LMP-1 recombinant gland related viral vectors as well as construction method and uses thereof

A viral vector, LMP-1 technology, applied in the direction of virus/phage, virus, application, etc., can solve the problems of low virus titer, unstable recombinant virus, limited capacity of therapeutic genes, etc., and achieve the effect of broad application prospects.

Inactive Publication Date: 2008-09-03
GUANGZHOU BOWOJIN BIOLOGICAL TECH
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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-1 recombinant gland related viral vectors as well as construction method and uses thereof
  • LMP-1 recombinant gland related viral vectors as well as construction method and uses thereof
  • LMP-1 recombinant gland related viral vectors as well as construction method and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. Construction and detection of recombinant adeno-associated virus vector rAAV / LMP-1 and rAAV / mLMP-1

[0048] Materials and their sources:

[0049] A. The pBR322 plasmid (named pBR-AAV2) carrying the whole genome DNA of AAV type 2: prepared by Professor Paul L. Hermonat, one of the main technical directors of the United States 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 mammalian tissue culture cells. Proc. Natl. Acad. Sci. U.S.A. 81: 6466-6470.).

[0050] B. Human nasopharyngeal carcinoma cells: isolated from cancer tissues of nasopharyngeal carcinoma patients or purchased from commercial sources, immunohistochemistry confirmed LMP-1 positive.

[0051] 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 purchase...

Embodiment 2

[0062] Embodiment 2, preparation of recombinant adeno-associated virus (rAAV) and virus titer determination

[0063] Materials and their sources:

[0064] A. The recombinant adeno-associated virus vector rAAV / LMP-1 carrying the LMP-1 gene constructed in Example 1 and the recombinant adeno-associated virus vector rAAV / mLMP-1 carrying the LMP-1 mutant gene (rAAV / AmLMP-1, rAAV / BmLMP-1, rAAV / CmLMP-1 and rAAV / DmLMP-1).

[0065] 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 University of Arkansas Medical College Hospital (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 humanpapillomavirus type 16 E6 antigen gene delivery into human dendritic cells by an adeno-associated virus vector.py Cancer 8: Gene Thera 948-957.).

[0066] C. AAV-HEK293 cells containing ...

Embodiment 3

[0086] Example 3. Tumor killing experiment of introducing tumor-associated antigen into monocyte-macrophage-dendritic cell line

[0087] Materials and their sources:

[0088] A. rAAV virus: rAAV / LMP-1 and rAAV / mLMP-1 (rAAV / AmLMP-1, rAAV / BmLMP-1, rAAV / CmLMP-1).

[0089] B. AIM-V cell culture medium: purchased from Invitrogen, USA.

[0090] 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.

[0091] 1. Tumor killing experiments

[0092] like 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-1 gene and mutant gene thereof) of the present invention infecting tumor patient's monocytes includes the following steps:

[0093] A. Take 50-150 ml of peripheral blood from a tumor patient, use a blood cell separator (or lymphocyte separation medium) to obtain peripheral bl...

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Abstract

The invention discloses a LMP-1 adeno related virus vector, constructing method and application thereof. The LMP-1 adeno related virus vector is adeno related virus vector obtained by substituting adeno related virus structure in adeno related virus vector with LMP-1 gene or mutation type gene thereof. The LMP-1 adeno related virus vector can convey LMP-1 gene of wild type or mutation type carried thereby into monocyte-macrophage-dendritic cell system, cells carried with these specific antigen gene can be used as effector cells to stimulate immune system. As proven by experiments, CTL infected by the rAAV and induced by DC can effectively inhibit malignant tumor cells or kill tumor cells in patient's body. Therefore, the LMP-1 adeno related virus vector or relevant products thereof can be used in preparation of medicament for nasopharyngeal carcinoma.

Description

technical field [0001] The present invention relates to a vector and its application, in particular to a recombinant adeno-associated virus vector and its construction method and its application in the preparation of antitumor drugs. 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' end segment, downstream 3' end 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, Labow MA, Wright R, Berns KI, Muzyczka N. Genetics ofadeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants. J Virol. 51: 329-339. Hermonat, P.L., and Muzyczka, N. Use o...

Claims

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Application Information

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IPC IPC(8): C12N15/86C12N15/38C12N7/01C12N5/10A61K39/245A61K48/00A61P35/00A61K39/00
CPCC07K14/82C12N15/86C12N2750/14143A61P35/00A61K39/4622A61K39/4634A61K39/4644A61K39/464406A61K2239/52A61K2239/55A61K39/4615A61K2239/49A61K39/464494A61K39/464482A61K39/464838A61K2239/58A61K39/464495A61K39/464481A61K2239/59A61K39/4611A61K2239/50A61K39/001106A61K39/001195A61K39/001182A61K39/0011A61K39/001194
Inventor 刘勇保罗·L·赫蒙纳特
Owner GUANGZHOU BOWOJIN BIOLOGICAL TECH
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