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NOVEL CARRIER MOLECULE AND METHOD BOTH FOR DELIVERING RNA INTO CYTOPLASM USING NEAR-INFRARED LIGHT

Foreign code F130007322
File No. S2011-0272-C0
Posted date Apr 26, 2013
Country WIPO
International application number 2011JP077075
International publication number WO 2012127739
Date of international filing Nov 24, 2011
Date of international publication Sep 27, 2012
Priority data
  • P2011-063953 (Mar 23, 2011) JP
Title NOVEL CARRIER MOLECULE AND METHOD BOTH FOR DELIVERING RNA INTO CYTOPLASM USING NEAR-INFRARED LIGHT
Abstract The purpose of the present invention is to provide a means for introducing RNA into a cell utilizing near-infrared light that has good biological tissue permeability (preferably having a wavelength of about 700-1000 nm) without causing the problem of cytotoxicity or heat generation. A carrier molecule according to the present invention is characterized by having a structure comprising: a carrier protein which comprises a cell-membrane-permeable peptide (CPP) and an RNA-binding protein (RBP); and a photosensitizer (PST) which is connected to the N-terminal side or the C-terminal side of the carrier protein and can act upon the irradiation with light having a wavelength falling within a near infrared region. A (carrier molecule)/RNA complex composed of the carrier molecule and RNA bound to the RBP in the carrier molecule is formed, the complex is brought into contact with a cell to localize the complex in an endosome in the cell, and subsequently the cell is irradiated with near infrared light with which the PST in the (carrier molecule)/RNA complex can act. In this manner, the (carrier molecule)/RNA complex can be dispersed in a cytoplasm, whereby the function of the introduced RNA can be exerted.
Outline of related art and contending technology BACKGROUND ART
Is RNAi, siRNA or shRNA depending on the nucleotide sequence of a particular gene expression is repressed phenomenon, disease-causing gene or suppression of expression of the viral genes can be used in applications such as disease treatment based on very recent years has attracted attention. In recent years, by generating ions and emitting, relieve stress or discomfort, virus and fungi, allergens cause allergic diseases or rendered harmless by the ion generator can be employed in various applications and is, for example, an unspecified number of persons used by the ion generating device placed in the elevator has been a growing demand for.
Is Patent Document 1, membrane permeable carrier peptide RNA binding protein and a fusion protein, the recognition sequence for the RNA-binding protein having a sequence and any bound RNA, the conjugate of any of the cells by mixing, the RNA introduced into the cell and method described.
Non-patent document 1 and 2 is, the carrier peptide RNA binding protein and membrane permeability in addition to the wavelength in the visible region excited with light of a fluorescent dye (for example Alexa Fluor 546, 530-550 nm excitation wavelength) to a fusion protein comprising siRNA bound to the complex is described. Then, contacting a cell with this conjugate, the conjugate incorporated into cells by endocytosis after localization to the endosome, by irradiating the excitation light, the fluorescent dye acts as a photosensitizer, is released into the cytoplasm from the endosome siRNA, a predetermined RNAi effect can be achieved (see Fig. 1) described. In addition, non-patent document 1 and 2 is, near-infrared region of wavelength excited with light of a fluorescent pigment may also be used to obtain the above-mentioned effect can be achieved is, not describe or suggest.
On the other hand, Patent Document 2 is, as a carrier of a functional molecule gold nano-rods, gold nano-rod complex aggregates DNA and added to the cultured cell, a near-infrared region (wavelength 1064 nm) by the pulse laser is irradiated, the gold nanorods to eliminate DNA, effective expression of the DNA of the gene from the described method (see Examples). However, the photothermal conversion function and has gold nano-rods may be, gold nano-rods are integrated on a photo thermal treatment of the surrounding tissue to the target site (tumor cells kill heat generated) is also used for the material (see for example Patent Document 3), as such is not intended for use in the cytotoxicity or concern for safety.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 細胞膜透過性ペプチド(CPP)およびRNA結合性蛋白質(RBP)を含むキャリア蛋白質と、該キャリア蛋白質のN末端側またはC末端側に連結した、近赤外線領域の波長を有する光で機能する光増感剤(PST)とからなる構造を有することを特徴とするキャリア分子。

[請求項2]
 前記光増感剤(PST)が700nm~1000nmの波長を有する光で機能するものである、請求項1に記載のキャリア分子。

[請求項3]
 前記光増感剤(PST)が、下記構造式で表される化合物(DY750)、図3に示す蛍光スペクトルを有する化合物(Alexa Fluor750)または図4に示す吸収スペクトルを有する化合物(IRDye800CW)である、請求項1または2に記載のキャリア分子。
[化1]

[請求項4]
 前記キャリア蛋白質が、さらにプロテアソーム分解シグナル配列タグ(PDS)および/またはHis-richタグ(HR)を含む、請求項1~3のいずれかに記載のキャリア分子。

[請求項5]
 前記キャリア蛋白質において、N末端側から、細胞膜透過性ペプチド(CPP)、RNA結合性蛋白質(RBP)、プロテアソーム分解シグナル配列タグ(PDS)および/またはHis-richタグ(HR)がこの順序で連結されており、かつ当該キャリア蛋白質のC末端側に前記光増感剤(PST)が連結されている、請求項1~4のいずれかに記載のキャリア分子。

[請求項6]
 前記CPPが、配列番号1で示されるアミノ酸配列を有する、ヒト免疫不全ウイルス由来Tat(trans-activator of transcription)蛋白質中の12アミノ酸からなるペプチドである、請求項1~5のいずれかに記載のキャリア分子。
YGRKKRRQRRRG              配列番号1

[請求項7]
 前記RBPが、配列番号4で示されるアミノ酸配列を有する、ヒト由来U1A(U1 small nuclear ribonucleoprotein A)のRNA結合ドメインである、請求項1~6のいずれかに記載のキャリア分子。
AVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQILDILVSRSLKMRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK                      配列番号4

[請求項8]
 請求項1~7のいずれかに記載のキャリア分子と、該RNAキャリア分子中のRBPに結合したRNAとからなる構造を有するキャリア分子/RNA複合体。

[請求項9]
 前記RNAが、short hairpin RNA(shRNA)、small interfering RNA(siRNA)またはmicroRNA(miRNA)である、請求項8に記載のRNA複合体。

[請求項10]
 請求項8または9に記載のキャリア分子/RNA複合体を含む遺伝子治療薬。

[請求項11]
 がんまたはウイルス疾患を対象とする、請求項10に記載の遺伝子治療薬。

[請求項12]
 生体外において、請求項8または9に記載のキャリア分子/RNA複合体を細胞と接触させて当該細胞内のエンドソームに局在化させる工程、および該キャリア分子/RNA複合体中のPSTが機能する近赤外線領域の波長を有する光を照射して該キャリア分子/RNA複合体を細胞質中に拡散させる工程を含むことを特徴とする、細胞質内へのRNAの送達方法。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION OKAYAMA UNIVERSITY
  • Inventor
  • OHTSUKI, Takashi
  • ISHIODORI, Yuka
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PE PG PH PL PT QA RO RS RU RW SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ MD RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

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