Liposome containing shRNA molecule for thymidylate synthase, and use for same

A technology of thymidylate synthase and plastids, which is applied in the field of chemotherapy drugs, can solve the problems of side effects and inability to obtain sufficient effects, and achieve the effect of inhibiting proliferation

Active Publication Date: 2014-02-05
DELTA FLY PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the lipoplex is repeatedly administered, the lipoplex is quickly eliminated due to the action of the immune system in the administered organism, and there are problems that a sufficient effect cannot be obtained and serious side effects occur.

Method used

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  • Liposome containing shRNA molecule for thymidylate synthase, and use for same
  • Liposome containing shRNA molecule for thymidylate synthase, and use for same
  • Liposome containing shRNA molecule for thymidylate synthase, and use for same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1: Preparation of RNAi molecules

[0092] The following siRNA and shRNA were synthesized according to well-known conventional methods.

[0093] (I) siRNA targeting TS

[0094] The siRNA targeting TS was synthesized based on the anti-TS siRNA (WO2010 / 113844) whose antitumor effect was confirmed, and consisted of the following sense strand and antisense strand.

[0095] Sense strand:

[0096] 5'-GUAACACCAUCGAUCAUGA-3' (SEQ ID NO: 1)

[0097] Antisense strand:

[0098] 5'-UCAUGAUCGAUGGUGUUAC-3' (SEQ ID NO: 2)

[0099] Hereinafter, siRNA targeting TS is referred to as "siTS".

[0100] (II) siRNA targeting luciferase

[0101] As a control siRNA, siRNA targeting luciferase was synthesized. This siRNA consists of a sense strand and an antisense strand as described below.

[0102] Sense strand:

[0103] 5'-CUUACGCUGAGUACUUCGATT-3' (SEQ ID NO: 9)

[0104] Antisense strand:

[0105] 5'-UCGAAGUACUCAGCGUAAGTT-3' (SEQ ID NO: 10)

[0106] Hereinafter, siRNA targ...

Embodiment 2

[0112] Example 2: Inhibition of TS expression by siRNA and shRNA

[0113]

[0114] Lipofectamine, a type of cationic liposome, is used as a transfection reagent TM RNAi MAX (hereinafter referred to as "Lf RNAi MAX").

[0115] The shRNA or siRNA prepared in Example 1 and Lf RNAi MAX were diluted and mixed with OptiMEM respectively, so that the ratio of shRNA or siRNA and Lf RNAi MAX reached 100 (pmol): 5( mu L). At this time, the amount of shRNA or siRNA solution is equal to that of Lf RNAi MNX solution. A complex (lipoplex) is formed by allowing the mixed solution to stand at room temperature for 10 to 20 minutes.

[0116] Each lipoplex was directly added to a 10 cm Petri dish previously filled with OptiMEM, and adjusted so that the total volume became 5 ml. Next, 10 ml of DLD-1 or DLD-1 / FU cell suspension was inoculated in the culture dish so as to reach 500,000 cells / dish, and the final total volume was 15 ml for transfection. At this time, adjust so that the fina...

Embodiment 3

[0128] Example 3 : Cancer cell (human colon adenocarcinoma) growth inhibitory effect of siRNA and shRNA

[0129] In this experiment, experiments were performed on a 96-well plate scale. The lipoplex prepared in the same way as in Example 2 above was directly added to the wells previously equipped with OptiMEM, and the total volume reached 50 mu l. Next, the cell suspension of human colon adenocarcinoma cells DLD-1 or DLD-1 / FU (2,000 cells / 100 mu l) Add in wells containing lipoplex (final total volume of 150 mu l), carry out transfection. Here, the final concentration of shRNA or siRNA in the wells was 5 nM.

[0130] 24 hours after the start of transfection, the medium was removed and 200 mu l New media with or without the existing chemotherapeutic drug 5-FU (fluorouracil). Here, relative to DLD-1, with 0.1 mu Add 5-FU at a concentration of g / mL, relative to DLD-1 / FU, at 10 mu 5-FU was added at a concentration of g / mL. Add new medium, remove medium after 0, 24, 4...

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Abstract

This method provides a method for delivering shRNA targeting TS in vivo. In addition, the following is provided: an antitumor agent, which comprises short hairpin RNA (shRNA) capable of inhibiting expression of thymidylate synthase by RNAi action and a PEG-modified cationic liposome, wherein the shRNA is bound to the surface of the PEG-modified cationic liposome and has an overhang comprising at least two nucleotides at the 3′ end.

Description

technical field [0001] The present invention relates to an antitumor drug and its use, especially its use in combination with a chemotherapeutic drug, which uses a liposome containing an anti-thymidylate synthase shRNA molecule as an active ingredient. Background technique [0002] In recent years, RNAi molecules that cause RNA interference (hereinafter referred to as "RNAi") have attracted attention as useful tools for treating tumors and the like, and various RNAi molecules that can suppress tumor growth are being developed. The present inventors previously reported an RNAi molecule targeting thymidylate synthase (hereinafter referred to as "TS") involved in DNA synthesis, and it was reported that the RNAi molecule had an antitumor effect by significantly inhibiting the expression of TS; in addition, It also enhances the antitumor effect of 5-FU antitumor drugs (especially the compound drug of tegafur-gimeracil-oteracil potassium) (Patent Document 1). [0003] Usually, ho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K9/127A61K31/513A61K31/713A61K45/00A61K47/18A61K47/24A61K47/28C12N15/113
CPCC12N2320/32A61K45/06A61K31/513C12N2310/14A61K31/7105C12N15/1137C12N2320/31A61K9/0019A61K9/1272A61P35/00A61K2300/00
Inventor 石田龙弘黃政龙和田洋巳
Owner DELTA FLY PHARMA
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