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Multifunctional anticancer recombinant adenovirus and use thereof for treating and preventing tumor

A technology of recombinant adenovirus and virus, which is applied in the direction of anti-tumor drugs, applications, antibody medical components, etc., can solve the problems of increased pain for patients, unsatisfactory anti-tumor effect, and poor tumor curative effect, so as to reduce toxic and side effects and increase Effect of Tolerated Dose

Inactive Publication Date: 2003-05-28
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 recombinant adenovirus and use thereof for treating and preventing tumor
  • Multifunctional anticancer recombinant adenovirus and use thereof for treating and preventing tumor
  • Multifunctional anticancer recombinant adenovirus and use thereof for treating and preventing tumor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Embodiment 1

[0075] Examples Example 1. Construction of Adenovirus E1B Gene Mutant A. Construction of Plasmid pXC-Δ1651

[0076] 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)]. The specific construction steps are as follows:

[0077] oligonucleotide chain

[0078] UCP1 (5'-TGAGACGCCCGACATCACCT-3')

[0079] UCP2(5,-CCG CTCGAG CGG TTAATTAA CCTAACACGCCATGCAAGTTAA-3')

[0080] XhoI PacI was used as a primer, and pXC-1 DNA was used 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

[0081] CC AATTAATT GGC GAGC...

Embodiment 2

[0117] Example 2. Construction of transgenic E1B gene mutant A. Construction of plasmid pXC-Δ1651 / GM-CSF

[0118] Take the oligonucleotide chain:

[0119] UCP5 (5'-CGC GGATCC GCGATGTGGCTGCAGAGCCTGCT-3')

[0120] BamHI

[0121] UCP6 (5'-CC GGAATT CCCCTCACTCCGGACTGGCTCCC-3')

[0122] EcoRI was used as a primer, and 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.

[0123] Take the oligonucleotide chain:

[0124] UCP7 (5'-CC TTAATTAA GGGTTGACATTGATTATTGACT-3')

[0125] PacI

[0126] UCP8 (5'-CC TTAATTAA GGATGCAATTTCCTCATTTTATT-3')

...

Embodiment 3

[0149] The PCR product K was obtained in the same way, digested with PacI, and connected to pXC-Δ2251 at the same site to obtain a new recombinant plasmid pXC-Δ2251 / GM-CSF / TK. Plasmid pXC-Δ2251 / GM-CSF / TK deletes E1B 55K gene, retains endogenous E1B promoter sequence and E1B 19K gene, and contains human GM-CSF gene sequence, HSV-TK gene sequence and bovine growth hormone (BGH) A polyadenylation signal, operatively linked between the human GM-CSF gene sequence and the HSV-TK gene sequence by a cerebromyocyte viroid internal ribosome entry site, and contains the CMV early promoter upstream of the GM-CSF gene , containing the BGH polyadenylation sequence downstream of the HSV-TK gene. Example 3. Production of Recombinant Adenovirus A. Production of Δ1651 Adenovirus

[0150] 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 deri...

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Abstract

A multifunctional anticancer recombinant adnovirus for deactivating viral proteins EIB 55K and E1B 19K at same time has two groups of exogenous genes, which are effectively linked by internal ribosome entry site. Said adnovirus can be greatly reproduced in tumor cell to generate cell pathogenic effect (CPE). The exogenous anticancer protein can be effectively expressed in tumor cell to finally kill it. The method for preventing and treating tumor by said adnovirus is also disclosed.

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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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/235A61P35/00C12N7/01C12N15/31
Inventor 陈瑜
Owner CHINESE PEPTIDE CO