Novel Method of Nucleic Acid Transfer

• US 20080318319A1
• Filed: 12/15/2004
• Published: 12/25/2008
• Est. Priority Date: 12/19/2003
• Status: Abandoned Application

• Current Assignee: National Cancer Center
• Original Assignee: National Cancer Center
• Inventors: Ochiya, Takahiro, Minakuchi, Yoshiko
• Application Number:
  US10/583,277
• Publication Number:
  US 20080318319A1
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1136 against growth factors, growth regulators, cytokines, lymphokines or hormones
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific

Abstract

The present invention provides a method of nucleic acid transfer comprising the following steps (a) and (b):

• • (a) contacting a nucleic acid with a cell in a medium; and
  • (b) following the step (a), contacting the medium of (a) with a high-concentration solution of a metal salt,
    a nucleic acid transfer agent comprising solid metal salt or a high-concentration solution of a metal salt as an ingredient, and the like.

Claims

1. A method of nucleic acid transfer comprising the following steps (a) and (b):

(a) contacting a nucleic acid with a cell in a medium; and

(b) following the step (a), contacting the medium of (a) with a high-concentration solution of a metal salt.

2. The method of nucleic acid transfer according to claim 1, wherein the nucleic acid is a single-stranded DNA, a double-stranded DNA, a single-stranded RNA, a double-stranded RNA, an oligonucleotide or a ribozyme.

3. The method of nucleic acid transfer according to claim 2, wherein the double-stranded DNA or the double-stranded RNA is in the linear or cyclic form.

4. The method of nucleic acid transfer according to claim 3, wherein the cyclic double-stranded DNA is in the form of expression plasmid.

5. The method of nucleic acid transfer according to claim 2, wherein the oligonucleotide is a deoxyribonucleotide, a ribonucleotide, a phosphorothioate oligodeoxynucleotide, a 2′

-O-(2-methoxy)ethyl-modified nucleic acid (2′

-MOE-modified nucleic acid), a small interfering RNA (siRNA), a cross-linked nucleic acid (locked nucleic acid;

LNA), a peptide nucleic acid (PNA) or a morpholino antisense nucleic acid.

6. The method of nucleic acid transfer according to claim 1, wherein the nucleic acid is in the form of a complex or an inclusion body with a biodegradable substance or a living body-derived substance.

7. The method of nucleic acid transfer according to claim 6, wherein the living body-derived substance is atelocollagen.

8. The method of nucleic acid transfer according to claim 1, wherein the concentration of the high-concentration solution of a metal salt to be contacted with the medium obtained in the step (a) is within the range of 0.1 M-3.0 M.

9. The method of nucleic acid transfer according to claim 8, wherein the concentration of the high-concentration solution of a metal salt to be contacted with the medium obtained in the step (a) is within the range of 0.5 M-2.0 M.

10. The method of nucleic acid transfer according to claim 1, wherein the volume of the high-concentration solution of a metal salt to be contacted with the medium obtained in the step (a) is within the range of 1 μ

L-20 μ

L per 500 μ

L of the medium of step (a).

11. The method of nucleic acid transfer according to claim 10, wherein the volume of the high-concentration solution of a metal salt to be contacted with the medium obtained in the step (a) is within the range of 2 μ

L-10 μ

L per 500 μ

L of the medium of step (a).

12. The method of nucleic acid transfer according to claim 1, wherein the solution of a metal salt is a solution of a divalent metal chloride.

13. The method of nucleic acid transfer according to claim 12, wherein the solution of a divalent metal chloride is a solution of calcium chloride.

14. A nucleic acid transfer agent comprising a solid metal salt or a high-concentration solution of a metal salt as an ingredient.

16. The nucleic acid transfer agent according to claim 14, wherein the concentration of the high-concentration solution of a metal salt is within the range of 0.1 M-6.0M.

17. The nucleic acid transfer agent according to claim 16, wherein the concentration of the high-concentration solution of a metal salt is within the range of 0.5 M-4.0 M.

18. The nucleic acid transfer agent according to claim 14, wherein the metal salt is a chloride of divalent metal.

19. The nucleic acid transfer agent according to 18, wherein the chloride of a divalent metal is calcium chloride.

20. A kit for nucleic acid transfer which comprises a nucleic acid transfer agent set forth in claim 14.

21. Use of a nucleic acid transfer agent or a kit set forth in claim 14 in the nucleic acid transfer.

15. (canceled)