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Multifunctional anticancer recombined adenovirus

A recombinant adenovirus and virus technology, applied in virus/bacteriophage, recombinant DNA technology, anti-tumor drugs, etc., can solve the problems of unsatisfactory anti-tumor effect, poor tumor curative effect, poor curative effect of pancreatic cancer, etc.

Inactive Publication Date: 2005-03-23
CHINESE PEPTIDE CO +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as a new class of anti-tumor drug, ONYX-015 has the following problems: First, the anti-tumor effect of ONYX-015 is not ideal
ONYX-015 lacks the E1B 55K protein, which greatly reduces the replication ability of adenovirus in tumor cells. Its replication ability in tumor cells is only about 10% of that of wild-type adenovirus. It is effective for superficial tumors such as head and neck cancer. Has a certain curative effect, but the curative effect is not good for deep tumors such as liver cancer and pancreatic cancer
Second, ONYX-015 has a certain killing effect on p53 gene mutant tumors, but has a poor effect on p53 gene normal type tumors
However, the combination of drugs will increase the side effects of chemotherapy drugs, such as bone marrow suppression, hair loss, etc., adding a lot of unnecessary pain to patients

Method used

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  • Multifunctional anticancer recombined adenovirus
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  • Multifunctional anticancer recombined adenovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1. Construction of Adenovirus E1B Gene Mutants

[0079] A. Construction of plasmid pXC-Δ1651

[0080] Plasmid pXC-1 was purchased from Microbix Biosystem Inc (Toronto), Canada. pXC-1 contains the gene sequence of human adenovirus type 5 (Ad5) from nt22-5790. An adenovirus left-end plasmid with deletion of E1B gene (from nt1651-2591) was constructed on pXC-1 by PCR method [refer to Molecular Cloning for detailed experimental steps: Experimental Manual, Second Edition, edited by Sambrook (1989)]. Specifically - the construction steps are as follows:

[0081] oligonucleotide chain

[0082] UCP1 (5'-TGAGACGCCCGACATCACCT-3')

[0083] UCP2 (5'-CCG CTCGAG CGG TTAATTAA CCTAACACGCCATGCAAGTTAA-3')

[0084] XhoI PacI

[0085] as a primer, and pXC-1 DNA as a template to obtain PCR product A. PCR product A contains the gene sequence of adenovirus from nt1316-1650 and contains oligonucleotides at the 3' end

[0086] CC AATTAATT GGC GAGCTC...

Embodiment 2

[0130] Example 2. Construction of transgenic E1B gene mutants

[0131] A. Construction of plasmid pXC-Δ1651 / GM-CSF

[0132] Take the oligonucleotide chain:

[0133] UCP5 (5'-CGC GGATCC GCGATGTGGCTGCAGAGCCTGCT-3')

[0134] BamHI

[0135] UCP6 (5'-CC GGAATT CCCCTCACTCCGGACTGGCTCCC-3')

[0136] EcoRI

[0137] As a primer, the plasmid PGT60hGM-CSF (purchased from Invivogen, USA) was used as a template to obtain PCR product F. PCR product F contains the complete human GM-CSF gene sequence, and contains BamHI and EcoRI restriction sites at the 5' end and 3' end. The PCR product F was digested with EcoRI and BamHI, and inserted into the plasmid pcDNA3 (purchased from Invitrogen, USA) at the same site to obtain a new recombinant plasmid pcDNA3-GM-CSF.

[0138] Take the oligonucleotide chain:

[0139] UCP7 (5'-CC TTAATTAA GGGTTGACATTGATTATTGACT-3')

[0140] PacI

[0141] UCP8 (5'-CC TTAATTAA GGATGCAATTTCCTCATTTTATT-3') ...

Embodiment 3

[0177] Example 3. Production of recombinant adenovirus

[0178] A. Generation of Δ1651 adenovirus

[0179] pBHGE3 was purchased from Microbix Biosystems Inc, Canada. pBHGE3 contains the Ad5 gene sequence but the E1 region is deleted from nt188 to 1339. pBHGE3 itself is not infectious, but pXC-1 or a plasmid derived from PXC-1 and pBHG-E3 co-transfect 293 cells, and can produce infectious viruses through homologous recombination (see Hitt, Construction and Propagation of detailed experimental steps Human Adenovirus Vectors, In: Cell Biology: ALaboratory Handbook, J. Celis, Academic Press, NY, 1995, or refer to Graham, Adenovirus Based Expression Vectors and Recombinant Vaccines, In: Vaccines: New Approaches to Immunological Problems. R.W. Eliss, Butterworth, pp363 -390, 1992). Δ1651 adenovirus was obtained by co-transfecting 293 cells with plasmid pXC-Δ1651 and plasmid pBHGE3 by homologous recombination. Single plaques were picked and amplified in 293 cells. Viral DNA was ...

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Abstract

The invention provides a recombinant adenovirus features that it can simultaneously inactivate the virus proteins able to combine with cell internal funcational tumor inhibition gene p53 and inhibit cell decline, respectively, and carries with a group of exogenous genes driven by exogenous stator. It can prolifically breed in P53 function defect-type and / or P53 function normality-type tumor cells, generating a cytopathic effect (CPE), and can make high-performance expression exogenous antitumor proteins in the tumor cells, and finally kills the tumor cells, but has no killing effect on P53 function normality-type non-tumor cells.

Description

technical field [0001] The invention belongs to the field of anticancer and describes a multifunctional anticancer recombinant adenovirus. This recombinant adenovirus can selectively replicate and reproduce in tumor cells with p53 function deficiency and / or normal p53 function, express exogenous anti-tumor proteins at high levels, and kill tumor cells, while in non-tumor cells Basically non-replicating. The invention also proposes a method for using the recombinant adenovirus to treat and prevent tumors. Background technique [0002] Malignant tumor is a common and frequently-occurring disease that seriously endangers human health and life. The number of new cancer cases in the world exceeds 10 million every year (the number of cancer patients exceeds 40 million). Cancer has surpassed cardiovascular and cerebrovascular diseases and has become the first cause of human death. At present, the conventional treatment for malignant tumors is still based on surgery, radiotherap...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K48/00A61P35/00C12N7/01C12N15/12C12N15/85C12N15/861
Inventor 陈瑜
Owner CHINESE PEPTIDE CO
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