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Recombinant oncolytic adenovirus, recombinant oncolytic adenovirus vector for preparing same, construction method of recombinant oncolytic adenovirus vector and applications of recombinant oncolytic adenovirus and recombinant oncolytic adenovirus vector

A technology of oncolytic adenovirus and construction method, which is applied in the field of recombinant oncolytic adenovirus vector and its construction, can solve the problems of tumor recurrence and metastasis, failure to fundamentally cure tumor, patient death, etc., and achieve reduction of recurrence and metastasis, good The effect of applying the foreground

Active Publication Date: 2018-08-21
BEIJING DOINGTIMES INST OF TRANSLATIONAL MEDICINE
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Malignant tumors are one of the diseases that seriously threaten human life. For most patients with malignant tumors, chemotherapy, radiotherapy, and biological immunotherapy can be used to kill most of the tumor cells, but they cannot fundamentally cure the tumor. Tumors will relapse and metastasize, resulting in the death of patients, because these conventional chemotherapy cannot eliminate drug-resistant cancer stem cells (cancer stem cells, CSCs), and cancer stem cells have high tumorigenicity, self-renewal replication and differentiation capabilities, so tumor relapse

Method used

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  • Recombinant oncolytic adenovirus, recombinant oncolytic adenovirus vector for preparing same, construction method of recombinant oncolytic adenovirus vector and applications of recombinant oncolytic adenovirus and recombinant oncolytic adenovirus vector
  • Recombinant oncolytic adenovirus, recombinant oncolytic adenovirus vector for preparing same, construction method of recombinant oncolytic adenovirus vector and applications of recombinant oncolytic adenovirus and recombinant oncolytic adenovirus vector
  • Recombinant oncolytic adenovirus, recombinant oncolytic adenovirus vector for preparing same, construction method of recombinant oncolytic adenovirus vector and applications of recombinant oncolytic adenovirus and recombinant oncolytic adenovirus vector

Examples

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Embodiment 1

[0051] Example 1 Construction, Amplification, Purification and Measurement of Particle Titer and Infection Titer of Recombinant Oncolytic Adenoviral Vector

[0052] 1. Modification of adenovirus backbone

[0053] In order to increase the infection efficiency of tumor cells with low Saatchi virus-adenovirus receptor (CAR) expression, the RGD-4C short peptide sequence was introduced into the fiber (Fiber) gene of the adenovirus plasmid, and the selected positions were 456T and 457P of HIloop In between, the targeting backbone plasmid pAdbone5RGD was constructed.

[0054] 2. Construction of oncolytic adenoviral vectors carrying therapeutic gene shRNA and PUMA and control adenoviral vectors

[0055] Construction strategy: clone the target gene (shRNA anti-tumor stem cell marker ALDH1A1 gene and pro-apoptosis gene PUMA gene) into the multiple cloning site of the shuttle plasmid pShuttle-CMV (the shuttle plasmid contains CMVp and pA signal sequences) to obtain Plasmid pSh5-Pumash;...

Embodiment 2

[0056] Example 2 Recombinant oncolytic adenovirus related experiments

[0057] 1. Amplification and purification of recombinant (oncolytic) adenovirus and determination of particle titer and infection titer

[0058] The prepared recombinant (oncolytic) adenovirus was massively amplified in human embryonic kidney 293 cells, purified by a special cesium chloride density gradient centrifugation method, and the number of adenovirus particles was measured by a digestion method, and the adenovirus concentration was measured by a limiting dilution method. Infectious titer, according to the ratio of particle titer / infectious titer to judge the proportion of live virus in pure adenovirus, the ratio of the above four recombinant oncolytic adenoviruses is <100, which is in line with the requirements of the US FDA and Chinese biological products on adenovirus requirements (as shown in Table 1).

[0059] Table 1 Particle titer, infection titer and ratio of recombinant (oncolytic) adenovir...

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Abstract

The invention discloses a recombinant oncolytic adenovirus, a recombinant oncolytic adenovirus vector for preparing the same, a construction method of the recombinant oncolytic adenovirus vector, andapplications of the recombinant oncolytic adenovirus and the recombinant oncolytic adenovirus vector. The construction method of the recombinant oncolytic adenovirus vector comprises following steps:a gene encoding RGD peptide is introduced into adenovirus plasmid, and targeting framework plasmid is formed; a gene sequence of a shRNA anti-tumor stem cell marker molecule ALDH1A1 and a gene sequence of an apoptosis-promoting gene PUMA are connected to first shuttle plasmid, and second shuttle plasmid is formed; a human telomerase reverse transcriptase promoter is connected to the second shuttleplasmid, and third shuttle plasmid is formed; the third shuttle plasmid and the targeting framework plasmid are recombined in escherichia coli to form the recombinant oncolytic adenovirus vector. Therecombinant oncolytic adenovirus vector constructed with the method is linearized and then transfected to virus packaging cells for packaging, amplification and purification, and then the recombinantoncolytic adenovirus is formed, so that tumor stem cells and common tumor cells of multiple malignant tumors can be effectively recognized and killed.

Description

technical field [0001] The invention relates to the field of gene therapy, in particular to a recombinant oncolytic adenovirus, a recombinant oncolytic adenovirus vector for preparing the recombinant oncolytic adenovirus, a construction method and application thereof. Background technique [0002] Malignant tumors are one of the diseases that seriously threaten human life. For most patients with malignant tumors, chemotherapy, radiotherapy, and biological immunotherapy can be used to kill most of the tumor cells, but they cannot fundamentally cure the tumor. Tumors will relapse and metastasize, resulting in the death of patients, because these conventional chemotherapy cannot eliminate drug-resistant cancer stem cells (cancer stem cells, CSCs), and cancer stem cells have high tumorigenicity, self-renewal replication and differentiation capabilities, so Tumors recur and metastasize. [0003] In recent years, cancer stem cell research and its application in tumor treatment ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N7/01A61K48/00A61P35/00A61P35/04
CPCA61K48/0025A61K48/0058A61P35/00A61P35/04C12N7/00C12N15/86C12N2710/10321C12N2710/10343C12N2710/10352C12N2800/107C12N2810/405
Inventor 孟二红
Owner BEIJING DOINGTIMES INST OF TRANSLATIONAL MEDICINE
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