Obtaining method and use of novel oncolytic adenovirus construct with selective tumor blockage STAT3

A technology for constructing and recombining adenovirus, which is applied in the field of d5 E3 region 29477-297, can solve the problems of lack of tumor specificity, restricting tumors, weak efficiency of adenovirus in lysing tumor cells, etc., and achieves increased amplification, copying The effect of number increase

Active Publication Date: 2009-05-27
南京江北新区生物医药公共服务平台有限公司
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Problems solved by technology

However, the second-generation Ad5 gene therapy vectors all have some common 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 adenoviruses obtained The effect of replicating and lysing tumor cells in tumor cells is far weaker than that of wt-Ad5. For example, the anticancer effective rate of ONYX-015 alone is only 0-14%.
Nevertheless, most of these new molecular targets and their molecular suppression methods lack tumor specificity, and the resulting dose-limiting toxicity is still a key problem that these therapies generally face and urgently need to be solved, which greatly limits the solved the tumor problem

Method used

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  • Obtaining method and use of novel oncolytic adenovirus construct with selective tumor blockage STAT3
  • Obtaining method and use of novel oncolytic adenovirus construct with selective tumor blockage STAT3

Examples

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example 1

[0024] Example 1. Construction of pXC1 series mutants (Δ920-946pXC1)

[0025] pXC1 was purchased from Microbix Biosystem Inc. (Toronato, Ontario, Canada, catalog number: PD-01-03), and this plasmid contains the 21-5790nt sequence of human adenovirus type 5 (Ad5).

[0026] Deletion of 920-946nt by 3 times PCR method, acquisition of fragment 1: primer 1=5′-CG GGA TCC GGG CCCCCA TTT CC-3', equivalent to 9883-9902nt, the underlined part is the BamHI restriction site; primer 2 = 5'- GTC ACT GGG TGG ATC GAT CAC CTC CGG TAC-3', corresponding to 922-905nt, the underlined part is complementary to primer 3;

[0027] Use pXC1 as a template for PCR reaction, the total volume of the reaction system is 100 μl, including:

[0028] Contains MgCl 2 10 μl of 10x PCR buffer

[0029] 2mM dNTP 10μl

[0030] 10 μM Primer 1 1 μl

[0031] 10 μM Primer 2 1 μl

[0032] pXC1 10ng / μl 1μl

[0033] pfu high fidelity Taq enzyme 2.5u

[0034] Add water to 100μl;

[0035] The reaction condition...

example 2

[0041] Example 2. Construction of Δ920-946 Ad5 recombinant adenovirus

[0042] 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).

[0043]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.

[0044] Homologous recombination method to obtain Δ920-946 Ad5 recombinant adenovirus construct, the method is as follows:

[0045] 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.

[0046] Prepare co-transfection DNA-calcium phosphate solution: 1600 μl st...

example 3

[0061] Example 3. Construction of subcloning vector pCDNA3.1-ΔADP of Ad5 E3 region

[0062] The Ad5 E3 region is called the Ad5 early region 3. Driven by the endogenous promoter, this region encodes 7 proteins. For the sequence, structure and function, see Figure 1: 12.5k, 27858-28179nt, the function is unknown; 6.7k, 27547-28736nt, together with the RID complex, inhibits the expression of TRAIL receptors 1 and 2 on the cell surface; gp19k, 28735-29215nt, binds to MHC class I antigens, inhibits its presentation to the cell surface, and escapes 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 lysis of TNF.

[0063] 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.

[006...

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Abstract

The invention discloses a proposal for constructing novel oncocytic adenovirus of selectively closed tumor STAT3 obtained by manual reconstruction of human 5 type adenovirus (Ad5), and particular application of a recombined adenovirus constructor in tumor treatment, and belongs to the technical field of medical genetic engineering. By techniques of PCR enlarging fixed point deletion, enzyme cutting, connection, cloning, homologous recombination, transfection, single cloning purification of adenovirus and the like, the recombination adenovirus constructor is obtained. The technical characteristics comprise that 27 basic groups are deleted in an E1A conservative sequence 2(CR2) zone of Ad5 genome; 29477 to 29714nt in an E3 zone of ADP gene are deleted; and partial STAT cDNA segments are oppositely inserted in the deletion zones. The constructor is a novel oncocytic adenovirus vector with higher tumor selective copy capability. The constructor has unique practical values in biological treatment of tumor, and also provides a reasonable gene target point specificity treatment mode for the genetic treatment of tumor.

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 29477-29714nt of the Ad5 E3 region is further deleted, and a ClaI restriction site is introduced in the above deletion region; a 770bp exogenous cleavage site is reversely inserted into the ClaI restriction site The specific STAT3 cDNA fragment is equivalent to the 960-190nt of STAT3 mRNA, so as to obtain the recombinant adenovirus construct Ad5 / ΔE1 / ΔADP / ASSTAT3 that has a therapeutic effect on tumors. The content of the invention belongs to the technical field of medical genetic engineering. 2. Background technology [0002] Malignant tumors are becoming the primary disease that endangers human health. Accord...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861A61K48/00A61K47/42A61P35/00
Inventor 马丁周剑峰王世宣卢运萍韩志强
Owner 南京江北新区生物医药公共服务平台有限公司
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