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Multifunctional anticancer recombinant adenovirus

A recombinant adenovirus and virus technology, applied in virus/bacteriophage, recombinant DNA technology, anti-tumor drugs, etc., can solve the problems of increased pain for patients, poor tumor efficacy, increased toxicity of chemotherapy drugs, etc., to reduce toxicity response, increasing the effect of the maximum tolerated dose

Inactive Publication Date: 2005-04-20
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
  • Multifunctional anticancer recombinant adenovirus
  • Multifunctional anticancer recombinant adenovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1. Construction of Adenovirus E1B Gene Mutants

[0072] A. Construction of plasmid pXC-Δ1651

[0073] 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:

[0074] oligonucleotide chain

[0075] UCP 1 (5'-TGAGACGCCCGACATCACCT-3')

[0076] UCP2 (5'-CCG CTCGAG CGG TTAATTAA CCTAACACGCCATGCAAGTTAA-3')

[0077] 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

[0078] CC AATTAATT GGC GAG...

Embodiment 2

[0117] Example 2. Construction of transgenic E1B gene mutants

[0118] A. Construction of plasmid pXC-Δ1651 / GM-CSF

[0119] Take the oligonucleotide chain:

[0120] UCP5 (5'-CGC GGATCC GCGATGTGGCTGCAGAGCCTGCT-3')

[0121] BamHI

[0122] UCP6 (5'-CC GGAATT CCCCTCACTCCGGACTGGCTCCC-3')

[0123] 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-derived GM-CSF gene sequence, and contains restriction sites of BamHI and EcoRI 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.

[0124] Take the oligonucleotide chain:

[0125] UCP7 (5'-CC TTAATTAA GGGTTGACATTGATTATTGACT-3')

[0126] PacI

[0127] UCP8 (5'-CC TTAATTAA GGATGCAATTTCCTCATTTTATT-3') ...

Embodiment 3

[0159] Example 3. Production of recombinant adenovirus

[0160] A. Generation of Δ1651 adenovirus

[0161] 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 is co-transfected with pBHG-E3 into 293 cells, and an infectious virus can be produced through homologous recombination (see Hitt for detailed experimental steps, Construction and Propagation of 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 ce...

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Abstract

Said invention provides a recombined adenovirus which can inactivate virus protein capable of combined with functional tumor suppressor gene p53 in cell, carry two groups of coded anti tumor protein exogenous gene and enter site through internal ribosome (IRES) effectly connected, two groups of exogenous gene can be cotransducted into one mRNA chain, enter site form internal ribosome to control the translation of second group of exogenous gene. Said recombined adenovirus can be greatly replication breeding in p53 functional fault type and / or p53 functional normal type tumor cell and produce cell pathogenesis effect (CPE), high-effectively express exogenous anti tumor protein, finally kill tumor cell without killing p53 functional normal type non-tumor cell.

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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