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Acquisition and application of a novel oncolytic adenovirus-thymidine kinase gene construct

An oncolytic adenovirus and construct technology, applied in the direction of virus/phage, gene therapy, genetic engineering, etc., can solve the problems of weak efficacy of lysing tumor cells, ineffective expression of therapeutic genes, and low efficacy of virus treatment, and achieve functional Complete, highly selective, broad anti-cancer effect

Inactive Publication Date: 2011-12-21
马丁
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, they still have the following disadvantages: Although the vectors designed based on the biological basis of adenoviruses have the therapeutic advantage of replicability in tumor conditions, due to various design deficiencies, the obtained adenoviruses cannot replicate in tumor cells and lyse tumors. The efficacy of the cells is far weaker than that of wild-type adenovirus; After the existing adenovirus vectors carrying therapeutic genes infect target cells, the therapeutic genes carried can obtain the ability to express, which inevitably produces toxicity to normal tissue cells effects, especially exogenous genes with greater toxicity or lethality; the exogenous promoters combined with the current adenovirus gene therapy vectors have unstable or low activity in vivo; even the existing adenovirus Gene therapy vectors have a strong oncolytic effect, but the premature lysis of tumor cells leads to low production of progeny viruses in tumor cells and the inability to effectively express the therapeutic genes carried. The effect is low; most of the current adenovirus vectors have deleted the immunoregulatory genes of the adenovirus itself, which accelerates the elimination of it by the immune system in the body

Method used

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  • Acquisition and application of a novel oncolytic adenovirus-thymidine kinase gene construct
  • Acquisition and application of a novel oncolytic adenovirus-thymidine kinase gene construct
  • Acquisition and application of a novel oncolytic adenovirus-thymidine kinase gene construct

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example 1

[0018] pXC1 was purchased from Microbix Biosystem Inc. (Toronato, Ontario, Canada, catalog number: PD-01-03). This plasmid contains the human adenovirus type 5 (Ad5) 22-5790nt sequence.

[0019] Deletion of 920-946nt by 3 times PCR method, acquisition of fragment 1: primer 1=5′-CG GGA TCC GGG CCC CCATTT CC-3', equivalent to 9883-9902nt, the underlined part is the BamHI restriction site; primer 2 = 5'- GTC ACT GGGTGG ATC GAT CAC CTC CGG TAC-3', corresponding to 922-905nt, the underlined part is complementary to primer 3;

[0020] Use pXC1 as a template for PCR reaction, the total volume of the reaction system is 100 μl, including:

[0021] Contains MgCl 2 10 μl of 10x PCR buffer

[0022] 2mM dNTP 10μl

[0023] 10 μM Primer 1 1 μl

[0024] 10 μM Primer 2 1 μl

[0025] pXC110ng / μl 1μl

[0026] pfu high fidelity Taq enzyme 2.5u

[0027] Add water to 100μl;

[0028] The reaction conditions were: 95°C for 30 seconds; 28 cycles of 95°C for 45 seconds, 60°C for 1 minute, ...

example 2

[0035] pBHGE3 was purchased from Microbix Biosystem Inc. (Toronato, Ontario, Canada, catalog number: PD-01-12), this plasmid contains the entire genome sequence except the ADd5 packaging signal (194-358nt).

[0036] When pBHGE3 is obtained from Microbix Biosystem Inc., the total amount is 10 μg. First, electroporate into competent bacteria, pick positive clones, and extract plasmids. The obtained plasmids are treated with CsCl 2 -EB purified by ultracentrifugation.

[0037] Homologous recombination method to obtain Δ920-946Ad5 recombinant adenovirus construct, the method is as follows:

[0038] Plant 7.5×10 in a 15cm petri dish 5 293 cells, the culture medium is 10% FBS DMEM, by the next day, the cells should be 1-1.5×10 6 , about 70% of the cells are confluent; 3-4 hours before transfection, replace with fresh culture medium.

[0039] Prepare co-transfection DNA-calcium phosphate solution: 1600 μl sterile 2×HBS (280mM NaCl, 43mM HEPES, 10mM KCl, 10mM Na 2 HPO 4 .7H 2 O,...

example 3

[0055] The Ad5E3 region is called the Ad5 early region 3. Driven by the endogenous promoter, this region encodes 7 proteins. For the sequence, structure and function, please refer to the appendix figure 1 : 12.5k, 27858-28179nt, function unknown; 6.7k, 27547-28736nt, together with RID complex, inhibits the expression of TRAIL receptor 1 and 2 on the cell surface; gp19k, 28735-29215nt, binds to MHC class I antigen, inhibits its Presented to the cell surface, evading the clearance of CTL; ADP, 29419-29770nt, lyses cells and releases virus; RIDα, 29784-30057nt, forms a complex with RIDβ, prevents the lysis of TNF, and clears FAS antigen; RIDβ, 30062-30458nt and 14.7k, 30453-30837, inhibits the cleavage of TNF.

[0056] The purpose of this experiment is to delete the 29477-29714nt region of the E3 region and insert an exogenous therapeutic gene into the E3ADP region of the recombinant adenovirus.

[0057] Deletion of 29477-29714nt in the Ad5E3 region by 3 PCRs:

[0058] Acquisit...

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Abstract

The invention discloses a construction scheme of artificially transforming human type 5 adenovirus (Ad5), and a specific application of a novel oncolytic adenovirus construct obtained by the scheme in tumor treatment, and belongs to the technical field of medical genetic engineering. A recombinant adenovirus construct obtained by PCR amplification site-directed deletion, enzyme digestion, ligation, cloning, homologous recombination, transfection, adenovirus monoclonal purification, etc., its technical characteristics are: Ad5 genome E1A conserved sequence 2 27 bases were deleted in the (CR2) region; 29477-29714nt of the ADP gene in the E3 region was deleted; and the full-length coding sequence (1131bp) of the HSV-TK gene was inserted in the deleted region. This construct is a new type of oncolytic adenoviral vector with higher tumor selective replication ability, which utilizes the suicide gene function of HSV-TK and the double killing effect of oncolytic virus to dissolve tumor cells, and has great potential in the biological treatment of tumors. Unique practical value.

Description

1. Technical field [0001] The present invention relates to a human adenovirus type 5 (Human adenovirus type 5, Ad5) recombinant construction scheme, which is characterized in that: the 920-946 nt of the Ad5 genome is directional deleted, that is, GAT CTT ACC TGC CAC GAG GCTGGC TTT, the The sequence encodes amino acids 121 to 129 of the E1A protein. On this basis, the Ad5E3 region 29477-29714nt is further deleted, and a ClaI restriction site is introduced in the above deletion region; at the same time, the full-length coding sequence of the HSV-TK gene is inserted in the deletion region (1131bp), so as to obtain a novel oncolytic adenovirus Ad5 / ΔE1A / ΔADP / HSV-TK which has a therapeutic effect on tumors. The content of the invention belongs to the technical field of medical genetic engineering. 2. Background technology [0002] At present, malignant tumors are one of the most serious public health problems facing the world, and my country is one of the areas with high incidenc...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/54C12N15/63A61K48/00A61P35/00
Inventor 马丁周剑峰韩志强卢运萍王世宣
Owner 马丁
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