example 1 was prepared by mixing 20 μL of the DNA-peptide complex with 10 μL of insulin (10 μg / μL) and reacting at room temperature for 5 minutes.

[0152] (Examples 2-3) Sample solutions containing the complexes of Examples 2-3 were prepared using the same procedure as in Example 1, except that the polynucleotide binder was diluted before mixing the plasmid and the polynucleotide binder. The dilution ratio when diluting the polynucleotide binder was set to the mixing ratio shown in Table 4 below. For example, when preparing the complex of Example 2, the plasmid and a 1 / 2 diluted polynucleotide binder (2.5 μg / μL) were mixed and reacted at room temperature for 5 minutes to prepare the DNA-peptide complex.

[0153] (Comparative Examples 1-2) A sample solution for Comparative Example 1 was prepared using the same procedure as in Example 1, except that purified water was used instead of the polynucleotide binder. A sample solution for Comparative Example 2 was prepared using the same procedure as in Example 1, except that purified water was used instead of the polynucleotide binder and insulin.

[0154]

[0155] The formation of complexes was confirmed by agarose gel electrophoresis of the sample solutions from Examples 1-3 and Comparative Examples 1-2. A 0.8% agarose gel was used, and electrophoresis was performed at 100V for 20 minutes. 6 μL of the sample solutions from Examples 1-3 and Comparative Examples 1-2, prepared according to the procedure described above, was loaded into each well. A DNA marker (Nippon Gene Co., Ltd., product number: OneSTEP Ladder 1kb) was used as the marker, and 1 μL of the marker was loaded into each well. The results are shown in Figure 4. Figure 4 shows the agarose gel electrophoresis images of the sample solutions from Examples 1-3 and Comparative Examples 1-2.

[0156] In Figure 4, the band appearing at the very bottom of the lane for Comparative Example 2 is considered to be the plasmid band. It was observed that as the amount of polynucleotide binder added increased, the plasmid band became fainter and the band at the top of the gel became darker. This indicates that a complex containing the polynucleotide binder, polynucleotide, and receptor-binding peptide of the present invention is formed by contacting the polynucleotide binder, polynucleotide, and receptor-binding peptide of the present invention. In addition, the bands appearing at the top of the gel in the lanes for Comparative Examples 1 and 2 are considered to be the residue of polynucleotides remaining in the wells.

[0157] [Experimental Example 3] The delivery efficiency of a complex containing a polynucleotide binder, polynucleotide, and receptor-binding protein to target cells was evaluated. The same polynucleotide binder, plasmid, and insulin as in Experimental Example 2 were used. HEK293F cells (CellsThermoFisher, catalog number: R79007) were used as the target cells.

[0158] (Example 4) A DNA-peptide complex was prepared by mixing 3 μL of plasmid (1 μg / μL) with 3 μL of polynucleotide binder (5 μg / μL) and reacting at room temperature for 5 minutes. A sample solution containing the complex from Example 4 was prepared by mixing 6 μL of the DNA-peptide complex with 3 μL of insulin (10 μg / μL) and reacting at room temperature for 5 minutes.

[0159] (Examples 5-8) Sample solutions containing the complexes of Examples 5-8 were prepared using the same procedure as in Example 4, except that the polynucleotide binder was diluted before mixing the plasmid and the polynucleotide binder in the procedure for preparing the complex of Example 4. The dilution ratio when diluting the polynucleotide binder was set to the mixing ratio shown in Table 5 below. For example, when preparing the sample solution of Example 5, the plasmid and a 0.9-fold diluted polynucleotide binder (4.5 μg / μL) were mixed and reacted at room temperature for 5 minutes to prepare the DNA-peptide complex.

[0160] (Example 9) A solution containing the complex of Example 9 was prepared using the same procedure as in Example 4, except that purified water was used instead of insulin.

[0161] (Comparative Examples 3-4) The sample solution for Comparative Example 3 was prepared using the same procedure as in Example 4, except that purified water was used instead of the polynucleotide binder. The sample solution for Comparative Example 4 was prepared using the same procedure as in Example 4, except that purified water was used instead of the polynucleotide binder and insulin.

[0162]

[0163] In the cell culture plate, the final concentration is 1 × 10⁻⁶. ５ HEK293F cells were seeded into each well to achieve a cell / well ratio. After seeding the HEK293F cells and removing the culture medium, 8 μL each of the sample solutions from Examples 4-9 and Comparative Examples 3-4 were added to the wells and allowed to stand at room temperature for 5 minutes.

[0164] 400 μL of HE100 medium (Gmep, catalog number: HE100-0010) was added to each well containing the sample solution for each example, and the mixture was heated at 37°C and 5% CO2 by volume. ２ The cells were cultured for three days under environmental conditions. After culturing, GFP in the target cells was observed by bright-field microscopy. Furthermore, GFP in the target cells was observed by fluorescence microscopy after culturing. The results are shown in Figure 5(A). Figure 5(A) shows microscopic images of GFP observed by bright-field microscopy and fluorescence microscopy.

[0165] The efficiency of introducing each example's complex into target cells was measured using the following procedure. The number of cells showing GFP fluorescence was measured using a flow cytometer, and the value of the number of GFP-positive cells, with the number of viable cells as the denominator, was defined as the introduction efficiency. The results are shown in Figure 5(B). Figure 5(B) is a graph showing the introduction efficiency when DNA was introduced into target cells using the sample solutions of Examples 4-9 and Comparative Examples 3-4.

[0166] Figures 5(A) and 5(B) show that the complexes in each example exhibited higher delivery efficiency to target cells compared to Comparative Examples 3 and 4, which did not contain a polynucleotide binder.

[0167] [Experimental Example 4] The delivery efficiency of a polynucleotide binder and a polynucleotide-containing complex to target cells was measured. As the polynucleotide binder, TRBP-NBP1, shown as binder amino acid number 42 (SEQ ID NO: 40) in Table 3A above, was used. TRBP-NBP1 is a polynucleotide binder in which the first and second binding sites bind directly, and there is no unbinding site between the C-terminus of the first binding site and the N-terminus of the second binding site. TRBP-NBP1 includes the first binding site shown as amino acid number 6 (SEQ ID NO: 6) in Table 1A above, and the second binding site shown as amino acid number 20 (SEQ ID NO: 18) in Table 2A above. The plasmid and target cells used were the same as in Experimental Example 3.

[0168] (Example 10) 3 μL of plasmid (1 μg / μL) and 3 μL of polynucleotide binder (5 μg / μL) were mixed and reacted at room temperature for 5 minutes. This prepared a sample solution containing the complex of Example 10, which includes the plasmid and the polynucleotide binder.

[0169] (Examples 11-12) Sample solutions containing the complexes of Examples 11-12 were prepared using the same procedure as in Example 10, except that the polynucleotide binder was diluted before mixing the plasmid and the polynucleotide binder. The dilution ratio when diluting the polynucleotide binder was set to the mixing ratio shown in Table 6 below. For example, when preparing the sample solution of Example 11, the plasmid and a 0.5-fold diluted polynucleotide binder (2.5 μg / μL) were mixed and reacted at room temperature for 5 minutes to prepare the complex.

[0170] (Comparative Examples 5-6) Sample solutions for Comparative Example 5 were prepared using the same procedure as in Example 10, except that purified water was used instead of a polynucleotide binder. Purified water was used as the sample solution for Comparative Example 6.

[0171]

[0172] Except for using the sample solutions from Examples 10-12 and Comparative Examples 5-6, the procedure was the same as in Experimental Example 3 to measure the introduction efficiency of each example's complex into target cells. The results are shown in Figure 6.

[0173] Figure 6 shows that all of the example complexes exhibited higher delivery efficiency to target cells compared to Comparative Examples 5-6, which did not contain a polynucleotide binder.

[0174] [Experimental Example 5] Plasmids with different base numbers were used, and the delivery efficiency to target cells was measured. As polynucleotide binders, IBP-NBP1, indicated by binder amino acid number 38 (SEQ ID NO: 36) in Table 3A above, and TRBP-GS-NBP1, indicated by binder amino acid number 43 (SEQ ID NO: 41), were used. The same insulin and target cells as in Experimental Example 3 were used.

[0175] TRBP-GS-NBP1 is a polynucleotide binder in which a first binding site and a second binding site are indirectly linked via a non-binding site. TRBP-GS-NBP1 includes a first binding site indicated by amino acid number 6 (SEQ ID NO: 6) in Table 1A above, and a second binding site indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A above. The non-binding site is located between the C-terminus of the first binding site and the N-terminus of the second binding site.

[0176] As plasmids, we used the following six plasmid vectors with different base numbers that are capable of expressing GFP: • Plasmid vector 1 (SEQ ID NO: 52, 4 kbp) • Plasmid vector 2 (SEQ ID NO: 53, 6.2 kbp) • Plasmid vector 3 (SEQ ID NO: 54, 10 kbp) • Plasmid vector 4 (SEQ ID NO: 55, 12 kbp) • Plasmid vector 5 (SEQ ID NO: 56, 14 kbp)

[0177]

[0178] (Example 13) A DNA-peptide complex was prepared by mixing 3 μL of plasmid vector 1 (1 μg / μL) and 3 μL of IBP-NBP1 (5 μg / μL) and reacting them at room temperature for 5 minutes. A sample solution containing the complex from Example 13 was prepared by mixing 6 μL of the DNA-peptide complex with 3 μL of insulin (10 μg / μL) and reacting them at room temperature for 5 minutes.

[0179] (Examples 14-17) Sample solutions containing the complex of Example 14 were prepared using the same procedure as in Example 13, except that plasmid vector 2 was used instead of plasmid vector 1. Sample solutions containing the complexes of Examples 14-17 were also prepared using plasmid vector 3 (Example 15), plasmid vector 4 (Example 16), and plasmid vector 5 (Example 17).

[0180] (Example 18) A sample solution containing the complex of Example 18 was prepared using the same procedure as in Example 13, except that TRBP-GS-NBP1 was used instead of IBP-NBP1 and purified water was used instead of insulin.

[0181] (Examples 19-22) Sample solutions containing the complex of Example 19 were prepared using the same procedure as in Example 18, except that plasmid vector 2 was used instead of plasmid vector 1. Sample solutions containing the complexes of Examples 19-22 were also prepared using plasmid vector 3 (Example 20), plasmid vector 4 (Example 21), and plasmid vector 5 (Example 22).

[0182] (Comparative Examples 7-8) The sample solution for Comparative Example 7 was prepared using the same procedure as in Example 14, except that purified water was used instead of IBP-NBP1. The sample solution for Comparative Example 8 was prepared using the same procedure as in Example 19, except that purified water was used instead of TRBP-GS-NBP1.

[0183] In the cell culture plate, the final concentration is 1 × 10⁻⁶. ５HEK293F cells were seeded into each well to achieve a density of cells / well. HEK293F cells were seeded and cultured in a CO ２ incubator at 37°C under an atmosphere containing 5% by volume of CO ２ for 24 to 27 hours. 3 μL of each of the sample solutions of Examples 13 to 22 and Comparative Examples 7 to 8 was added to each well from which the medium had been removed, followed by the addition of 400 μL of medium (HE100), followed by culturing in CO ２ incubator at 37°C under an atmosphere containing 5% by volume of CO ２ for 48 hours.

[0184] The transfection efficiency of each example into target cells was measured by the following procedure. The number of cells positive for GFP fluorescence was measured by flow cytometry, the number of viable cells was counted, and the value of the number of GFP-positive cells relative to the total number of viable cells as the denominator was defined as the transfection efficiency. The results obtained using the sample solutions of Examples 13 to 17 and Comparative Example 7 are shown in Figure 7. The results obtained using the sample solutions of Examples 19 to 22 and Comparative Example 8 are shown in Figure 8.

[0185] As shown in Figures 7 to 8, the sample solutions of all Examples exhibited higher transfection efficiency than that obtained when using the sample solutions of the Comparative Examples.

[0186] [Experimental Example 6] Changes over time in the number of viable target cells into which a polynucleotide had been introduced and the number of GFP-positive cells were confirmed. As a sample solution containing the complex, a sample solution prepared under the same experimental conditions as in Example 17 (plasmid vector 5 (SEQ ID NO: 56, 14 kbp)) was used. The same plasmid, receptor-binding protein, and target cells as those used in Example 17 were employed.

[0187] (Comparative Example 9) 1 μL of plasmid (1 μg / μL) and 1 μL of PEIpro (registered trademark, manufactured by POLYplus, product number: 101000017) were mixed in 40 μL of medium (Opti-MEM, product number: 31985062), and the mixture was allowed to stand at room temperature for 15 minutes, thereby preparing the sample solution of Comparative Example 9.

[0188] HEK293F cells were seeded on a cell culture plate to a final concentration of 1 × 10 ５HEK293F cells were seeded in each well to achieve a cell / well ratio. HEK293F cells were seeded, CO ２ Incubator at 37°C, 5% CO2 by volume ２ The cultures were incubated in the environment for 24 to 27 hours. 3 μL each of the sample solutions from Example 17 and Comparative Example 9 were added to the wells from which the culture medium had been removed, and then 400 μL of medium (HE100) was added. ２ Incubator at 37°C, 5% CO2 by volume ２ The culture was performed under environmental conditions for 13 days, with half of the culture medium being replaced every 2-3 days.

[0189] The number of viable cells and GFP-positive cells were measured over time using a flow cytometer up to 13 days after gene transfer. Figure 9 shows the results of measuring the number of viable cells up to 13 days after gene transfer, and Figure 10 shows the results of measuring the number of GFP-positive cells up to 13 days after gene transfer.

[0190] Figure 9 shows that target cells into which plasmids were introduced using the polynucleotide binder of the example showed a higher number of viable cells 5 days after plasmid introduction compared to target cells using the sample solution of the comparative example. Furthermore, while the number of viable cells decreased over time after plasmid introduction in target cells using the sample solution of the comparative example, the number of viable cells increased over time after plasmid introduction in target cells using the polynucleotide binder of the example.

[0191] Figure 10 shows that target cells into which plasmids were introduced using the polynucleotide binder of the example showed a higher number of GFP-positive cells after 13 days of culture compared to target cells using the sample solution of the comparative example. Furthermore, while the number of GFP-positive cells decreased over time after plasmid introduction in target cells using the sample solution of the comparative example, the number of GFP-positive cells was maintained after plasmid introduction in target cells using the polynucleotide binder of the example.

[0192] [Experimental Example 7] The efficiency of polynucleotide introduction into HEK293F cells was measured using polynucleotide binders with different amino acid sequences. The polynucleotide binders used were those listed in Tables 3A to 3C above. The plasmids used were the same as in Experimental Example 3. The receptor-binding proteins used were insulin and transferrin (Fujifilm Wako Pure Chemical Industries, Ltd., catalog number: 200-19771), the same as in Experimental Example 3. The target cells used were HEK293F cells, the same as in Experimental Example 3, and Jurkat cells, a human immortalized cell line that mimics the function of T cells.

[0193] (Example 23) IBP-NBP1, indicated by amino acid number 38 (SEQ ID NO: 36) in Table 3A above, was used as the polynucleotide binder in Example 23. IBP-NBP1 has a C-terminus of the first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above, and a N-terminus of the second binding site indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A above, which are directly bound. IBP-NBP1 can indirectly bind to the insulin receptor via insulin, which is a receptor-binding protein.

[0194] A DNA-peptide complex was prepared by mixing 3 μL of plasmid (1 μg / μL) and 3 μL of IBP-NBP1 (5 μg / μL) and reacting them at room temperature for 5 minutes. A sample solution containing the complex of Example 23 was prepared by mixing 6 μL of the DNA-peptide complex with 3 μL of insulin (10 μg / μL) and reacting them at room temperature for 5 minutes.

[0195] (Example 24) The sample solution for Example 24 was prepared using the same procedure as in Example 23, except that IBP-GS-NBP1, indicated by amino acid number 39 (SEQ ID NO: 37), was used instead of IBP-NBP1. IBP-GS-NBP1 has the C-terminus of the first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above and the N-terminus of the second binding site indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A above, indirectly bound via a non-binding site. IBP-GS-NBP1 can indirectly bind to the insulin receptor via insulin, which is a receptor-binding protein.

[0196] (Example 25) The sample solution for Example 25 was prepared using the same procedure as in Example 23, except that NBP1-GS-IBP, indicated by amino acid number 47 (SEQ ID NO: 45) as the binder, was used instead of IBP-NBP1. NBP1-GS-IBP has a second binding site located on the N-terminal side of the polynucleotide binder, and the N-terminal side of the first binding site located on the C-terminal side, which are indirectly bound via a non-binding site. NBP1-GS-IBP includes a second binding site indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A and a first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A. NBP1-GS-IBP can indirectly bind to the insulin receptor via insulin, which is a receptor-binding protein.

[0197] (Example 26) The sample solution for Example 26 was prepared using the same procedure as in Example 23, except that NBP1-GS-RDG, indicated by amino acid number 48 (SEQ ID NO: 46), was used instead of IBP-NBP1, and purified water was used instead of insulin. NBP1-GS-RDG has an indirect binding site between the C-terminus of the second binding site indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A and the N-terminus of the first binding site indicated by amino acid number 7 in Table 1A. NBP1-GS-RDG can directly bind to integrins.

[0198] (Example 27) The sample solution for Example 27 was prepared using the same procedure as in Example 26, except that NBP1-GS-EILDV, indicated by amino acid number 49 (SEQ ID NO: 47), was used instead of NBP1-GS-RDG. NBP1-GS-EILDV is indirectly bound to the C-terminus of the second binding site, indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A, and to the N-terminus of the first binding site, indicated by amino acid number 8 (SEQ ID NO: 7) in Table 1A, via a non-binding site. NBP1-GS-EILDV can directly bind to integrins.

[0199] (Example 28) The sample solution for Example 28 was prepared using the same procedure as in Example 26, except that RDG-GS-NBP1, indicated by amino acid number 44 (SEQ ID NO: 42), was used instead of NBP1-GS-RDG. RDG-GS-NBP1 has indirect binding between the C-terminus of the first binding site, indicated by amino acid number 7 in Table 1A, and the N-terminus of the second binding site, indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A, via non-binding sites. RDG-GS-NBP1 can directly bind to integrins.

[0200] (Example 29) The sample solution for Example 29 was prepared using the same procedure as in Example 26, except that EILDV-GS-NBP1, indicated by amino acid number 45 (SEQ ID NO: 43), was used instead of NBP1-GS-RDG. EILDV-GS-NBP1 has its C-terminus of the first binding site, indicated by amino acid number 8 (SEQ ID NO: 7) in Table 1A, and its N-terminus of the second binding site, indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A, indirectly bound via non-binding sites. EILDV-GS-NBP1 can directly bind to integrins.

[0201] (Example 30) The sample solution for Example 30 was prepared using the same procedure as in Example 23, except that TfBP-GS-NBP1, indicated by amino acid number 41 (SEQ ID NO: 39), was used instead of IBP-NBP1, and transferrin was used instead of insulin. TfBP-GS-NBP1 has the C-terminus of the first binding site, indicated by amino acid number 5 (SEQ ID NO: 5) in Table 1A, and the N-terminus of the second binding site, indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A, indirectly bound via a non-binding site. TfBP-GS-NBP1 can indirectly bind to the transferrin receptor via transferrin, which is a receptor-binding protein.

[0202] (Example 31) The sample solution for Example 31 was prepared using the same procedure as in Example 30, except that TRBP-GS-NBP1, indicated by amino acid number 43 (SEQ ID NO: 41), was used instead of TfBP-GS-NBP1, and purified water was used instead of transferrin. TRBP-GS-NBP1 has the C-terminus of the first binding site, indicated by amino acid number 6 (SEQ ID NO: 6) in Table 1A above, and the N-terminus of the second binding site, indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A above, indirectly bound via non-binding sites. TRBP-GS-NBP1 can directly bind to the transferrin receptor.

[0203] (Example 32) The sample solution for Example 32 was prepared using the same procedure as in Example 30, except that nEDT-NBP2-EDTc, indicated by amino acid number 50 (SEQ ID NO: 48), was used instead of TfBP-GS-NBP1. nEDT-NBP2-EDTc includes a first binding site indicated by amino acid number 16 (SEQ ID NO: 14) in Table 1A, a second binding site indicated by amino acid number 21 (SEQ ID NO: 19) in Table 2A, a first binding site indicated by amino acid number 17 (SEQ ID NO: 15) in Table 1A, and an unbound sequence. The C-terminus of the first binding site (amino acid number 16), located on the N-terminal side, and the N-terminus of the second binding site (amino acid number 21) are indirectly bound via the unbound site. Furthermore, the C-terminus of the second binding site (amino acid number 21) and the N-terminus of the first binding site (amino acid number 17), located on the C-terminal side, are indirectly bound via a non-binding site. nEDT-NBP2-EDTc can directly bind to the endothelin receptor.

[0204] (Comparative Example 10) A sample solution for Comparative Example 10 was prepared using the same procedure as in Example 25, except that purified water was used instead of IBP-NBP1 and insulin.

[0205] In the cell culture plate, the final concentration is 2 × 10 ５ HEK293F cells were seeded in each well to achieve a cell / well ratio. HEK293F cells were seeded, CO ２ Incubator at 37°C, 5% CO2 by volume ２ The cultures were incubated in the environment for 24 to 27 hours. To the wells from which the culture medium had been removed, 3 μL each of the sample solutions from Examples 23-31 and Comparative Example 10 were added and allowed to stand for 10 minutes. Then, 400 μL of culture medium (HE100) was added, and CO2 was added. ２ Incubator at 37°C, 5% CO2 by volume ２ The samples were cultured for 48 hours under environmental conditions.

[0206] The transduction efficiency of each sample into HEK293F cells was measured using the following procedure. Measurements were performed using a flow cytometer, and the transduction efficiency was defined as the number of cells showing positive GFP fluorescence relative to the number of viable cells. The results are shown in Figure 11.

[0207] In the cell culture plate, the final concentration is 2 × 10 ５ Jurkat cells were seeded in each well to achieve a cell / well ratio. Jurkat cells were seeded, CO ２ Incubator at 37°C, 5% CO2 by volume ２ The cultures were incubated in the environment for 24 to 27 hours. In the wells from which the culture medium had been removed, 3 μL each of the sample solutions from Examples 21, 24-27, 29-32, and Comparative Example 10 were added. After standing for 10 minutes, 400 μL of culture medium (RPMI1640) was added, and CO2 was added. ２ Incubator at 37°C, 5% CO2 by volume ２ The samples were cultured for 48 hours under environmental conditions.

[0208] The transduction efficiency of each example to Jurkat cells was measured using the following procedure. Measurements were performed using a flow cytometer, and the number of cells showing positive GFP fluorescence was defined as the transduction efficiency, with the number of cells in Comparative Example 10 (which did not contain a polynucleotide binder) as the denominator. The results are shown in Figures 12-13.

[0209] Figure 11 shows that the sample solutions in each example were more efficient at introducing HEK293F cells compared to the sample solutions in the comparative examples.

[0210] Figures 12-13 show that the sample solutions in each example were more efficient at introducing the cells into Jurkat cells compared to the sample solutions in the comparative examples.

[0211] [Experimental Example 8] The efficiency of introducing the complex into wild-type cells and Cav1-overexpressing cells was measured using the following procedure. As the polynucleotide binder, NBP1-IBP, indicated by amino acid number 46 (SEQ ID NO: 44) in Table 3B above, was used. NBP1-IBP has a direct binding between the C-terminus of the second binding site, indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A above, and the N-terminus of the first binding site, indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above. NBP1-IBP can indirectly bind to the insulin receptor via insulin, which is a receptor-binding protein. The same polynucleotides and insulin as in Experimental Example 3 were used.

[0212] Wild-type Jurkat cells and Cav1-overexpressing Jurkat cells were used as target cells. The wild-type Jurkat cells used were the same as those used in Experimental Example 7. Cav1-overexpressing Jurkat cells were prepared using the following procedure.

[0213] Overexpression strains of Jurkat cells were created by introducing a Cav1 expression plasmid vector (pEF1-CAV1-puro, SEQ ID NO: 57) into Jurkat cells (manufactured by ECACC (The European Collection of Authenticated Cell Cultures)).

[0214] (Example 33) A DNA-peptide complex was prepared by mixing 3 μL of plasmid (1 μg / μL) and 3 μL of NBP1-IBP (5 μg / μL) and reacting them at room temperature for 5 minutes. A sample solution containing the complex from Example 33 was prepared by mixing 6 μL of the DNA-peptide complex with 3 μL of insulin (10 μg / μL) and reacting them at room temperature for 5 minutes.

[0215] In the cell culture plate, the final concentration is 2 × 10 ５ Wild-type Jurkat cells and Cav1-overexpressing Jurkat cells were seeded in separate wells to achieve a cell / well ratio. 8 μL each of the sample solutions from Example 33 and Comparative Example 10 were added to the wells from which the culture medium had been removed, and the mixtures were allowed to stand at room temperature for 5 minutes.

[0216] Add 400 μL of RPMI1640 medium to each well containing the sample solution, and then heat at 37°C and 5% CO2. ２ Cells were cultured for three days under environmental conditions. After culturing, the plasmid delivery efficiency to target cells was calculated by measuring GFP-positive cells and viable cells at a flow site. The results are shown in Figures 14(A) and 14(B).

[0217] Figures 14(A) and 14(B) show that the polynucleotide binder of the present invention exhibits higher gene transfer efficiency when the target cells overexpress Cav1.

[0218] [Experimental Example 9] The efficiency of introducing the complex into wild-type cells, Cav1-overexpressing cells, Cav2-overexpressing cells, and Cavin1-overexpressing cells was measured using the following procedure. The same wild-type Jurkat cells and Cav1-overexpressing Jurkat cells were used as in Experimental Example 8. Cav2-overexpressing Jurkat cells were prepared using the same procedure as in Experimental Example 8, except that a Cav2-expressing plasmid vector (pEF1-CAV2-puro, SEQ ID NO: 58) was used instead of a Cav1-expressing plasmid vector. Cavin1-overexpressing Jurkat cells were prepared using the same procedure as in Experimental Example 8, except that a Cavin1-expressing plasmid vector (pEF1-Cavin1-puro, SEQ ID NO: 59) was used instead of a Cav1-expressing plasmid vector.

[0219] The sample solutions used were those from Examples 23, 28, 29, and 31.

[0220] In the cell culture plate, the final concentration is 2 × 10 ５ Jurkat cells of the wild-type, Cav1-overexpressing, Cav2-overexpressing, and Cavin1-overexpressing strains were seeded in separate wells to achieve a cell / well ratio. 8 μL of the sample solution containing the complexes from Examples 23, 28, 29, and 31 was added to each well (after removing the culture medium), and the mixture was allowed to stand at room temperature for 5 minutes.

[0221] Add 400 μL of RPMI1640 medium to each well containing the sample solution, and then heat at 37°C and 5% CO2. ２The cells were cultured for three days under environmental conditions. After culturing, the plasmid delivery efficiency to the target cells was calculated by measuring with a flow cytometer. Figure 15 shows the results after adding the sample solution from Example 23, Figure 16 shows the results after adding the sample solution from Example 28, Figure 17 shows the results after adding the sample solution from Example 29, and Figure 18 shows the results after adding the sample solution from Example 31. In Figures 15-18, "Jurkat-WT" refers to wild-type Jurkat cells. "Jurkat-Cav1" refers to Jurkat cells overexpressing Cav1. "Jurkat-Cav2" refers to Jurkat cells overexpressing Cav2. "Jurkat-Cavin1" refers to Jurkat cells overexpressing Cavin1.

[0222] Figures 15-18 show that when using the polynucleotide binder of the present invention, the efficiency of introducing polynucleotides into the cytoplasm is improved when the target cells overexpress one of Cav1, Cav2, or Cavin1.

[0223] [Experimental Example 10] Plasmid introduction into human peripheral blood unicellular cells (PBMCs) and human preadipocytes (hPADs) was observed using the following procedure. The same sample solution as in Example 4 was used. PBMCs (CTL, catalog number: CTL-UP1) and hPADs (Lonza, catalog number: PT-5006) were used as target cells.

[0224] In the cell culture plate, the final concentration is 2 × 10 ５ PBMC and hPAD were seeded in separate wells to achieve a cell / well ratio. 8 μL of the sample solution containing the complex from Example 4 was added to each well from which the culture medium had been removed, and the mixture was allowed to stand at room temperature for 5 minutes.

[0225] PBMC added 400 μL of TexMax medium (Miltenyi Biotec, catalog number 130-097-196) to each well containing the sample solution, and then heated it at 37°C and 5% CO2. ２The cells were cultured for 3 days under environmental conditions. For hPAD, 400 μL of ADSC-BM medium (Lonza, PT-3273) was added to each well, and the culture was performed at 37°C and 5% CO2. ２ The cells were cultured for three days under environmental conditions. After culturing, bright-field fluorescence microscopy and bright-field fluorescence microscopy were used to observe bright-field fluorescence and GFP levels within the target cells, respectively. The results are shown in Figures 19-20. Figure 19 is a microscopic image of DNA introduced into PBMCs using a polynucleotide binder according to one embodiment of the present invention. Figure 20 is a microscopic image of DNA introduced into hPADs using a polynucleotide binder according to one embodiment of the present invention.

[0226] Figures 19-20 demonstrate that plasmids can be introduced into target cells using the polynucleotide binder of the present invention.

[0227] [Experimental Example 11] RNA introduction into human peripheral blood unicellular cells (PBMCs) was observed using the following procedure. Except for using RNA (EGFP mRNA, TriLink BioTechnologies, catalog number L-7201-100) instead of plasmid and using only PBMCs as target cells, the procedure was the same as in Experimental Example 10, and GFP expression in the target cells was observed. The results are shown in Figure 21. Figure 21 is a microscopic image of PBMCs after RNA introduction using a polynucleotide binder according to one embodiment of the present invention. As is clear from the results shown in the figure, EGFP expression could be confirmed by RNA introduction into PBMCs.

[0228] [Experimental Example 12] RNA introduction into Jurkat cells was observed using the following procedure. Except for using Jurkat cells similar to those in Experimental Example 7 instead of PBMCs, the procedure was the same as in Experimental Example 11, and GFP expression in the target cells was observed. The results are shown in Figure 22. Figure 22 is a microscopic image of RNA introduction into Jurkat cells using a polynucleotide binder according to one embodiment of the present invention. As is clear from the results shown in the figure, EGFP expression was confirmed by RNA introduction into Jurkat cells.

[0229] Figures 21-22 demonstrate that RNA can be introduced into target cells using the polynucleotide binder of the present invention.

[0230] [Experimental Example 13] The storage stability of the polynucleotide binder was confirmed by the following procedure. The plasmid and target cells used were the same as those in Experimental Example 3.

[0231] (Example 34) As the polynucleotide binder, IBP-NBP22, indicated by amino acid number 78 (SEQ ID NO: 87) in Table 3D above, was used. IBP-NBP22 includes a first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above, and a second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above. Specifically, in IBP-NBP22, the C-terminus of the first binding site indicated by amino acid number 1 (SEQ ID NO: 1) and the N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) are directly bound. IBP-NBP22 can indirectly bind to the insulin receptor via insulin, which is a receptor-binding protein.

[0232] A DNA-peptide complex was prepared by mixing 3 μL of plasmid (1 μg / L) and 3 μL of IBP-NBP22 (5 μg / μL) and reacting them at room temperature for 5 minutes. A sample solution containing the complex of Example 34 was prepared by mixing 6 μL of the DNA-peptide complex with 3 μL of insulin (10 μg / μL) and reacting them at room temperature for 5 minutes.

[0233] (Comparative Example 11) The sample solution for Comparative Example 11 was prepared in the same manner as in Comparative Example 9, except that TransIT-293 (manufactured by Mirus, product code: MIR2704) was used instead of PEIpro.

[0234] (Comparative Example 12) The sample solution for Comparative Example 12 was prepared in the same manner as in Comparative Example 9, except that the volume of PEIpro was changed from 1 μL to 3 μL.

[0235] The prepared sample solutions of Example 34 and Comparative Examples 11-12 were stored at room temperature for 15 min, 4.5 h, 8.5 h, and 24 h, respectively. This resulted in the preparation of sample solutions of Example 34 and Comparative Examples 11-12 that were stored for 15 min, 4.5 h, 8.5 h, and 24 h, respectively, after preparation.

[0236] Except for using the sample solutions from Example 34 and Comparative Examples 11-12, which were stored after preparation, as sample solutions, target cells were cultured using the same procedure as in Experimental Example 3, and the introduction efficiency was calculated. The results are shown in Figure 23.

[0237] In Figure 23, the introduction efficiency shown on the vertical axis represents the relative value with respect to the introduction efficiency when using a sample solution that has been stored for 15 minutes after preparation. For example, the value shown at 4.5h in Example 34 represents the relative value of the introduction efficiency when using a sample solution that has been stored for 4.5 hours after preparation, with respect to the introduction efficiency when using the sample solution of Example 34 that has been stored for 15 minutes after preparation.

[0238] Figure 23 shows that the sample solution in the example exhibited high storage stability, as the decrease in introduction efficiency was suppressed even after long-term storage. In contrast, the sample solutions in the comparative example all showed a significant decrease in introduction efficiency during storage, indicating low storage stability.

[0239] [Experimental Example 14] The storage stability of the polynucleotide binder was confirmed by the following procedure. The plasmid and target cells used were the same as those used in Experimental Example 13. The sample solutions used were those from Example 34 and Comparative Examples 11-12.

[0240] The prepared sample solutions of Example 34 and Comparative Examples 11-12 were stored at room temperature for 15 minutes, at room temperature for 24 hours, at 5°C for 24 hours, and at -30°C for 24 hours, respectively. This prepared sample solutions of Example 34 and Comparative Examples 11-12 stored under each condition.

[0241] Except for using the sample solutions from Example 34 and Comparative Examples 11-12, which were stored after preparation, as sample solutions, target cells were cultured using the same procedure as in Experimental Example 13, and the introduction efficiency was calculated. The results are shown in Figure 24.

[0242] In Figure 24, the introduction efficiency shown on the vertical axis represents the relative value with respect to the introduction efficiency when using a sample solution that has been stored at room temperature (RT) for 15 minutes after preparation. For example, the value shown for 24h (5°C) in Example 34 represents the relative value of the introduction efficiency when using a sample solution stored at 5°C for 24 hours, with respect to the introduction efficiency when using the sample solution of Example 34 that has been stored at room temperature for 15 minutes after preparation.

[0243] Figure 24 shows that the sample solution in the example exhibited high storage stability, as the decrease in introduction efficiency was suppressed even under conditions of high storage temperature and long storage time. In contrast, the sample solutions in the comparative example all showed a significant decrease in introduction efficiency during storage, indicating low storage stability.

[0244] [Experimental Example 15] The efficiency of polynucleotide introduction into HEK293F cells was measured using polynucleotide binders with different amino acid sequences. The polynucleotide binders used were those listed in Table 3D above. The plasmids and target cells used were the same as those in Experimental Example 3.

[0245] (Example 35) IBP-NBP21, indicated by amino acid number 77 (SEQ ID NO: 86) in Table 3D above, was used as the polynucleotide binder in Example 35. IBP-NBP21 has a C-terminus of the first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above, and a N-terminus of the second binding site indicated by amino acid number 69 (SEQ ID NO: 78) in Table 2B above, which are directly bound. IBP-NBP21 can indirectly bind to the insulin receptor via insulin, which is a receptor-binding protein. The sample solution of Example 35 was prepared in the same manner as in Example 34, except that the polynucleotide binder of Example 35 was used.

[0246] (Example 36) IBP-NBP19, indicated by amino acid number 84 (SEQ ID NO: 93) in Table 3D above, was used as the polynucleotide binder in Example 36. IBP-NBP19 has a C-terminus of the first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above, and a N-terminus of the second binding site indicated by amino acid number 67 (SEQ ID NO: 76) in Table 2B above, which are directly bound. IBP-NBP19 can indirectly bind to the insulin receptor via insulin, which is a receptor-binding protein. The sample solution of Example 36 was prepared in the same manner as in Example 34, except that the polynucleotide binder of Example 36 was used.

[0247] (Example 37) IBP-NBP20, indicated by amino acid number 85 (SEQ ID NO: 94) in Table 3D above, was used as the polynucleotide binder in Example 37. IBP-NBP20 has a direct binding at the C-terminus of the first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above, and a direct binding at the N-terminus of the second binding site indicated by amino acid number 68 (SEQ ID NO: 77) in Table 2B above. IBP-NBP20 can indirectly bind to the insulin receptor via insulin, which is a receptor-binding protein. The sample solution of Example 37 was prepared in the same manner as in Example 34, except that the polynucleotide binder of Example 37 was used.

[0248] Except for using the sample solutions from Examples 35-37 and Comparative Example 2 as sample solutions, target cells were cultured using the same procedure as in Experimental Example 3, and the translocation efficiency was calculated. The sample solution for Comparative Example 2 was a sample solution that did not contain a polynucleotide binder or receptor-binding protein. The results are shown in Figure 25.

[0249] Figure 25 shows that the sample solutions from each example exhibited higher delivery efficiency to target cells compared to Comparative Example 2, which did not contain a polynucleotide binder.

[0250] [Experimental Example 16] Based on the amino acid sequence of the second binding site, which showed high transfection efficiency in Experimental Example 15, polynucleotide binders with different amino acid sequences were prepared, and the transfection efficiency of polynucleotides into HEK293F cells was measured. The polynucleotide binders used were those listed in Table 3D above. The plasmids and target cells used were the same as in Experimental Example 3.

[0251] (Example 38) IBP-NBP23, indicated by amino acid number 79 (SEQ ID NO: 88) in Table 3D above, was used as the polynucleotide binder in Example 38. IBP-NBP23 has a C-terminus of the first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above, and a N-terminus of the second binding site indicated by amino acid number 71 (SEQ ID NO: 80) in Table 2B above, both of which are directly bound. IBP-NBP23 can indirectly bind to the insulin receptor via insulin, which is a receptor-binding protein. The sample solution for Example 38 was prepared in the same manner as in Example 34, except that the polynucleotide binder for Example 38 was used.

[0252] (Example 39) IBP-NBP24, indicated by amino acid number 80 (SEQ ID NO: 89) in Table 3D above, was used as the polynucleotide binder in Example 39. IBP-NBP24 has a C-terminus of the first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above, and a N-terminus of the second binding site indicated by amino acid number 72 (SEQ ID NO: 81) in Table 2B above, which are directly bound. IBP-NBP24 can indirectly bind to the insulin receptor via insulin, which is a receptor-binding protein. The sample solution of Example 39 was prepared in the same manner as in Example 34, except that the polynucleotide binder of Example 39 was used.

[0253] (Example 40) IBP-NBP26, indicated by amino acid number 82 (SEQ ID NO: 91) in Table 3D above, was used as the polynucleotide binder in Example 40. IBP-NBP26 has a C-terminus of the first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above, and a N-terminus of the second binding site indicated by amino acid number 74 (SEQ ID NO: 83) in Table 2B above, which are directly bound. IBP-NBP26 can indirectly bind to the insulin receptor via insulin, which is a receptor-binding protein. The sample solution for Example 40 was prepared in the same manner as in Example 34, except that the polynucleotide binder for Example 40 was used.

[0254] (Example 41) IBP-NBP25, indicated by amino acid number 81 (SEQ ID NO: 90) in Table 3D above, was used as the polynucleotide binder in Example 41. IBP-NBP25 has a direct binding at the C-terminus of the first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above, and a direct binding at the N-terminus of the second binding site indicated by amino acid number 73 (SEQ ID NO: 82) in Table 2B above. IBP-NBP25 can indirectly bind to the insulin receptor via insulin, which is a receptor-binding protein. The sample solution of Example 41 was prepared in the same manner as in Example 34, except that the polynucleotide binder of Example 41 was used.

[0255] (Example 42) IBP-NBP27, indicated by amino acid number 83 (SEQ ID NO: 92) in Table 3D above, was used as the polynucleotide binder in Example 42. IBP-NBP27 has a C-terminus of the first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above, and a N-terminus of the second binding site indicated by amino acid number 75 (SEQ ID NO: 84) in Table 2B above, both of which are directly bound. IBP-NBP27 can indirectly bind to the insulin receptor via insulin, which is a receptor-binding protein. The sample solution of Example 42 was prepared in the same manner as in Example 34, except that the polynucleotide binder of Example 42 was used.

[0256] Except for using the sample solutions from Examples 23, 34-35, 38-42, and Comparative Example 2 as sample solutions, target cells were cultured using the same procedure as in Experimental Example 3, and the introduction efficiency was calculated. The results are shown in Figures 26-27.

[0257] Figures 26-27 show that the sample solutions from each example exhibited higher delivery efficiency to target cells compared to Comparative Example 2, which did not contain a polynucleotide binder.

[0258] [Experimental Example 17] Polynucleotide binders with different amino acid sequences at the first binding site were prepared, and the efficiency of introducing polynucleotides into HEK293F cells was measured. The polynucleotide binders used were those listed in Tables 3D to 3E above. The plasmids and target cells used were the same as in Experimental Example 3.

[0259] (Example 43) IBC20-NBP1, indicated by amino acid number 86 (SEQ ID NO: 95) in Table 3D above, was used as the polynucleotide binder in Example 43. IBC20-NBP1 has a direct binding between the C-terminus of the first binding site indicated by amino acid number 51 (SEQ ID NO: 60) in Table 1B above and the N-terminus of the second binding site indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A above. IBC20-NBP1 can directly bind to the insulin receptor. The sample solution of Example 43 was prepared in the same manner as in Example 34, except that the polynucleotide binder of Example 43 was used and a receptor-binding protein was not used.

[0260] (Example 44) IBC2429-NBP1, indicated by amino acid number 87 (SEQ ID NO: 96) in Table 3D above, was used as the polynucleotide binder in Example 44. IBC2429-NBP1 has a direct binding at the C-terminus of the first binding site indicated by amino acid number 52 (SEQ ID NO: 61) in Table 1B above, and a direct binding at the N-terminus of the second binding site indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A above. IBC2429-NBP1 can directly bind to the insulin receptor. The sample solution for Example 44 was prepared in the same manner as in Example 43, except that the polynucleotide binder for Example 44 was used.

[0261] (Example 45) IBC20-NBP22, indicated by amino acid number 88 (SEQ ID NO: 97) in Table 3D above, was used as the polynucleotide binder in Example 45. IBC20-NBP22 has a direct binding at the C-terminus of the first binding site indicated by amino acid number 51 (SEQ ID NO: 60) in Table 1B above, and a direct binding at the N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above. IBC20-NBP22 can directly bind to the insulin receptor. The sample solution of Example 45 was prepared in the same manner as in Example 43, except that the polynucleotide binder of Example 45 was used.

[0262] (Example 46) IBC2429-NBP22, indicated by amino acid number 89 (SEQ ID NO: 98) in Table 3D above, was used as the polynucleotide binder in Example 46. IBC2429-NBP22 has a direct binding at the C-terminus of the first binding site indicated by amino acid number 52 (SEQ ID NO: 61) in Table 1B above, and a direct binding at the N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above. IBC2429-NBP22 can directly bind to the insulin receptor. The sample solution for Example 46 was prepared in the same manner as in Example 43, except that the polynucleotide binder for Example 46 was used.

[0263] (Example 47) IBC20-NBP21, indicated by amino acid number 90 (sequence number 99) in Table 3D above, was used as the polynucleotide binder in Example 47. IBC20-NBP21 has a direct binding at the C-terminus of the first binding site indicated by amino acid number 51 (sequence number 60) in Table 1B above, and a direct binding at the N-terminus of the second binding site indicated by amino acid number 69 (sequence number 78) in Table 2B above. IBC20-NBP21 can directly bind to the insulin receptor. The sample solution of Example 47 was prepared in the same manner as in Example 43, except that the polynucleotide binder of Example 47 was used.

[0264] (Example 48) TRBP-NBP22, indicated by amino acid number 91 (SEQ ID NO: 100) in Table 3D above, was used as the polynucleotide binder in Example 48. TRBP-NBP22 has a direct binding at the C-terminus of the first binding site indicated by amino acid number 6 (SEQ ID NO: 6) in Table 1A above, and a direct binding at the N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above. TRBP-NBP22 can directly bind to the transferrin receptor. The sample solution for Example 48 was prepared in the same manner as in Example 43, except that the polynucleotide binder for Example 48 was used.

[0265] (Example 49) In Table 3D above, TfBP-NBP22, indicated by amino acid number 92 (SEQ ID NO: 101), was used as the polynucleotide binder in Example 49. TfBP-NBP22 has a C-terminus of the first binding site indicated by amino acid number 5 (SEQ ID NO: 5) in Table 1A above, and a N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above, which are directly bound. TfBP-NBP22 can indirectly bind to the transferrin receptor via transferrin, which is a receptor-binding protein. The sample solution of Example 49 was prepared in the same manner as in Example 43, except that the polynucleotide binder of Example 49 was used and transferrin was used instead of insulin.

[0266] (Example 50) In Table 3E, T7-NBP28, indicated by amino acid number 93 (SEQ ID NO: 102), was used as the polynucleotide binder in Example 50. T7-NBP28 has a direct binding at the C-terminus of the first binding site, indicated by amino acid number 53 (SEQ ID NO: 62) in Table 1B, and a direct binding at the N-terminus of the second binding site, indicated by amino acid number 76 (SEQ ID NO: 85) in Table 2B. T7-NBP28 can directly bind to the transferrin receptor. The sample solution for Example 50 was prepared in the same manner as in Example 43, except that the polynucleotide binder for Example 50 was used.

[0267] (Example 51) SA21-NBP22, indicated by amino acid number 94 (SEQ ID NO: 103) in Table 3E above, was used as the polynucleotide binder in Example 51. SA21-NBP22 has a direct binding at the C-terminus of the first binding site indicated by amino acid number 54 (SEQ ID NO: 63) in Table 1B above, and a direct binding at the N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above. SA21-NBP22 can indirectly bind to the albumin receptor via albumin, which is a receptor-binding protein. The sample solution of Example 51 was prepared in the same manner as in Example 34, except that the polynucleotide binder of Example 51 was used and albumin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog number: 010-27601) was used instead of insulin.

[0268] (Example 52) HSABP-NBP22, indicated by amino acid number 95 (SEQ ID NO: 104) in Table 3E above, was used as the polynucleotide binder in Example 52. HSABP-NBP22 has a C-terminus of the first binding site indicated by amino acid number 55 (SEQ ID NO: 64) in Table 1B above, and a N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above, which are directly bound. HSABP-NBP22 can indirectly bind to the albumin receptor via albumin, which is a receptor-binding protein. The sample solution of Example 52 was prepared in the same manner as in Example 51, except that the polynucleotide binder of Example 52 was used.

[0269] (Example 53) In Table 3E, PepL1-NBP22, indicated by amino acid number 96 (SEQ ID NO: 105), was used as the polynucleotide binder in Example 53. PepL1-NBP22 has a direct binding at the C-terminus of the first binding site indicated by amino acid number 56 (SEQ ID NO: 65) in Table 1B, and a direct binding at the N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B. PepL1-NBP22 can directly bind to the albumin receptor. The sample solution for Example 53 was prepared in the same manner as in Example 43, except that the polynucleotide binder for Example 53 was used.

[0270] (Example 54) ITGBP1-NBP22, indicated by amino acid number 97 (SEQ ID NO: 106) in Table 3E above, was used as the polynucleotide binder in Example 54. ITGBP1-NBP22 has a direct binding at the C-terminus of the first binding site indicated by amino acid number 7 in Table 1A above, and a direct binding at the N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above. ITGBP1-NBP22 can directly bind to the integrin receptor. The sample solution of Example 54 was prepared in the same manner as in Example 43, except that the polynucleotide binder of Example 54 was used.

[0271] (Example 55) Lep1-NBP1, indicated by amino acid number 98 (SEQ ID NO: 107) in Table 3E above, was used as the polynucleotide binder in Example 55. Lep1-NBP1 has a direct binding between the C-terminus of the first binding site indicated by amino acid number 57 (SEQ ID NO: 66) in Table 1B above and the N-terminus of the second binding site indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A above. Lep1-NBP1 can directly bind to the leptin receptor. The sample solution of Example 55 was prepared in the same manner as in Example 43, except that the polynucleotide binder of Example 55 was used.

[0272] (Example 56) Lep1-NBP22, indicated by amino acid number 99 (SEQ ID NO: 108) in Table 3E above, was used as the polynucleotide binder in Example 56. Lep1-NBP22 has a direct binding at the C-terminus of the first binding site indicated by amino acid number 57 (SEQ ID NO: 66) in Table 1B above, and a direct binding at the N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above. Lep1-NBP22 can directly bind to the leptin receptor. The sample solution for Example 56 was prepared in the same manner as in Example 43, except that the polynucleotide binder for Example 56 was used.

[0273] (Example 57) LDLR1-NBP28, indicated by amino acid number 100 (SEQ ID NO: 109) in Table 3E above, was used as the polynucleotide binder in Example 57. LDLR1-NBP28 has a direct binding at the C-terminus of the first binding site indicated by amino acid number 58 (SEQ ID NO: 67) in Table 1B above, and a direct binding at the N-terminus of the second binding site indicated by amino acid number 76 (SEQ ID NO: 85) in Table 2B above. LDLR1-NBP28 can directly bind to the LDL receptor. The sample solution for Example 57 was prepared in the same manner as in Example 43, except that the polynucleotide binder for Example 57 was used.

[0274] (Example 58) LDLR2-NBP28, indicated by amino acid number 101 (SEQ ID NO: 110) in Table 3E above, was used as the polynucleotide binder in Example 58. LDLR2-NBP28 has a direct binding at the C-terminus of the first binding site, indicated by amino acid number 59 (SEQ ID NO: 68) in Table 1B above, and a direct binding at the N-terminus of the second binding site, indicated by amino acid number 76 (SEQ ID NO: 85) in Table 2B above. LDLR2-NBP28 can directly bind to the LDL receptor. The sample solution for Example 58 was prepared in the same manner as in Example 43, except that the polynucleotide binder for Example 58 was used.

[0275] (Example 59) In Table 3E, Cub1-NBP28, indicated by amino acid number 102 (SEQ ID NO: 111), was used as the polynucleotide binder in Example 59. Cub1-NBP28 has a direct bond between the C-terminus of the first binding site, indicated by amino acid number 60 (SEQ ID NO: 69) in Table 1B, and the N-terminus of the second binding site, indicated by amino acid number 76 (SEQ ID NO: 85) in Table 2B. Cub1-NBP28 can directly bind to cubilin. The sample solution for Example 59 was prepared in the same manner as in Example 43, except that the polynucleotide binder for Example 59 was used.

[0276] (Example 60) Mega1-NBP28, indicated by amino acid number 103 (sequence number 112) in Table 3E above, was used as the polynucleotide binder in Example 60. Mega1-NBP28 has a direct bond between the C-terminus of the first binding site, indicated by amino acid number 61 (sequence number 70) in Table 1B above, and the N-terminus of the second binding site, indicated by amino acid number 76 (sequence number 85) in Table 2B above. Mega1-NBP28 can directly bind to megalin. The sample solution for Example 60 was prepared in the same manner as in Example 43, except that the polynucleotide binder for Example 60 was used.

[0277] (Example 61) CD4a-NBP22, indicated by amino acid number 104 (SEQ ID NO: 113) in Table 3E above, was used as the polynucleotide binder in Example 61. CD4a-NBP22 has a C-terminus of the first binding site indicated by amino acid number 62 (SEQ ID NO: 71) in Table 1B above, and a N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above, both of which are directly bound. CD4a-NBP22 can directly bind to the CD4 receptor. The sample solution of Example 61 was prepared in the same manner as in Example 43, except that the polynucleotide binder of Example 61 was used.

[0278] (Example 62) CD4b-NBP22, indicated by amino acid number 105 (SEQ ID NO: 114) in Table 3E above, was used as the polynucleotide binder in Example 62. CD4b-NBP22 has a direct binding at the C-terminus of the first binding site indicated by amino acid number 63 (SEQ ID NO: 72) in Table 1B above, and a direct binding at the N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above. CD4b-NBP22 can directly bind to the CD4 receptor. The sample solution for Example 62 was prepared in the same manner as in Example 43, except that the polynucleotide binder for Example 62 was used.

[0279] (Example 63) CD28a-NBP22, indicated by amino acid number 106 (SEQ ID NO: 115) in Table 3E above, was used as the polynucleotide binder in Example 63. CD28a-NBP22 has a C-terminus of the first binding site indicated by amino acid number 64 (SEQ ID NO: 73) in Table 1B above, and a N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above, both of which are directly bound. CD28a-NBP22 can directly bind to the CD28 receptor. The sample solution for Example 63 was prepared in the same manner as in Example 43, except that the polynucleotide binder for Example 63 was used.

[0280] (Example 64) CD28b-NBP22, indicated by amino acid number 107 (SEQ ID NO: 116) in Table 3E above, was used as the polynucleotide binder in Example 64. CD28b-NBP22 has a direct binding at the C-terminus of the first binding site indicated by amino acid number 65 (SEQ ID NO: 74) in Table 1B above, and a direct binding at the N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above. CD28b-NBP22 can directly bind to the CD28 receptor. The sample solution for Example 64 was prepared in the same manner as in Example 43, except that the polynucleotide binder for Example 64 was used.

[0281] (Example 65) CD25-NBP22, indicated by amino acid number 108 (SEQ ID NO: 117) in Table 3E above, was used as the polynucleotide binder in Example 65. CD25-NBP22 has a C-terminus of the first binding site indicated by amino acid number 66 (SEQ ID NO: 75) in Table 1B above, and a N-terminus of the second binding site indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above, both of which are directly bound. CD25-NBP22 can directly bind to the CD25 receptor. The sample solution for Example 65 was prepared in the same manner as in Example 43, except that the polynucleotide binder for Example 65 was used.

[0282] Except for using the sample solutions from Examples 10, 43-65, and Comparative Example 2 as sample solutions, target cells were cultured using the same procedure as in Experimental Example 3, and the introduction efficiency was calculated. The results are shown in Figure 28.

[0283] Figure 28 shows that the sample solutions from each example exhibited higher delivery efficiency to target cells compared to Comparative Example 2, which did not contain a polynucleotide binder.

[0284] [Experimental Example 18] The efficiency of polynucleotide introduction and the number of viable cells were compared with and without the presence of the first binding site. The sample solution from Example 34 was used as the sample solution containing the polynucleotide binder having the first binding site.

[0285] (Comparative Example 13) As a molecule lacking a first binding site, NBP22, indicated by amino acid number 70 (SEQ ID NO: 79) of the second binding site in Table 2B above, was used. 3 μL of plasmid (1 μg / L) and 3 μL of NBP22 (5 μg / μL) were mixed and allowed to stand at room temperature for 5 minutes. 6 μL of the resulting solution was mixed with 3 μL of insulin (10 μg / μL) and allowed to stand at room temperature for 5 minutes to prepare the sample solution for Comparative Example 13.

[0286] Except for using the sample solutions from Example 34 and Comparative Example 13 as sample solutions, the gene transfer efficiency and the number of viable cells were measured using a flow cytometer in the same procedure as in Experimental Example 5. The results are shown in Figure 29. Figure 29(A) shows the value of the number of GFP-positive cells (gene transfer efficiency) with the number of viable cells as the denominator. Figure 29(B) shows the relative value of the number of viable cells in Comparative Example 13 when the number of viable cells in Example 34 is set to 100%. Figure 29(C) shows the relative value of the gene transfer efficiency in Comparative Example 13 when the gene transfer efficiency in Example 34 is set to 100%.

[0287] Figure 29 shows that the sample solution of the example resulted in a higher number of viable cells and higher gene transfer efficiency compared to the sample solution of Comparative Example 13, which did not have a first binding site.

[0288] [Experimental Example 19] The efficiency of plasmid vector introduction was compared with and without a polynucleotide binder. As polynucleotide binders, IBP-NBP22, indicated by binder amino acid number 78 (SEQ ID NO: 87) in Table 3D above, and Lep1-NBP22, indicated by binder amino acid number 99 (SEQ ID NO: 108) in Table 3E above, were used.

[0289] As plasmids, we used the following two plasmid vectors with different base numbers that are capable of expressing GFP: • Plasmid vector 4 (SEQ ID NO: 55, 12 kbp) • Plasmid vector 6 (SEQ ID NO: 118, 16 kbp)

[0290] (Example 66) A DNA-peptide complex was prepared by mixing 3 μL of plasmid vector 4 (1 μg / μL) and 3 μL of IBP-NBP22 (5 μg / μL) and reacting them at room temperature for 5 minutes. A sample solution containing the complex from Example 66 was prepared by mixing 6 μL of the DNA-peptide complex with 3 μL of insulin (10 μg / μL) and reacting them at room temperature for 5 minutes.

[0291] (Example 67) A sample solution containing the complex of Example 67 was prepared in the same manner as in Example 66, except that Lep1-NBP22 was used instead of IBP-NBP22.

[0292] (Example 68) A sample solution containing the complex of Example 68 was prepared in the same manner as in Example 66, except that plasmid vector 6 was used instead of plasmid vector 4.

[0293] (Example 69) A sample solution containing the complex of Example 69 was prepared in the same manner as in Example 67, except that plasmid vector 6 was used instead of plasmid vector 4.

[0294] (Comparative Example 14) The sample solution for Comparative Example 14 was prepared in the same manner as in Example 66, except that purified water was used instead of plasmid vector 4.

[0295] (Comparative Example 15) The sample solution for Comparative Example 15 was prepared in the same manner as in Example 68, except that purified water was used instead of plasmid vector 6.

[0296] Gene transfer efficiency was measured using a flow cytometer in the same procedure as in Experimental Example 5, except that the sample solutions from Examples 66-69 and Comparative Examples 14-15 were used as sample solutions. The results are shown in Figures 30-31. Figure 30 shows the results when a 14 kbp plasmid vector 4 was used. Figure 31 shows the results when a 16 kbp plasmid vector 6 was used.

[0297] Figures 30-31 show that the sample solutions in each example exhibited higher introduction efficiency compared to the sample solutions in the comparative examples.

[0298] [Experimental Example 20] An experiment was conducted to produce a virus by introducing polynucleotides into cells using a polynucleotide binder. As the polynucleotide binder, TRBP-GS-NBP1, indicated by binder amino acid number 43 (SEQ ID NO: 41) in Table 3A above, was used.

[0299] The following three plasmid vectors were used as polynucleotides: (1) Adeno-associated virus gene vector: pRC2-mi342 Vector (contains adeno-associated virus genes (AAV2 REP gene, AAV2 CAP gene) and an hsa-miR-342 expression cassette. Manufactured by Takara Bio Inc.) (2) Helper gene vector: pHelper Vector (contains adenovirus E2A gene, adenovirus E4 gene, and adenovirus VA gene. Manufactured by Takara Bio Inc.) (3) Target gene vector: pAAV-U6-ZsGreen1 Vector (contains the ZsGreen1 gene as the target gene between two ITR sequences. Manufactured by Takara Bio Inc.)

[0300] (Example 70) 0.5 μL of adeno-associated virus gene vector (1 μg / μL), 0.5 μL of helper gene vector (1 μg / μL), and 0.5 μL of target gene vector (1 μg / μL) were mixed. 1.5 μL of TRBP-GS-NBP1 (5 μg / μL) was added to the mixed polynucleotide solution and mixed, and the mixture was allowed to react at room temperature for 3 minutes. Then, 177 μL of Opti-MEM (Takara Bio Inc.) was added. This prepared the sample solution for Example 70.

[0301] (Comparative Example 16) 0.166 μL of adeno-associated virus gene vector (1 μg / μL), 0.166 μL of helper gene vector (1 μg / μL), and 0.166 μL of target gene vector (1 μg / μL) were mixed. 0.5 μL of PEIpro® (manufactured by Takara Bio Inc.) and 59 μL of serum-free Opti-MEM (manufactured by Takara Bio Inc.) were added to the mixed polynucleotide solution at room temperature and mixed. The mixture was allowed to react at room temperature for 15 minutes to prepare a sample solution.

[0302] (Comparative Example 17) The sample solution for Comparative Example 17 was prepared in the same manner as in Comparative Example 16, except that the amount of PEIpro added was 1.5 μL.

[0303] In the cell culture plate, the final concentration is 1 × 10⁻⁶. ５ HEK293F cells were seeded into each well to achieve a cell / well ratio. In the wells from which the HEK293F cells had been seeded and the culture medium had been removed, 60 μL each of the sample solutions from Example 70 and Comparative Examples 16-17 were added, and the mixture was allowed to stand at room temperature for 5 minutes.

[0304] To each well containing the sample solutions from Example 70 and Comparative Examples 16-17, 340 μL of HE100 medium (Gmep, product code: HE100-0010) was added until the final volume reached 400 μL. The mixture was then heated at 37°C and 5% CO2 by volume. ２ The cells were cultured for three days under environmental conditions.

[0305] Virus extraction from cultured cells, i.e., cells into which the complex was introduced, was performed using the TAKARABIO AAVpro® Helper Free System (manufactured by Takara Bio Inc.). Specifically, after cell culture, the recovered cell pellet was thoroughly loosened by a vortex mixer or tapping. Then, 50 μL of AAV. Extraction. Solution A was added and suspended in a vortex mixer for 15 seconds. After standing at room temperature for 5 minutes, it was suspended in a vortex mixer for another 15 seconds. The mixture was centrifuged at 10,000 × g at 4°C for 10 minutes, and the supernatant was collected in a new tube. 5 μL of AAV. Extraction. Solution B was added to the supernatant and mixed by pipetting to obtain the virus extracts of Example 70 and Comparative Examples 16-17.

[0306] The titers of the virus extracts from Example 70 and Comparative Examples 16-17 were measured using the AAV qPCR (Real-Time PCR) Rapid Titer Measurement Kit (AAVpro® Titation Kit (for Real Time PCR) Ver. 2, manufactured by Takara Bio Inc.) according to the kit's protocol. The results are shown in Figure 32. In Figure 32, the titer of Comparative Example 16 is set to 100%, and the titers of Example 70 and Comparative Example 17 are expressed as relative values.

[0307] Figure 32 shows that the virus extract from the example had a higher titer than all of the comparative examples.

[0308] [Experimental Example 21] An experiment was conducted to infect cells with viruses using the virus extracts from Example 70 and Comparative Examples 16-17. A mixture of 10 μL of each virus extract from Example 70 and Comparative Examples 16-17 and 10 μL of HE100 medium was used to infect HEK293F cells (1 × 10) that had been seeded the previous day. ５ Cells / well (48-well plate) was added dropwise. After standing for 10 minutes, HE100 medium was added to bring the total volume to 400 μL, and then the mixture was left at 37°C and 5% CO2 by volume. ２The cells were cultured in a controlled environment for three days. After culturing, the cells were observed using bright-field and fluorescence microscopy. To determine the number of virus-infected cells, HEK293F cells cultured on day 3 were analyzed by flow cytometry, and the number of green fluorescence-positive cells was counted. The results are shown in Figures 33-34.

[0309] Figure 33 shows, from left to right, untreated (i.e., cells not infected with the virus), cells treated with the virus extract from Comparative Example 16, cells treated with the virus extract from Comparative Example 17, and cells treated with the virus extract from Example 70. (A) shows a microscopic image obtained using fluorescence microscopy, (B) shows a microscopic image obtained using bright-field microscopy, and (C) shows an image obtained by overlaying the fluorescence microscopy and bright-field microscopy images. Figure 34 is a graph showing the number of virus-infected cells in Example 70 and Comparative Examples 16-17. In Figure 34, the number of infected cells is a graph showing the relative value of the number of virus-infected cells in Example 70 and Comparative Example 17, with the number of virus-infected cells in Comparative Example 16 set to 100%.

[0310] Figures 33 and 34 show that the expression of the fluorescent protein encoded by the target gene was confirmed when using the viral extracts from Example 70 and Comparative Examples 16-17, indicating that the virus had infected the cells. The viral extract from Example 70 was used to show a higher number of infected cells compared to the viral extracts from Comparative Examples 16-17, in which polynucleotides were introduced using a cationic polymer.

[0311] [Experimental Example 22] The viability of cells into which polynucleotides were introduced was investigated using the sample solutions from Example 70 and Comparative Examples 16-17. HEK293F cells into which the complex was introduced on day 3 of culture were analyzed by flow cytometry. The flow cytometry analysis results show the ratio of viable cells to the total number of cells. Gating was performed by gating the portion corresponding to viable cells based on the forward scattered light (FCSC) and side scattered light (SSC) values ​​when laser light was irradiated onto the cells into which the complex was introduced. The results are shown in Figures 35 and 36.

[0312] Figure 35 is a graph showing the flow cytometry analysis results for (A) Example 70, (B) Comparative Example 16, and (C) Comparative Example 17. In Figure 35, R1 indicates the region corresponding to living cells due to gating. Figure 36 is a graph showing the relative number of living cells in Example 70 and Comparative Example 17, with the number of living cells in Comparative Example 16 set to 100%.

[0313] As shown in Figure 36, the cell viability of Example 70 was found to be higher compared to Comparative Examples 16-17, in which polynucleotides were introduced using cationic polymers. This result suggests that the polynucleotide binder of the present invention has lower toxicity than the polynucleotides introduced using cationic polymers.

[0314] [Experimental Example 23] The introduction of polynucleotides encoding cell membrane-localized CARs (chimeric antigen receptors) into human peripheral blood unicellular cells (PBMCs) containing CD3-positive T cells was observed using the following procedure. As the polynucleotide binder, TfBP-GS-NBP1, indicated by binder amino acid number 41 (SEQ ID NO: 39) in Table 3A above, was used. PBMCs (CTL Corporation, catalog number: CTL-UP1) were used as target cells.

[0315] As the plasmid, mRNA encoding anti-hCD19-hBBzCAR was used. The mRNA was commissioned to Vectorbuilder Japan, and plasmid DNA (SEQ ID NO: 121) with the sequence shown in SEQ ID NO: 119 introduced under the T7 promoter was used to synthesize the mRNA using the IVT mRNA Synthesis Service. Plasmid DNA (SEQ ID NO: 121) contains the sequence shown in SEQ ID NO: 120. The sequence shown in SEQ ID NO: 120 includes a 5' UTR, an open reading frame shown in SEQ ID NO: 119, a 3' UTR, and a poly-A tail downstream of the T7 promoter.

[0316] As antibodies, the following three types of fluorescently labeled antibodies capable of binding to CD19-CAR, IgG antibody, and CD3 were used: • CD19-CAR-PE (Anti-FMC63 Idiotype, Catalog No. 130-127-342, Milltenyi Biotec) • IgG-PE (REA Control Antibody, human IgG1, PE, REAfinity®, Catalog No. 130-113-450, Milltenyi Biotec) • CD3-FITC (BD® CD3 FITC, Catalog No. 349201, BD)

[0317] (Example 71) A complex containing mRNA, a polynucleotide binder, and transferrin was prepared by mixing 1 μL of mRNA (1 μg / μL) with 1 μL of TfBP-GS-NBP1 (5 μg / μL), reacting at room temperature for 5 minutes, and then mixing in 1 μL of transferrin (10 μg / μL) and reacting at room temperature for 5 minutes. A sample solution containing the complex from Example 71 was prepared by mixing 2 μL of the complex containing mRNA and the polynucleotide binder with 57 μL of Alys 705 medium.

[0318] (Comparative Example 18) The sample solution for Comparative Example 18 was prepared in the same manner as in Example 71, except that purified water was used instead of TfBP-GS-NBP1.

[0319] Each well of the 48-well plate contains a final concentration of 5 × 10⁻⁶ ５ Target cells were seeded to a cell / well ratio. 0.4 mL of Allys® 705 (Cell Science Institute Co., Ltd.) medium was added to each well of a 48-well plate, and CO2 was added. ２ Incubator at 37°C, 5% CO2 by volume ２ The samples were incubated in the environment for 48 hours. 60 μL each of the sample solutions from Example 71 and Comparative Example 18 were added to the wells from which the culture medium had been removed, and the mixture was left to stand at room temperature for 5 minutes. 340 μL of Alys 705 medium was added to each well containing the sample solutions from Example 71 and Comparative Example 18, and the mixture was incubated at 37°C and 5% CO2 by volume. ２ The cells were cultured for four days under environmental conditions.

[0320] Cells 1 × 10⁶ days after sample solution addition (introduction treatment) ５ One μL of each antibody was added to the cells, and the cells were allowed to stand at 4°C for 30 minutes before staining. Subsequently, a gate representing living cells was set using a flow cytometer, and the expression of CD3 and CD19-CAR was evaluated by antibody staining. The results are shown in Figures 37-40.

[0321] Figure 37 shows the results when CD19-CAR-PE and CD3-FITC were used as antibodies. Figure 37(A) shows the results when the sample solution of Example 71 was used, and Figure 37(B) shows the results when the sample solution of Comparative Example 18 was used. Figure 38 shows the results when IgG-PE and CD3-FITC, which are negative controls of CD19-CAR, were used as antibodies. Figure 38(A) shows the results when the sample solution of Example 71 was used, and Figure 38(B) shows the results when the sample solution of Comparative Example 18 was used. Figure 39 shows the results when no antibodies were used. Figure 39(A) shows the results when the sample solution of Example 71 was used, and Figure 39(B) shows the results when the sample solution of Comparative Example 18 was used. Figure 40 shows the comparison of cell numbers under each condition obtained by flow cytometry, and shows the relative value when the number of CD19-CAR positive and CD3 positive cells when using the sample solution of Example 71 is set to 100%. For example, in Figure 40, "Control (+ / +)" indicates the relative number of CD19-CAR positive and CD3 positive cells when no antibody is used. "IgG / CD3 (+ / +)" indicates the relative number of IgG positive and CD3 positive cells. "CAR / CD3 (+ / +)" indicates the relative number of CD19-CAR positive and CD3 positive cells.

[0322] Figures 37-40 show that using the sample solution of Example 71 introduced a polynucleotide encoding the CD19-CAR sequence into CD3-positive T cells, and that the subsequently translated protein (CD19-CAR) localized to the cell membrane. Furthermore, it was shown that using the polynucleotide binder in the example resulted in greater CD19-CAR expression compared to using the sample solution of Comparative Example 18, which did not contain the polynucleotide binder.

[0323] [Experimental Example 24] The size of the plasmid vector used in this example was confirmed by agarose gel electrophoresis.

[0324] Plasmid vectors 2 (6.2 kbp), 3 (10 kbp), 4 (12 kbp), 5 (14 kbp), and 6 (16 kbp) were single-site cleaved with restriction enzymes and loaded into 100 ng of each well of an agarose gel. Xho I was used as the restriction enzyme for plasmid vectors 2, 3, 5, and 6. EcoRV was used for plasmid vector 4. A 0.5% agarose gel was used, and a DNA marker (Takara Bio, 2.5 kbp DNA Ladder) was used as the marker, loaded into 1 μL wells. Electrophoresis was performed at 100 mA for approximately 150 minutes. The results are shown in Figure 41.

[0325] Plasmid vector 6 (16 kbp) and plasmid vector 7 (SEQ ID NO: 150, 22 kbp) were single-site cleaved with restriction enzymes and loaded into 50 ng of each well of an agarose gel. Xho I was used as the restriction enzyme for plasmid vector 6, and Bln I was used for plasmid vector 7. A 0.5% agarose gel was used. A DNA marker (Takara Bio, 2.5 kbp DNA Ladder) was used as a marker, and the marker was loaded into 1 μL wells. Electrophoresis was performed at 50 mA for approximately 4 hours. The results are shown in Figure 42.

[0326] Figures 41 and 42 show that the actual size of the plasmid vector used in this embodiment is the expected size.

[0327] [Experimental Example 25] The binding of polynucleotides to polynucleotide binders was confirmed by the following procedure. Insulin was used as the receptor-binding protein.

[0328] (Example 72) IBP-NBP32, indicated by amino acid number 119 (SEQ ID NO: 132) in Table 3F above, was used as the polynucleotide binder in Example 72. IBP-NBP32 has a direct bond between the C-terminus of the first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above and the N-terminus of the second binding site indicated by amino acid number 118 (SEQ ID NO: 131) in Table 2C above.

[0329] (Example 73) IBP-NBP30, indicated by amino acid number 120 (SEQ ID NO: 132) in Table 3F above, was used as the polynucleotide binder in Example 73. IBP-NBP30 has a direct bond between the C-terminus of the first binding site indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above and the N-terminus of the second binding site indicated by amino acid number 116 (SEQ ID NO: 129) in Table 2C above.

[0330] (Example 74) Mega1-NBP22, indicated by amino acid number 121 (sequence number 134) in Table 3F above, was used as the polynucleotide bond in Example 74. Mega1-NBP22 has a direct bond between the C-terminus of the first binding site indicated by amino acid number 61 (sequence number 70) in Table 1B above and the N-terminus of the second binding site indicated by amino acid number 70 (sequence number 79) in Table 2B above.

[0331] (Example 75) In Table 3F above, Mega1-NBP24, indicated by amino acid number 122 (SEQ ID NO: 135), was used as the polynucleotide bond in Example 75. Mega1-NBP24 has a direct bond between the C-terminus of the first binding site, indicated by amino acid number 61 (SEQ ID NO: 70) in Table 1B above, and the N-terminus of the second binding site, indicated by amino acid number 72 (SEQ ID NO: 81) in Table 2C above.

[0332] (Comparative Example 19) In Table 9 below, IBP-WRAP1, indicated by amino acid number 137 (SEQ ID NO: 150), was used as the polynucleotide bond in Comparative Example 19. IBP-WRAP1 has a direct bond between the C-terminus of the first binding site, indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above, and the N-terminus of the second binding site, indicated by amino acid number 130 (SEQ ID NO: 143) in Table 8 below.

[0333] (Comparative Example 20) IBP-RICK, indicated by amino acid number 138 (SEQ ID NO: 151) in Table 9 below, was used as the polynucleotide bond in Comparative Example 20. IBP-RICK has a direct bond between the C-terminus of the first binding site, indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above, and the N-terminus of the second binding site, indicated by amino acid number 128 (SEQ ID NO: 141) in Table 8 below.

[0334] (Comparative Example 21) In Table 9 below, IBP-TAT, indicated by amino acid number 139 (SEQ ID NO: 152), was used as the polynucleotide bond in Comparative Example 21. IBP-TAT has a direct bond between the C-terminus of the first binding site, indicated by amino acid number 1 (SEQ ID NO: 1) in Table 1A above, and the N-terminus of the second binding site, indicated by amino acid number 129 (SEQ ID NO: 142) in Table 8 below.

[0335] (Comparative Example 22) IBP-H1-2, indicated by amino acid number 140 (sequence number 153) in Table 9 below, was used as the polynucleotide bond in Comparative Example 22. IBP-H1-2 has a direct bond between the C-terminus of the first binding site, indicated by amino acid number 1 (sequence number 1) in Table 1A above, and the N-terminus of the second binding site, indicated by amino acid number 132 (sequence number 145) in Table 8 below.

[0336] (Comparative Example 23) IBP-GS-KALA, indicated by comparative amino acid number 141 (sequence number 154) in Table 9 below, was used as the polynucleotide bond in Comparative Example 23. IBP-GS-KALA is indirectly bound to the C-terminus of the first binding site, indicated by amino acid number 1 (sequence number 1) in Table 1A above, and to the N-terminus of the second binding site, indicated by comparative amino acid number 131 (sequence number 144) in Table 8 below, via a non-binding site.

[0337] (Comparative Example 24) IBP-TP10, indicated by comparative amino acid number 142 (sequence number 155) in Table 9 below, was used as the polynucleotide bond in Comparative Example 24. IBP-TP10 has a direct bond between the C-terminus of the first binding site indicated by amino acid number 1 (sequence number 1) in Table 1A above and the N-terminus of the second binding site indicated by comparative amino acid number 133 (sequence number 146) in Table 8 below.

[0338] (Comparative Example 25) NBP1, indicated by amino acid number 20 (SEQ ID NO: 18) in Table 2A above, was used as the peptide in Comparative Example 25. In other words, the peptide in Comparative Example 25 consists only of the second binding site.

[0339] (Comparative Example 26) NBP22, indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above, was used as the peptide in Comparative Example 26.

[0340] (Comparative Example 27) NBP21, indicated by amino acid number 69 (SEQ ID NO: 78) in Table 2B above, was used as the peptide in Comparative Example 27.

[0341] (Comparative Example 28) NBP19, indicated by amino acid number 67 (SEQ ID NO: 76) in Table 2B above, was used as the peptide in Comparative Example 28.

[0342] (Comparative Example 29) WRAP1, indicated by amino acid number 130 (SEQ ID NO: 143) in Table 8 below, was used as the peptide in Comparative Example 29.

[0343] (Comparative Example 30) NBP20, indicated by amino acid number 68 (SEQ ID NO: 77) in Table 2B above, was used as the peptide for Comparative Example 30.

[0344] (Comparative Example 31) The MPG indicated by amino acid number 134 (SEQ ID NO: 147) in Table 8 below was used as the peptide in Comparative Example 31.

[0345] (Comparative Example 32) Pep-1, indicated by amino acid number 135 (SEQ ID NO: 148) in Table 8 below, was used as the peptide in Comparative Example 32.

[0346] (Comparative Example 33) In Table 8 below, TAT (47-57), indicated by amino acid number 129 (SEQ ID NO: 142), was used as the peptide for Comparative Example 33.

[0347]

[0348]

[0349] The polynucleotide binders of Examples 1, 34-36, 38, 39, 41, 72, and 73, and the polynucleotide binders of Comparative Examples 19-24 and 38 were used. A complex was prepared by mixing 4 μL of plasmid vector 2 (1 μg / μL) with 4 μL of the polynucleotide binder of each example (5 μg / μL) and reacting at room temperature for 5 minutes. A sample solution for each example was prepared by mixing 8 μL of the complex with 4 μL of insulin (10 μg / μL) and reacting at room temperature for 5 minutes.

[0350] The polynucleotide binders from Examples 10, 48, 74, and 75, and the peptides from Comparative Examples 25 to 33 were used. A complex was prepared by mixing 4 μL of plasmid vector 2 (1 μg / μL) with 4 μL of the polynucleotide binder or peptide from each example (5 μg / μL) and reacting at room temperature for 5 minutes. A sample solution for each example was prepared by mixing 8 μL of the complex with 4 μL of PBS and reacting at room temperature for 5 minutes.

[0351] The formation of the complex was confirmed by agarose gel electrophoresis. A 0.5% agarose gel was used, and electrophoresis was performed at 100 mV for 150 minutes. 6 μL of the sample solution for each example, prepared according to the procedure described above, was loaded into each well. A DNA marker (Nippon Gene, Inc., catalog number: OneSTEP Ladder 1kb) was used as the marker, and the marker was loaded so that 100 ng of DNA was present in each lane of the well. In addition, 4 μL of plasmid vector 2 was loaded. The results are shown in Figures 43 and 44. In Figures 43 and 44, "Marker" indicates the location where the DNA marker was loaded, and "DNA" indicates the location where plasmid vector 2 was loaded.

[0352] Figures 43 and 44 show that in the lanes loaded with the sample solutions for each example, the plasmid vector band either disappeared or became fainter, indicating that the plasmid vector formed a complex by binding with the polynucleotide binder or peptide.

[0353] [Experimental Example 26] The introduction efficiency of polynucleotides into HEK293F cells was confirmed using the following procedure.

[0354] (Example 76) In Table 3F above, IBC(B25-30)-NBP22, indicated by amino acid number 123 (SEQ ID NO: 136), was used as the polynucleotide bond in Example 76. IBC(B25-30)-NBP22 has a direct bond between the C-terminus of the first binding site, indicated by amino acid number 109 (SEQ ID NO: 122) in Table 1C above, and the N-terminus of the second binding site, indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above.

[0355] (Example 77) Mega3-NBP22, indicated by amino acid number 130 (sequence number 143) in Table 3F above, was used as the polynucleotide bond in Example 77. Mega3-NBP22 has a direct bond between the C-terminus of the first binding site indicated by amino acid number 111 (sequence number 124) in Table 1C above and the N-terminus of the second binding site indicated by amino acid number 70 (sequence number 79) in Table 2B above.

[0356] (Comparative Example 34) A sample solution for Comparative Example 34 was prepared by mixing 1 μL of plasmid (1 μg / μL) with 1 μL of purified water.

[0357] The polynucleotide binders from Examples 10, 48, 53, 55, and 74-77, and the peptides from Comparative Examples 25-33 were used. Plasmid vectors 2 and 7 were used as polynucleotides. A mixture for each example was prepared by mixing 0.5 μL of plasmid vector (1 μg / μL) with 0.5 μL of the polynucleotide binder or peptide (5 μg / μL) from each example and reacting at room temperature for 5 minutes. A sample solution for each example was prepared by mixing 1 μL of the resulting mixture with 0.5 μL of insulin (10 μg / μL), letting it stand for 5 minutes, and then adding 58.50 μL of HE100 cell culture medium. Target cells were cultured using the same procedure as in Experimental Example 5, except that the sample solutions from Examples 10, 48, 53, 55, and 74-77 and Comparative Examples 25-33 were used, and the translocation efficiency was calculated. The results are shown in Figure 45.

[0358] The polynucleotide binders of Examples 1, 34-36, and Comparative Examples 19-24 and 38 were used. Plasmid vectors 2 and 7 were used as polynucleotides. 0.5 μL of plasmid vector (1 μg / μL) and 0.5 μL of the polynucleotide binder or peptide (5 μg / μL) of each example were mixed and reacted at room temperature for 5 minutes. 59.00 μL of HE100 cell culture medium was then added to prepare the sample solution for each example. Except for using the sample solutions of Examples 1, 34-36, and Comparative Examples 19-24, 38 and 34, target cells were cultured using the same procedure as in Experimental Example 5, and the translocation efficiency was calculated. The results are shown in Figure 45.

[0359] Figure 45 shows that the sample solutions of each example had higher introduction efficiency compared to the sample solutions of the comparative examples. Comparative Example 19, which had 16 amino acid residues at the second binding site and a tryptophan content of 25%, had low introduction efficiency. Comparative Example 20, which had 19 amino acid residues at the second binding site and a tryptophan content of 21.1%, also had low introduction efficiency. Comparative Examples 21 and 22, which had 8 cationic amino acid residues at the second binding site, also had low introduction efficiency. Comparative Example 23, which had 30 amino acid residues at the second binding site and 7 cationic amino acid residues, also had low introduction efficiency. Comparative Example 24, which had 21 amino acid residues at the second binding site, also had low introduction efficiency. Comparative Example 38, which had 6 cationic amino acid residues at the second binding site, also had low introduction efficiency.

[0360] Graphs were created for HEK293F cells into which polynucleotides were introduced using the polynucleotide binders of Examples 1, 34-36, 53, 74, and 75, and the peptides of Comparative Examples 25-28. The vertical axis represents the introduction efficiency, and the horizontal axis represents the relative value of the cell number. Figure 46 shows the case when plasmid vector 2 was used as the polynucleotide, and Figure 47 shows the case when plasmid vector 7 was used. In Figures 46 and 47, the relative value of the cell number shown on the horizontal axis represents the percentage of the cell number when using each example's polynucleotide binder or peptide, with the number of cells after culture when using the polynucleotide binder of Example 1 being set to 100%.

[0361] Figures 46 and 47 show that when the polynucleotide binder of the example was used, the introduction efficiency was higher compared to when the peptide of the comparative example was used. Furthermore, since the number of cells did not decrease significantly, it was shown that the cytotoxicity to target cells was low. Comparative examples 25 to 28, which used only the second binding site, showed lower introduction efficiency and a significant decrease in the number of cells compared to the example.

[0362] [Experimental Example 27] The introduction efficiency of 6.2 kbp and 22 kbp polynucleotides into HEK293F cells was confirmed by the following procedure.

[0363] (Comparative Example 35) In Table 8 below, 9R (non-arginine), represented by amino acid number 136 (SEQ ID NO: 149), was used as the peptide for Comparative Example 35.

[0364] (Comparative Example 36) IBP-9R, represented by amino acid number 143 (sequence number 156) in Table 9 below, was used as the polynucleotide binder in Comparative Example 36. IBP-9R has a direct bond between the C-terminus of the first binding site, indicated by amino acid number 1 (sequence number 1) in Table 1A above, and the N-terminus of the second binding site, indicated by amino acid number 136 (sequence number 149) in Table 8 below.

[0365] (Comparative Example 37) IBC(B25-30)-9R, represented by amino acid number 144 (sequence number 157) in Table 9 below, was used as the polynucleotide binder in Comparative Example 37. IBC(B25-30)-9R has a direct bond between the C-terminus of the first binding site, indicated by amino acid number 109 (sequence number 122) in Table 1C above, and the N-terminus of the second binding site, indicated by amino acid number 136 (sequence number 149) in Table 8 below.

[0366] The polynucleotide binders of Examples 1 and 34 and the polynucleotide binder of Comparative Example 36 were used. Plasmid vectors 2 and 7 were used as polynucleotides. A mixture for each example was prepared by mixing 0.5 μL of plasmid vector (1 μg / μL) with 0.5 μL of the polynucleotide binder or peptide (5 μg / μL) of each example and reacting at room temperature for 5 minutes. A sample solution for each example was prepared by mixing 1 μL of the resulting mixture with 0.5 μL of insulin (10 μg / μL), letting it stand for 5 minutes, and then adding 58.50 μL of HE100 cell culture medium. Target cells were cultured using the same procedure as in Experimental Example 5, except that the sample solutions of Examples 1, 34 and Comparative Example 36 were used, and the translocation efficiency was calculated. The results are shown in Figure 48.

[0367] The polynucleotide binders from Example 76 and Comparative Example 37, and the peptide from Comparative Example 35 were used. Plasmid vectors 2 and 7 were used as polynucleotides. 0.5 μL of plasmid vector (1 μg / μL) and 0.5 μL of the polynucleotide binder or peptide from each example (5 μg / μL) were mixed and reacted at room temperature for 5 minutes. 59.00 μL of HE100 cell culture medium was then added to prepare the sample solution for each example. Except for using the sample solutions from Example 76, Comparative Examples 34, 35, and 37, target cells were cultured using the same procedure as in Experimental Example 5, and the translocation efficiency was calculated. The results are shown in Figure 48.

[0368] Figure 48 shows that both 9R and the comparative examples containing 9R as the second binding site exhibited lower introduction efficiency compared to the example. In other words, it was shown that when the number of cationic amino acid residues in the second binding site is 9, the introduction efficiency is low.

[0369] [Experimental Example 28] An experiment was conducted to produce a virus by introducing polynucleotides into cells using a polynucleotide binder. The polynucleotide binders used were the same as those in Examples 1, 10, 34, and 48, and the same peptides as those in Comparative Examples 25 and 26.

[0370] As the polynucleotide, we used the all-in-one vector pAAV2-ZsGreen1 One-Vector (containing the ZsGreen1 gene as the target gene between two ITR sequences, manufactured by Takara Bio, catalog number: 6264).

[0371] Instead of HEK293F cells used in Experimental Example 20, VPC cells (Viral Production Cells 2.0, Thermo Fisher Sciences, Catalog No.: A49784) and Viral Production Medium (Gibco, Product No.: A4817901) were used. Instead of polynucleotide binders, the same polynucleotide binders as in Examples 1, 10, 34, and 48, and the same peptides as in Comparative Examples 25 and 26 were used. Furthermore, plasmid vectors were introduced into VPC cells and cultured using the same procedure as in Experimental Example 20, except that an all-in-one vector was used as the polynucleotide. In addition, viral extracts for each example were obtained using the same procedure as in Experimental Example 20.

[0372] For the virus extracts of Examples 1, 10, 34, 48 and Comparative Examples 25, 26, the titer of the virus extracts was measured using the AAV qPCR (Real-Time PCR) Rapid Titer Measurement Kit (AAVpro® Titation Kit (for Real Time PCR) Ver. 2, manufactured by Takara Bio Inc.) according to the kit's protocol to determine the amount of virus (AAV) produced. The results are shown in Figures 49 and 50. In Figures 49 and 50, the relative value of the AAV production shown on the vertical axis represents the percentage of the AAV production in each example, with the AAV production in Example 10 set to 100%.

[0373] Figures 49 and 50 show that using the polynucleotide binder of the example resulted in a higher viral production compared to using only the second binding site.

[0374] [Experimental Example 29] An experiment was conducted to produce a virus by introducing polynucleotides into cells using a polynucleotide binder. The sample solutions used were those from Examples 53, 75, 76, and Comparative Examples 25-35 and 38. The plasmid vector used was the same all-in-one vector as in Experimental Example 28.

[0375] (Comparative Example 38) A sample solution for Comparative Example 38 was prepared by mixing 1 μL of plasmid (1 μg / μL) and 3 μL of PEIpro (registered trademark, manufactured by POLYplus, catalog number: 101000017) in 56 μL of culture medium (Opti-MEM, catalog number: 31985062) and allowing it to stand at room temperature for 15 minutes.

[0376] Plasmid vectors were introduced into VPC cells using the same procedure as in Experimental Example 28, and the cells were cultured to obtain viral extracts for each example. The titer of the virus (AAV) was measured using the same procedure as in Experimental Example 20. The results are shown in Figures 51 and 52. Furthermore, the number of virus-infected cells was measured using the same procedure as in Experimental Example 21, and the infection efficiency was determined. The results are shown in Figure 53. In Figure 53, the AAV vector infection efficiency represents the ratio of the number of virus-infected cells in each example to 100% of the number of virus-infected cells in Example 76.

[0377] Figures 51 and 52 show that the virus extract from the example had a higher titer than the virus extract from the comparative example. Figure 53 shows that when the virus extract from Example 76 was used, the number of infected cells was higher compared to when the virus extracts from Comparative Examples 25 and 26 were used, which were obtained by introducing the plasmid vector using only the second binding site.

[0378] [Experimental Example 30] The introduction of polynucleotides into target cells, induced pluripotent stem cells (iPS cells, STEMCELL Technologies, SCTi003A), was observed using the following procedure. StemFit® AK02N medium (Ajinomoto Co., Inc.) was used as the culture medium. Plasmid vector (Plasmid Vector 2) and mRNA (TriLnk Biotechnologies, L-7201-100) were used as the polynucleotides.

[0379] (Comparative Example 39) A sample solution for Comparative Example 39 was prepared by mixing 1 μL of mRNA with 3 μL of Lipofectamine Stem Transfer Reagent (Thermo Fisher Scientific, STEM00003).

[0380] (Comparative Example 40) A sample solution for Comparative Example 40 was prepared by mixing 1 μL of mRNA with 1 μL of purified water.

[0381] An mRNA-peptide complex was prepared by mixing 1 μL of mRNA (1 μg / μL) with 1 μL of the polynucleotide binder from Example 34 and reacting at room temperature for 5 minutes. A sample solution containing the complex from Example 34 was prepared by mixing 2 μL of the mRNA-peptide complex with 1 μL of insulin (10 μg / μL), reacting at room temperature for 5 minutes, and then mixing with 57 μL of Opti-MEM medium (GIbco, product number 31985062). An mRNA-peptide complex was prepared by mixing 1 μL of mRNA (1 μg / μL) with 1 μL of the polynucleotide binder from Example 49 and reacting at room temperature for 5 minutes. A sample solution containing the complex of Example 49 was prepared by mixing 2 μL of the mRNA-peptide complex with 1 μL of transferrin (10 μg / μL), reacting at room temperature for 5 minutes, and then mixing with 57 μL of Opti-MEM medium (GIbco, product no. 31985062).

[0382] In the cell culture plate, the final concentration is 2 × 10 ４ iPS cells were seeded to achieve a cell / well ratio. In the wells from which the iPS cells had been seeded and the culture medium had been removed, 60 μL each of the mRNA-containing sample solutions from Examples 34 and 49 and Comparative Examples 39 and 40 were added and left to stand at room temperature for 5 minutes.

[0383] 340 μL of StemFit® AK02N medium (manufactured by Ajinomoto Co., Inc.) was added to each of the wells to which the sample solution had been added, and the mixture was incubated at 37°C and 5% CO2. ２ Cells were cultured for two days under environmental conditions. After culturing, bright-field fluorescence and bright-field fluorescence were used to observe GFP levels in target cells, respectively. The results are shown in Figure 54. In addition, the efficiency of mRNA delivery to target cells was calculated by measuring GFP-positive cells and viable cells at a flow site after culturing. The results are shown in Figure 55.

[0384] The efficiency of DNA delivery to target cells was calculated using the same procedure as when introducing mRNA, except that plasmid vector 2 was used instead of mRNA. The results are shown in Figure 56.

[0385] Figures 54 and 55 show that Examples 34 and 49 had a higher efficiency in introducing mRNA into iPS cells compared to Comparative Example 40. Figure 56 shows that Examples 34 and 49 had a higher efficiency in introducing plasmid vectors into iPS cells compared to Comparative Examples 39 and 40.

[0386] [Experimental Example 31] The introduction of polynucleotides into target cells, bone marrow-derived mesenchymal stem cells (BM-MSCs, STEMCELL Technologies, 70071), was observed using the following procedure. The culture medium used was R: STEM Medium for hMSC High Growth medium (Rohto Pharmaceutical Co., Ltd., catalog number: EM-1-500).

[0387] (Example 78) In Table 3F above, GE11-NBP24, indicated by amino acid number 125 (SEQ ID NO: 138), was used as the polynucleotide bond in Example 78. GE11-NBP24 has a direct bond between the C-terminus of the first binding site, indicated by amino acid number 113 (SEQ ID NO: 126) in Table 1C above, and the N-terminus of the second binding site, indicated by amino acid number 72 (SEQ ID NO: 81) in Table 2C above.

[0388] (Example 79) In Table 3F above, D4-NBP22, indicated by amino acid number 126 (SEQ ID NO: 139), was used as the polynucleotide bond in Example 79. D4-NBP22 has a direct bond between the C-terminus of the first binding site, indicated by amino acid number 114 (SEQ ID NO: 127) in Table 1C above, and the N-terminus of the second binding site, indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above.

[0389] The sample solutions for each example were prepared using the same procedure as in Experimental Example 30, except that the polynucleotide binders of Examples 48, 53, 75, 76, 78, and 79 were used instead of the polynucleotide binders of Examples 34 and 49, and purified water was used instead of the receptor-binding protein.

[0390] The introduction of mRNA into target cells was confirmed using the same procedure as in Experimental Example 30, except that the sample solutions from Examples 48, 53, 74, 76, 78, and 79 were used instead of the sample solutions from Examples 34 and 49, and BM-MSCs were used as the target cells and R:STEM Medium for hMSC High Growth medium was used as the culture medium. The efficiency of mRNA and plasmid vector introduction into target cells was also confirmed. The results of confirming mRNA introduction into target cells are shown in Figure 57. The results of confirming mRNA introduction efficiency into target cells are shown in Figure 58. The results of confirming plasmid vector introduction efficiency into target cells are shown in Figure 59.

[0391] Figures 57 and 58 show that Examples 48, 53, 74, 76, 78, and 79 demonstrated higher mRNA introduction efficiency into BM-MSCs compared to Comparative Example 40. Figure 59 shows that Examples 76 and 79 demonstrated higher plasmid vector introduction efficiency into BM-MSCs compared to Comparative Example 40.

[0392] [Experimental Example 32] The introduction of polynucleotides into target cells, adipose-derived stem cells (ADSCs, manufactured by Lonza Corporation, catalog number: PT-5006 HADSCs), was observed using the following procedure. PT-4505 medium (manufactured by Lonza Corporation) was used as the culture medium.

[0393] (Example 80) In Table 3F above, PepL1-NBP22, indicated by amino acid number 127 (SEQ ID NO: 140), was used as the polynucleotide bond in Example 80. PepL1-NBP22 has a direct bond between the C-terminus of the first binding site, indicated by amino acid number 56 (SEQ ID NO: 65) in Table 1B above, and the N-terminus of the second binding site, indicated by amino acid number 70 (SEQ ID NO: 79) in Table 2B above.

[0394] The sample solutions for each example were prepared using the same procedure as in Experimental Example 30, except that the polynucleotide binders of Examples 48, 53, 74, 76, 78, and 80 were used instead of the polynucleotide binders of Examples 34 and 49, and purified water was used instead of the receptor-binding protein.

[0395] The introduction of mRNA into target cells was confirmed using the same procedure as in Experimental Example 30, except that the sample solutions from Examples 48, 53, 74, 76, 78, and 80 were used instead of the sample solutions from Examples 34 and 49, and ADSC was used as the target cell and PT-4505 medium as the culture medium. The efficiency of mRNA and plasmid vector introduction into target cells was also confirmed. The results of confirming mRNA introduction into target cells are shown in Figure 60. The results of confirming mRNA introduction efficiency into target cells are shown in Figure 61. The results of confirming plasmid vector introduction efficiency into target cells are shown in Figure 62.

[0396] Figures 60 and 61 show that Examples 53, 74, 76, 78, and 80 demonstrated higher mRNA introduction efficiency into ADSCs compared to Comparative Example 40. Figure 62 shows that Examples 48, 53, 74, 76, and 78 demonstrated higher plasmid vector introduction efficiency into ADSCs compared to Comparative Example 40.

[0397] [Experimental Example 33] The introduction of polynucleotides into target cells, human fibroblasts (HDF, Takara Bio, D10051), was observed using the following procedure. Fibroblast Growth Medium 2 (Promocell, catalog number: C-23020) was used as the culture medium.

[0398] The sample solution for Example 53 was prepared using the same procedure as in Experimental Example 30, except that the polynucleotide binder from Example 53 was used instead of the polynucleotide binders from Examples 34 and 49, and purified water was used instead of the receptor binding protein.

[0399] The introduction of mRNA into target cells was confirmed using the same procedure as in Experimental Example 30, except that the sample solution from Example 53 was used instead of the sample solutions from Examples 34 and 49, and HDF was used as the target cells and Fibroblast Growth Medium 2 medium was used as the culture medium. The results are shown in Figure 63.

[0400] After culturing, the number of GFP-positive cells was measured at the flow site. The results are shown in Figure 64. In Figure 64, the relative value of the number of GFP-positive cells shown on the vertical axis represents the relative value of the number of GFP-positive cells in Example 53 and Comparative Example 39, with the number of GFP-positive cells in Comparative Example 40 set to 100%.

[0401] Figure 63 shows that Example 53 demonstrated higher plasmid vector introduction efficiency into HDF compared to Comparative Examples 39 and 40.

[0402] [Experimental Example 34] The expression of Cav1, Cav2, and Cavin1 was confirmed in the Cav1 overexpression strain, Cav2 overexpression strain, and Cavin1 overexpression strain prepared in Experimental Examples 8 and 9. The expression levels of Cav1, Cav2, and Cavin1 were also confirmed.

[0403] To confirm the high expression of target proteins in Cav1-highly expressing cells, Cav2-highly expressing cells, and Cavin1-highly expressing cells, the molecular weight of the proteins was measured by Western blotting. The Western blotting conditions were as follows: The electrophoresis sample was prepared by mixing an equal volume of the protein sample with a 2x concentrated DTT-containing sample buffer (Ato Corporation, AE-1430 EzApply (2x concentrate)). Polyacrylamide gel: EHR-T10L (Ato Corporation) Electrophoresis buffer: Electrophoresis buffer (x10) (Fujifilm Wako Pure Chemical Industries, Ltd.) Molecular weight marker: WSE-7020 EzProtein Ladder (Ato Corporation) Staining solution: EzStain AQua (Ato Corporation)

[0404] Figure 65 shows images of Western blotting results using Cav1 antibody, Cav2 antibody, and Cavin1 antibody on Cav1-highly expressing cells (Jurkat+CAV1), Cav2-highly expressing cells (Jurkat+CAV2), and Cavin1-highly expressing cells (Jurkat+Cavin1). In Figure 65, wild-type Jurkat cells are indicated as "Jurkat-WT".

[0405] As shown in Figure 65, Western blotting was also performed on wild-type Jurkat cells as a control example, but no signal was detected. Comparison with wild-type Jurkat cells confirmed that Cav1-highly expressing cells, Cav2-highly expressing cells, and Cavin1-highly expressing cells were indeed high in Cav1, Cav2, and Cavin1, respectively.

[0406] Figure 66 is a graph showing the evaluation results of mRNA levels in cells overexpressing caveolin 1, caveolin 2, and Cavin 1, respectively. mRNA levels were evaluated by PCR (Takara Bio, 3735S). In Figure 66, Jurkat+CAV1 represents cells into which pEF1-CAV1-puro has been introduced (i.e., cells overexpressing caveolin 1). Jurkat+CAV2 represents cells into which pEF1-CAV2-puro has been introduced (i.e., cells overexpressing caveolin 2). Jurkat+CAVIN1 represents cells into which pEF1-Cavin1-puro has been introduced (i.e., cells overexpressing Cavin 1). Since mRNA could not be detected in Jurkat (wild strain), the amount of mRNA equivalent to caveolin 1, caveolin 2, or cabin 1 in each cell is shown as a relative value, with the amount equivalent to caveolin 1, caveolin 2, or cabin 1 in HEK293F (wild strain) set to 1.

[0407] Figure 66 shows that cells overexpressing caveolin 1, caveolin 2, and cabin 1 respectively had mRNA levels more than 200 times higher than HEK293F cells (wild-type).

[0408] 1,2...complex, 10...polynucleotide, 20a,20b...polynucleotide binder, 21,22...first binding site, 23,24...second binding site, 30...receptor-binding protein, 40...receptor

Claims

1. A polynucleotide binder comprising a first binding site and a second binding site, wherein the first binding site comprises an amino acid sequence capable of binding to a receptor-binding protein that binds to a receptor on a target cell, and the second binding site comprises an amino acid sequence capable of binding to a polynucleotide.

2. The polynucleotide binder according to claim 1, wherein the molecular weight of the polynucleotide is 350 kDa or more.

3. The polynucleotide binder according to claim 1, wherein the number of amino acid residues contained in the first binding site and the second binding site is 5 or more and 100 or less.

4. The polynucleotide binder according to claim 1, wherein the amino acid sequence of the first binding site has 80% or more sequence identity with the amino acid sequence represented by SEQ ID NOs: 1 to 17, SEQ ID NOs: 60 to 75, or SEQ ID NOs: 122 to 127.

5. The polynucleotide binder according to claim 1, wherein the second binding site contains a cationic amino acid.

6. The polynucleotide binder according to claim 1, wherein the polynucleotide is at least one of single-stranded or double-stranded DNA and RNA.

7. The polynucleotide binder according to claim 1, wherein the polynucleotide is at least one of circular DNA and linear DNA.

8. The polynucleotide binder according to claim 1, wherein the polynucleotide is a double-stranded polynucleotide forming a complementary chain, and the number of base pairs contained in the polynucleotide is 1 kbp or more.

9. A polynucleotide transport composition comprising the polynucleotide binder according to any one of claims 1 to 8, and a polynucleotide.

10. The polynucleotide transport composition according to claim 9, wherein the content of cationic polyethyleneimine or a derivative thereof, or a cationic lipid, is 0.1% by mass or less.

11. The polynucleotide transport composition according to claim 9, further comprising a receptor-binding protein.

12. A polynucleotide transport composition comprising the polynucleotide binder, polynucleotide, and target cell according to any one of claims 1 to 8.

13. The polynucleotide transport composition according to claim 12, wherein the content of cationic polyethyleneimine or cationic lipid is 0.1% by mass or less.

14. The polynucleotide transport composition according to claim 12, further comprising a receptor-binding protein.

15. A method for producing a virus, comprising the step of introducing at least a polynucleotide from the cell membrane into the cytoplasm by contacting a target cell with a polynucleotide binder according to any one of claims 1 to 8 or a polynucleotide transport composition according to any one of claims 9 to 14, and producing a virus in the target cell.

16. The method for producing a virus according to claim 15, wherein the virus is a virus expressed from an adeno-associated virus gene.

17. A method for producing cells, comprising the step of introducing at least a polynucleotide from the cell membrane into the cytoplasm by contacting a target cell with a polynucleotide binder according to any one of claims 1 to 8 or a polynucleotide transport composition according to any one of claims 9 to 14, wherein the target cell expresses an exogenous membrane protein or overexpresses an intrinsic membrane protein on the cell membrane.

18. The cell production method according to claim 17, wherein the foreign membrane protein is a chimeric antigen receptor.

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