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Nucleic acid-encapsulating polymer micelle complex and method for producing same

Foreign code F170009181
File No. K10503WO
Posted date Sep 12, 2017
Country United States of America
Application number 201414909162
Gazette No. 20160184457
Date of filing Aug 5, 2014
Gazette Date Jun 30, 2016
International application number JP2014070567
International publication number WO2015020026
Date of international filing Aug 5, 2014
Date of international publication Feb 12, 2015
Priority data
  • P2013-163106 (Aug 6, 2013) JP
  • 2014JP70567 (Aug 5, 2014) WO
Title Nucleic acid-encapsulating polymer micelle complex and method for producing same
Abstract A nucleic acid-encapsulating polymer micelle complex is formed of a block copolymer containing an uncharged hydrophilic polymer chain block and a cationic polymer chain block; and two single-stranded DNA molecules having mutually complementary base sequences of 1000 or more bases in length, double-stranded DNA of 1000 or more base pairs in length in which at least a part of a double helix structure is dissociated and forms a single-stranded structure, or one single-stranded DNA molecule of 1000 or more bases in length.
Outline of related art and contending technology BACKGROUND ART
As a next-generation treatment, gene therapy for treating diseases by controlling gene expression has been greatly anticipated.The biggest problem with gene therapy is that introduction efficiency at the time when genes are introduced into target cells or tissues is insufficient.Particularly, in order to realize gene therapy through systemic administration, it is necessary that genes be stably circulated in the blood and accumulated on target tissues and that gene expression be effectively performed after genes have entered target tissues.Here, in order to solve these problems, development of gene carriers having better introduction efficiency to target cells or the like, and gene expression efficiency in target cells has been actively promoted.
For example, it is known that a polymer in which a primary structure is precisely controlled is spontaneously organized and may form a higher-order structure such as a micelle or a vesicle and use of a structure obtained by a polymer being self-organized in such a manner has been previously examined in various fields including drug delivery systems and material science.For example, PTL 1 discloses an electrostatic binding type polymer micelle drug carrier formed of a block copolymer including an uncharged segment (uncharged polymer chain block) and a charged segment (charged polymer chain block) and capable of encapsulating a drug having an opposite charge to that of the charged segment, in a core portion.When a cationic segment is used as the charged segment, it is possible to encapsulate DNA in the core portion.
Furthermore, research performed for stabilization of a polymer micelle in various manners has been reported.For example, in regard to an electrostatic binding type polymer micelle drug carrier.PTL 2 discloses an electrostatic binding type polymer micelle drug carrier stabilized by crosslinking block copolymers through a crosslinking agent.In addition, PTL 3 discloses a block copolymer formed by containing an uncharged hydrophilic polymer chain block and a cationic polyamino acid chain block in which a hydrophobic group is introduced into a part of the side chain thereof.By virtue of a hydrophobic group introduced into the side chain of the block copolymer, interfacial energy is increased, thereby the cohesive force in a micelle becomes higher and the core becomes smaller, and thus, the polymer micelle is stabilized.
Scope of claims [claim1]
1. A nucleic acid-encapsulating polymer micelle complex formed of a block copolymer containing an uncharged hydrophilic polymer chain block and a cationic polymer chain block; and two single-stranded DNAs having mutually complementary base sequences of 1000 or more bases in length, double-stranded DNA of 1000 or more base pairs in length in which at least a part of a double helix structure is dissociated and forms a single-stranded structure, or one single-stranded DNA of 1000 or more bases in length.
[claim2]
2. The nucleic acid-encapsulating polymer micelle complex according to claim 1, formed of a block copolymer containing an uncharged hydrophilic chain block and a cationic polymer chain block; and two single-stranded DNAs having mutually complementary base sequences of 1000 or more bases in length or double-stranded DNA of 1000 or more base pairs in length in which at least a part of a double helix structure is dissociated and forms a single-stranded structure.
[claim3]
3. The nucleic acid-encapsulating polymer micelle complex according to claim 1,
wherein the single-stranded DNA is 2000 or more bases in length, and
the double-stranded DNA is 2000 or more base pairs in length.
[claim4]
4. The nucleic acid-encapsulating polymer micelle complex according to claim 1, wherein the average particle diameter thereof in an aqueous medium measured according to a dynamic light scattering method is 100 nm or less.
[claim5]
5. The nucleic acid-encapsulating polymer micelle complex according to claim 1,
wherein the DNA and the cationic polymer chain block bonded to the DNA due to an electrostatic interaction form a core portion, and
the uncharged hydrophilic polymer chain block forms a shell portion.
[claim6]
6. The nucleic acid-encapsulating polymer micelle complex according to claim 5, wherein the average particle diameter of the core portion is 50 nm or less.
[claim7]
7. The nucleic acid-encapsulating polymer micelle complex according to claim 1, wherein the complex is spherical.
[claim8]
8. The nucleic acid-encapsulating polymer micelle complex according to claim 1, wherein the single-stranded DNA or the double-stranded DNA is linear.
[claim9]
9. The nucleic acid-encapsulating polymer micelle complex according to claim 1, wherein at least a part of the block copolymer is mutually cross-linked.
[claim10]
10. The nucleic acid-encapsulating polymer micelle complex according to claim 1, wherein a hydrophobic group is covalently bonded to a main chain or a side chain of the cationic polymer chain block.
[claim11]
11. The nucleic acid-encapsulating polymer micelle complex according to claim 1, wherein the cationic polymer chain block has an ethylamine structure or a propylamine structure in the side chain thereof.
[claim12]
12. A method for producing a nucleic acid-encapsulating polymer micelle complex which accommodates DNA, the method comprising:
a process of mixing a block copolymer containing an uncharged hydrophilic polymer chain block and a cationic polymer chain block with double-stranded DNA of 1000 or more base pairs in a state in which at least a part of a double helix structure is dissociated, in an aqueous medium.
[claim13]
13. The method for producing a nucleic acid-encapsulating polymer micelle complex according to claim 12, wherein the double-stranded DNA is 2000 or more base pairs in length.
[claim14]
14. The method for producing a nucleic acid-encapsulating polymer micelle complex according to claim 12, wherein the double-stranded DNA is linear.
[claim15]
15. The method for producing a nucleic acid-encapsulating polymer micelle complex according to claim 12, wherein the double-stranded DNA has been denatured at 60° C. or higher.
  • Inventor, and Inventor/Applicant
  • KATAOKA Kazunori
  • OSADA Kensuke
  • TOCKARY Theofilus Agrios
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
IPC(International Patent Classification)
Reference ( R and D project ) PRESTO Molecular technology and creation of new functions AREA
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