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Drug- or gene-carrier composition having lowered hemagglutinin activity

A composition and carrier technology, applied in the field of drug carrier composition or gene carrier composition with reduced blood coagulation activity, can solve the problem of drug delivery system with reduced blood coagulation activity

Inactive Publication Date: 2008-01-02
DNAVEC RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Therefore, so far there is no known drug delivery system based on negative-strand RNA viral envelope proteins whose hemagglutination activity is significantly reduced while the ability to deliver drugs into cells remains unchanged

Method used

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  • Drug- or gene-carrier composition having lowered hemagglutinin activity
  • Drug- or gene-carrier composition having lowered hemagglutinin activity
  • Drug- or gene-carrier composition having lowered hemagglutinin activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] [Example 1] Preparation and purification of SeV vector containing NLS-LacZ gene

[0127] The NLS-LacZ / SeV carrying the LacZ gene containing a nuclear localization signal was obtained by a previously published method (Kato et al., Genes Cells, 1996, 1, 569-579; Hasan et al., J.Gen.Virol., 1997, 78 , 2813-2820) prepared. The vector was inoculated into eggs containing 10-day-old embryos. After three days of incubation at 35.3°C, the allantoic fluid was harvested and centrifuged at 4,000 rpm for 15 minutes. The resulting supernatant was then centrifuged at 10,000 rpm for one hour to pellet the vector. After resuspension in PBS, the vector was layered on a sucrose density gradient (30% / 50%) and centrifuged at 25,000 rpm for one hour (in a Beckmanrotor SW28). The vector at the sucrose interface was recovered, centrifuged, pelleted, and then resuspended in PBS to prepare a stock solution of the purified vector (hereinafter described as SeV vector). The protein concentratio...

Embodiment 2

[0128] [Example 2] Preparation of TPEG-modified SeV vector

[0129] The SeV vector stock solution in Example 1 was diluted to 2 mg protein / ml in PBS. To 0.5 ml of this solution was added 0.5 ml of 0.5 M boric acid buffer (pH 8.5) to prepare a solution of 1 mg protein / ml at pH 8.5. 1 mg of Tresyl-activated PEG reagent (TPEG, MW: 5000) (Shearwater Polymers) was added in a small amount to the above solution while stirring, and reacted at room temperature for 90 minutes ( FIG. 1 ). After the reaction was completed, the reaction mixture was diluted with ice-cold PBS (14 ml), and then the carrier was recovered by centrifugation at 15,000 rpm for one hour (in a Beckman rotor SW28.1). The vector was resuspended in PBS (0.8 ml), and a TPEG-modified SeV vector was obtained. The protein concentration and HAU of the modified carrier were determined as in Example 1.

Embodiment 3

[0130] [Example 3] Utilize TPEG-modified SeV vector to carry out gene expression in vitro

[0131] The SeV vector stock solution in Example 1 was diluted to 1×10 in PBS 5pfu / ml. The protein concentrations of the TPEG-modified SeV vector solution and the non-modified SeV vector solution in Example 2 were standardized, so that the two vectors provided equal numbers of virus particles. The day before the experiment, HeLa cells (from human cervical cancer) were mixed with 5×10 4 The cells / well were cultured (1 ml / well of MEM medium containing 10% inactivated fetal calf serum (FCS) was added) on a 12-well plate (Sumitomo Bakelite). After reducing the medium to 0.5ml, add 50μl of the above-mentioned diluted carrier solution (equivalent to 5×10 3 pfu / well, moi=0.1), in 5% CO 2 Infections were performed at 37°C when present. After one hour, the carrier was removed and the cells were washed twice with medium. Add fresh medium (2ml) to the cells, then in 5% CO 2 Incubate further ...

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Abstract

The present invention provides pharmaceutical- or gene-carrier compositions with reduced hemagglutinating activity. By attaching a compound to a minus-strand RNA virus envelope protein having hemagglutinating activity, a pharmaceutical- or gene-carrier composition with lower hemagglutinating activity than a composition to which the compound has not been attached can be successfully constructed. For example, an embodiment of the present invention provides a viral vector whose erythrocyte agglutination activity and hemolytic activity are significantly lowered, and whose stability in blood is remarkably elevated. The pharmaceutical- or gene-carrier compositions provided in this invention can be preferably used for transferring pharmaceuticals or genes in vivo.

Description

technical field [0001] The present invention relates to vectors for drug or gene delivery into cells whose hemagglutination activity is reduced by modifying the envelope protein of a negative-strand RNA virus. Background technique [0002] Sendai virus (SeV) includes two types of outer envelope proteins: hemagglutinin-sialidase (HN) and fusion protein (F). The HN protein binds to the sialic acid on the cell surface, and then fuses with the cell through the mediation of the F protein, so that the SeV can directly, rapidly and efficiently infect and enter the cytoplasm. Moreover, because sialic acid is present on the surface of almost all cells, SeV is capable of infecting a wide range of cells. In order to apply these highly infectious SeVs to gene therapy, a recombinant SeV vector carrying various genes has been developed (Shiotani et al., Gene Therapy, 2001, 8, 1043-1050), and a drug delivery system Research is also underway on a system in which genes and various drugs ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/48A61K35/76A61K48/00A61P7/02C12N15/86
CPCA61K47/48776A61K48/0008A61K47/48215C12N2760/18845C12N2760/18832C12N2810/40C12N15/86A61K35/76A61K47/60A61K47/6901A61P43/00A61P7/02A61K47/50A61K48/00
Inventor 榊原裕幸原裕人上田泰次长谷川护游军
Owner DNAVEC RES
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