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LMP-2 recombinant adeno-associated virus vector and construction method and application thereof

A technology of LMP-2 and viral vectors, applied in the direction of virus/bacteriophage, application, virus, etc., can solve the problems of low virus titer, limited therapeutic gene capacity, rAAV stability damage, etc., and achieve the effect of broad application prospects

Pending Publication Date: 2018-09-18
GUANGDONG TOPHEALTH BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, rAAV still has some shortcomings, such as the instability of recombinant virus, low virus titer, and limited capacity for accepting therapeutic genes (generally, the maximum exogenous gene fragment that can be inserted is about 2000 base pairs (bp), otherwise the stability of rAAV sex will be destroyed)

Method used

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  • LMP-2 recombinant adeno-associated virus vector and construction method and application thereof
  • LMP-2 recombinant adeno-associated virus vector and construction method and application thereof
  • LMP-2 recombinant adeno-associated virus vector and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Construction and detection of recombinant adeno-associated virus vector rAAV / LMP-2 and rAAV / mLMP-2

[0046] Materials and their sources:

[0047] A. The pBR322 plasmid (named pBR-AAV2) carrying the whole genome DNA of AAV type 2: It was prepared by Professor Paul L. Hermonat, one of the main technical directors of Broadway Gene International Co., Ltd. (Hermonat, P.L., and Muzyczka, N .Use of adeno-associated virus as a mammalian DNA cloning vector:transduction of neomycin resistance into mam malian tissue culture cells.Proc.Natl.Acad.Sci.U.S.A.81:6466-6470.).

[0048] B. Human nasopharyngeal carcinoma cells: isolated from cancer tissues of patients with nasopharyngeal carcinoma, immunohistochemically confirmed LMP-2 positive (or purchased from commercial channels).

[0049] C. The pCI-neo plasmid carrying the CMV promoter was purchased from Promega Corporation in the United States, and the plasmid pSG424 carrying the SV40 early promoter was purchased from ...

Embodiment 2

[0060] Embodiment 2, preparation of recombinant adeno-associated virus (rAAV) and virus titer determination

[0061] Materials and their sources:

[0062] A. The recombinant adeno-associated virus vector rAAV / LMP-2 carrying the LMP-2 gene constructed in Example 1 and the recombinant adeno-associated virus vector rAAV / mLMP-2 carrying the LMP-2 mutant gene (rAAV / AmLMP-2, rAAV / BmLMP-2, rAAV / CmLMP-2 and rAAV / DmLMP-2).

[0063] B. Auxiliary plasmid pHelper containing the Rep gene and Lip / Cap gene of AAV: constructed by Professor Liu Yong from the Gene Therapy Center of the Affiliated Hospital of the University of Arkansas School of Medicine (Liu, Y., Chiriva-Internati, M., Grizzi, F. Salati , E., Roman, J.J., Lim S., and Hermonat, P.L. Rapid induction of cytotoxic T cell response against cervical cancer cells by human papilloma virus type16E6 antigen gene delivery into human dendritic cells by an aden o-associated virus vector. Cancer 8: Gene The 948-957.).

[0064] C. AAV-HEK...

Embodiment 3

[0084] Example 3. Tumor killing experiment of introducing tumor-associated antigen into monocyte-macrophage-dendritic cell line

[0085] Materials and their sources:

[0086] A. rAAV viruses: rAAV / LMP-2 and rAAV / mLMP-2 (rAAV / AmLMP-2, rAAV / BmLMP-2, rAAV / CmLMP-2).

[0087] B. AIM-V cell culture medium: purchased from Invitrogen, USA.

[0088] C. Cytokines: Colony cell stimulating factor (GM-CSF), interleukins 2, 4, 7 (IL-2, 4, 7) and tumor necrosis factor (TNF-α) were purchased from American R&D Corporation.

[0089] 1. Tumor killing experiment

[0090] Such as Figure 5 As shown, the whole process of the tumor-killing experiment based on one or more rAAV viruses carrying tumor-associated antigen genes (LMP-2 gene and mutant gene thereof) of the present invention infecting tumor patient's monocytes includes the following steps:

[0091] A. Take 50-150 ml of peripheral blood from a tumor patient, use a blood cell separator (or lymphocyte separation medium) to obtain periphe...

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Abstract

The invention discloses an LMP-2 recombinant adeno-associated virus vector and a construction method and an application thereof. The LMP-2 recombinant adeno-associated virus vector is obtained by replacing an adeno-associated virus structure gene in an adeno-associated virus vector with an LMP-2 gene or a mutant gene thereof. A wild type or mutant type LMP-2 gene carried by the LMP-2 recombinant adeno-associated virus vector can be transported in a monocyte-macrophage-dendritic cell line by the LMP-2 recombinant adeno-associated virus vector, and cells carrying specific antigen genes are usedto stimulate effector cells of an immune system. Experiments show that CTL induced by DC and infected by rAAV of the invention can effectively inhibit the growth of malignant tumor cells or kill tumorcells in the body of patients, and therefore, the LMP-2 recombinant adeno-associated virus vector of the invention or products related to the LMP-2 recombinant adeno-associated virus vector can be used for preparing antitumor drugs.

Description

technical field [0001] The invention belongs to vectors and applications thereof, in particular to a LMP-2 recombinant adeno-associated virus vector and its construction method and application. Background technique [0002] The genetic structure of adeno-associated virus (AAV) has been identified. In 1983, Samulski et al. described the terminal repeat segment of AAV (upstream 5' segment, downstream 3' segment) (Samulski RJ, Srivastava A, Berns KI, Muzyczka N. Rescue of adeno-associated virus from recombinant plasmamids: gene correction within the terminal repeats of AAV. Cell. 33:135-143.). In 1984, Hermonat et al. described the low infectious particle (lip) gene and envelope (cap) gene of AAV (Hermonat PL, LabowMA, Wright R, Berns KI, Muzyczka N. Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants. JVirol.51:329-339. Hermonat, P.L., and Muzyczka, N. Use of adeno-associated virus as a mammalian DNA cloning...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N15/66A61K35/15A61P35/00
CPCA61P35/00A61K35/15C12N15/66C12N15/86C12N2750/14143
Inventor 刘勇
Owner GUANGDONG TOPHEALTH BIOTECH
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