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Novel recombinant plasma membrane-based vesicle, for treating cancer

A technology of vesicles and plasma membranes, applied in the field of vesicles based on recombinant plasma membranes, which can solve the problems of ineffective killing of cancer cells and difficulties in the supply of neural stem cells

Inactive Publication Date: 2020-09-25
天腾有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, the method disclosed in the prior art is a technique that is difficult to apply to an actual clinical setting because the supply of neural stem cells is difficult and the killing effect of cancer cells is not very significant

Method used

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  • Novel recombinant plasma membrane-based vesicle, for treating cancer
  • Novel recombinant plasma membrane-based vesicle, for treating cancer
  • Novel recombinant plasma membrane-based vesicle, for treating cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Preparation of VSV-G H162R construct

[0083] In order to prepare recombinant exosomes including the VSV-G H162R mutein, the inventors first prepared a gene construct encoding the H162R mutein, in which the 162nd amino acid residue of VSV-G (based on the protein disclosed in GenBank number CAC47944 No. 178 of ), the composition is replaced by arginine. Specifically, for this purpose, site-directed mutagenesis was performed using a plasmid DNA (pCMV-VSV-G envelope vector, RV-110, Cell Biolabs, hereinafter abbreviated as "VSV-G construct") that included as a template The polynucleotide (SEQ ID NO: 2) encoding the wild-type VSV-G protein (SEQ ID NO: 1), together with the forward primer of SEQ ID NO: 3 (5'-ATA TGC CCC ACT GTC CGC AAC TCT ACA ACC TGG-3') and the reverse primer of SEQ ID NO: 4 (3'-TAT ACG GGG TGA CAG GCG TTG AGA TGT TGG ACC-5') (bold letters correspond to the mutated amino acid arginine).

Embodiment 2

[0084] Example 2: Preparation of recombinant exosomes including VSV-G H162R

[0085] The inventors made the pCMV-VSV-G H162R plasmid vector (hereinafter abbreviated as "VSV-G H162R construct", Figure 2a and 2b ) was transfected into HEK293T cells and cultured for 48 hours, and the gene encoding the VSV-G H162R protein (SEQ ID NO: 5) prepared in Example 1 above was inserted into the pCMV-VSV-G H162R plasmid vector (SEQ ID NO: 6). Subsequently, the cell culture was recovered, sequentially centrifuged at 300 g for 10 minutes, 2,000 g for 10 minutes, and 10,000 g for 30 minutes, filtered using a 0.2 μm filter, and recovered by ultrafiltration at 150,000 g for 3 hours clumps ( image 3 ).

[0086] Subsequently, in order to confirm whether the VSV-G H162R mutant protein was included in the exosomes, a fraction of the recovered exosomes was disrupted, and an anti-VSV-G antibody (Abcam, ab50549) and an anti-Alix antibody (exosome marker, Santacruz, sc99010) performed Western b...

Embodiment 3

[0090] Example 3: Preparation of recombinant exosomes in which doxorubicin is encapsulated

[0091] In order to load doxorubicin (DOX), an immunogenic cell death inducer, into the recombinant exosomes prepared in Example 2 above, the purified exosomes (approximately 10 11 exosomes) were mixed with DOX in 1 mL PBS. Subsequently, the DOX recombinant exosome mixture was sonicated using a Model 505 Sonic Dismembrator with a 0.25-inch tip at the following settings: 20% amplitude, 6 cycles of on / off 30 seconds for 3 minutes, with 2 minute cool down period between cycles. After sonication, the Exo-DOX solution was incubated at 37 °C for 60 min to recover exosome membranes. Excess free drug was removed by size exclusion chromatography using a NAP-10 Sephadex G25 column (GE Healthcare, Buckinghamshire, UK).

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Abstract

The present invention relates to a recombinant plasma membrane-based vesicle, and more specifically to a recombinant plasma membrane-based vesicle comprising a VSV-G mutated protein in which histidine, which is the 162nd amino acid in the membrane, has been replaced with arginine, and a pharmaceutical composition, for treating cancer, comprising the recombinant plasma membrane-based vesicle.

Description

technical field [0001] The present invention relates to novel recombinant plasma membrane-based vesicles and, more particularly, to novel recombinant plasma membrane-based vesicles for use in the treatment of cancer. Background technique [0002] Cancer refers to a group of diseases associated with abnormal cell growth with the potential to invade and metastasize to other parts of the body. As of 2015, more than 90 million cancer patients are known to exist worldwide, of which about 14 million new cancer patients occur each year. Cancer accounts for 15.7% of human deaths. The most frequently occurring cancers are lung, prostate, colon, and stomach cancers in men, and breast, colon, lung, and cervix cancers in women. [0003] For cancer treatment, various treatment methods including chemotherapy using various anticancer drugs, radiation therapy by radiation, antibody therapy targeting a specific in vivo molecule related to cancer, etc. have been tried. However, anticancer ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/127A61K47/42A61K47/46A61K35/766A61K39/395A61K31/69A61K45/06A61P35/00C07K14/005
CPCA61K39/395C07K14/005A61K9/5068A61K9/0019A61K31/337A61K31/136A61K31/555C12N2760/20022A61K35/12A61P35/00A61K38/162A61K2300/00A61K9/1271A61K47/42A61K47/46A61K35/766A61K31/69A61K45/06C12N2760/20233C07K16/2827C07K16/2863
Inventor 金起范梁有秀金仁山南其勋
Owner 天腾有限公司
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