# US 7,745,596 B2 · Nuclear transport nucleic acid delivery vector

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- Number: US 7,745,596 B2
- Document ID: US7745596B2
- Status: Granted
- Kind: B2
- Application number: 11/632,138
- Filed: 2005-07-11
- Published: 2007-12-06
- Granted: 2010-06-29
- Earliest claimed: 2004-07-12
- Assignees: Japan Science and Technology Agency
- Inventors: Seiji Shinkai; Takeshi Nagasaki; Takeshi Kawazu; Shinji Kakimoto
- Web page: https://patentgrep.com/patent/US7745596B2

## CPC

- C12N 15/88: using microencapsulation, e.g. using {amphiphile} liposome vesicle
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C07K 2319/09: containing a nuclear localisation signal
- C07K 2319/23: containing a GST-tag
- C12N 2810/6072: negative strand RNA viruses
- C12N 2810/85: mammalian
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

To provide a new technique by which efficient transfection is ensured in delivering a target gene into a cell, disclosed is a nucleic acid construct for nuclear import, which comprises a ternary complex consisting of a nucleic acid substance containing a gene to be delivered into the nucleus of a cell, an importin protein (for example, importin-β) capable of passing through the nuclear pore and involved in the nuclear transport, and a binding substance (for example, polyethyleneimine) bound to both of the nucleic acid substance and the importin protein. Nucleic acid transport from outside of a cell into the cell nucleus can be particularly promoted by administering the nucleic acid construct bound to a cell membrane receptor binding factor and/or a membrane fusing substance, or administering the nucleic acid construct encapsulated in a non-viral vector (for example, Sendai virus envelope) having cell membrane permeability and membrane fusing properties.

## Claims

### Claim 1

A nucleic acid complex for nuclear import comprising a nucleic acid encoding a gene to be delivered into the nucleus of a cell, an importin protein capable of passing through a nuclear pore and involved in nuclear transport, and a polycationic binding substance bound to both said nucleic acid and said importin protein, wherein said nucleic acid is bound to said polycationic binding substance via a non-covalent bond such that the gene remains intact, and wherein the binding between said binding substance and said importin protein is via a biotin-avidin interaction in which the polycationic binding substance is biotinylated and bound to the importin protein via a biotin-avidin interaction.

### Claim 2 (depends on 1)

The nucleic acid complex for nuclear import as claimed in claim 1, wherein the polycationic binding substance is polyethyleneimine.

### Claim 3 (depends on 1)

The nucleic acid complex for nuclear import as claimed in claim 1, wherein the polycationic binding substance is a cationic polymer which is responsive to an intracellular reductant or light so as to decompose into lower molecular weight substances.

### Claim 4 (depends on 3)

The nucleic acid complex for nuclear import as claimed in claim 3, wherein the polycationic binding substance is polyethyleneimine having a disulfide crosslinkage.

### Claim 5 (depends on 1)

The nucleic acid complex for nuclear import as claimed in claim 1, wherein the importin protein is a protein selected from importin-β, importin 7, transportin, transportin SR, and CAS protein.

### Claim 6 (depends on 5)

The nucleic acid complex for nuclear import as claimed in claim 5, wherein the importin protein is importin-β.

### Claim 7 (depends on 1)

The nucleic acid complex for nuclear import as claimed in claim 1, wherein the nucleic acid is a plasmid DNA.

### Claim 8

A nucleic acid complex for nuclear import consisting of:
a) nucleic acid encoding a gene to be delivered into the nucleus of a cell,
b) an importin protein capable of passing through a nuclear pore and involved in nuclear transport,
c) at least one of:
i) a cell membrane receptor binding factor, and
ii) a membrane fusing substance, and
d) a polycationic binding substance bound to said nucleic acid, said importin protein, and said at least one of the cell membrane binding factor and the membrane fusing substance, wherein said nucleic acid is bound to said polycationic binding substance via a non-covalent bond such that the gene remains intact, and wherein the binding of the polycationic substance with each of said importin protein and said at least one of the cell membrane receptor binding factor and the membrane fusing substance is via a covalent bond or non-covalent interaction.

### Claim 9 (depends on 8)

The nucleic acid complex for nuclear import as claimed in claim 8, wherein the cell membrane receptor binding factor is present in said complex and is selected from transferrin, EGF (epidermal growth factor), FGF (fibroblast growth factor), HGF (hepatocyte growth factor), NGF (nerve growth factor), TGF (transforming growth factor), LDL (low density lipoprotein), insulin, folic acid, diphtheria toxin, integrin binding factor, and asialoglycoprotein receptor binding factor.

### Claim 10 (depends on 8)

The nucleic acid complex for nuclear import as claimed in claim 8, wherein the membrane fusing substance is present in said complex and is selected from influenza virus hemagglutinin HA-2, human immunodeficiency virus Tat, GALA, and diphtheria toxin T-domain.

### Claim 11 (depends on 9)

The nucleic acid complex for nuclear import as claimed in claim 9, wherein the cell membrane receptor binding factor is transferrin.

### Claim 12 (depends on 10)

The nucleic acid complex for nuclear import as claimed in claim 10, wherein the membrane fusing substance is GALA.

### Claim 13

A nucleic acid complex for nuclear import comprising a) a nucleic acid encoding a gene to be delivered into the nucleus of a cell, b) an importin protein capable of passing through a nuclear pore and involved in nuclear transport, c) optionally, at least one of a cell membrane receptor binding factor and a membrane fusing substance, and d) a binding substance bound to said nucleic acid, said importin protein, and said at least one of the cell membrane receptor binding factor and the membrane fusing substance, where present, said nucleic acid being bound to said binding substance via a non-covalent bond such that the gene remains intact, the binding of said binding substance with said importin protein being via a covalent bond or non-covalent interaction, the binding of said binding substance with said at least one of the cell membrane receptor binding factor and the membrane fusing substance, where present, being via a covalent bond or non-covalent interaction, and said complex being encapsulated in an envelope or capsid of viral origin.

### Claim 14 (depends on 13)

The nucleic acid complex for nuclear import as claimed in claim 13, wherein the complex is encapsulated in a Sendai virus envelope.

### Claim 15 (depends on 1)

An in vitro method of delivering a gene into cells comprising contacting a nucleic acid complex as claimed in claim 1 with the cells in vitro, wherein the nucleic acid is introduced into the cells and the gene is expressed.

### Claim 16 (depends on 8)

The nucleic acid complex for nuclear transport as claimed in claim 8, wherein the binding of the polycationic binding substance with said importin protein and the at least one of the cell membrane receptor binding factor and the membrane fusing substance is via a biotin-avidin interaction in which said polycationic binding substance, said importin protein, and said at least one of the cell membrane receptor binding factor and the membrane fusing substance are biotinylated to form a complex via streptavidin interaction.