CN111909962A - Virus construct for treating liver cancer and application and construction method thereof - Google Patents

Virus construct for treating liver cancer and application and construction method thereof Download PDF

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CN111909962A
CN111909962A CN202010718678.9A CN202010718678A CN111909962A CN 111909962 A CN111909962 A CN 111909962A CN 202010718678 A CN202010718678 A CN 202010718678A CN 111909962 A CN111909962 A CN 111909962A
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flag
rgd
ade
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宫晓艳
秦苗苗
刘进稳
周海涛
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Yaoding Beijing International Cell Medical Technology Co ltd
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Abstract

The application relates to a virus construct for treating liver cancer, and application and a construction method thereof. The invention also relates to a novel oncolytic virus for individualized targeted therapy of liver cancer and a construction method thereof. The constructs of the invention comprise, in an operable linkage, an AFP promoter, an RGD-4C gene and a GMSF-flag gene, and optionally a glutamyltranspeptidase gene and a tumor-associated antigen gene.

Description

Virus construct for treating liver cancer and application and construction method thereof
Technical Field
The present invention relates to the fields of biotechnology and gene therapy. Specifically, the invention relates to a virus construct for treating liver cancer, and application and a construction method thereof. The invention also relates to a novel oncolytic virus for individualized targeted therapy of liver cancer and a construction method thereof.
Background
Liver cancer refers to malignant tumor of liver, and is generally divided into two main categories: namely primary liver cancer and secondary liver cancer. The primary liver cancer originates from the epithelium or mesenchymal tissue of the liver and belongs to high-grade cancer in China; compared with primary liver cancer, secondary or metastatic liver cancer mainly refers to cancer in which tumors of various organs of a body have metastasized invade the liver. Liver cancer is induced by many factors, such as hepatitis B and C virus infection, drinking water pollution, carcinogenesis of chemical substances, immune disorder and other complex factors. Currently, there are several main approaches for cancer treatment: surgical treatment, palliative surgical treatment, multi-mode comprehensive treatment, absolute alcohol intratumoral injection, radiotherapy, guide treatment, hepatic artery embolism chemotherapy, radiotherapy, biological treatment and the like. The biological treatment is the fourth major liver cancer treatment method except operation, chemotherapy and radiotherapy at present. But the biological treatment means can be counted, wherein the immunotherapy method is to achieve the effect of autoimmunity by separating anticancer immune cells, carrying out in-vitro culture and then reinjecting the cells into the body of a patient; also targeted drugs for liver cancer, such as lenvatinib, approved by FDA in 2016, 5 months, have been used as first line drugs for systemic treatment of liver cancer. The Levatinib has main targets of VEGFR-1, VEGFR-2, VEGFR-3, FGFR1, PDGFR, cKit and Ret, and inhibits tumorigenesis and development by inhibiting cell activities such as tumorigenesis, angiogenesis and the like. The effective rate of lenvatinib is 24%, the median progression-free survival time is 7.4 months, and the median overall survival time is 13.6 months, which are shown according to clinical effects. Nevertheless, the mortality rate of liver cancer remains high worldwide, and the research on more effective methods for treating liver cancer is in the direction of necessity.
The invention relates to an Alpha Fetoprotein (AFP) which is a secretory protein mainly expressed in liver cancer cells, and the invention utilizes the characteristic of the liver cancer specific expression AFP to construct a novel oncolytic virus Adv-PDC316-AFP-RGD-4C-GMSF-Flag-GT-TAA which targets the infection and propagation of the liver cancer cells, the virus does not infect normal liver cells and other normal tissues, the targeting medication has the characteristics of high efficiency/strong specificity and the like, and the virus has the characteristics of increasing virus replication, increasing cell immunity and the like.
Oncolytic virus therapy is a novel tumor treatment that kills tumors by the selective infection of tumor cells with the virus. Oncolytic viruses are replication-competent tumor-killing viruses, but such viruses do not affect normal cells, and kill tumors primarily by stimulating a body's residual tumor-specific immune response. At present, oncolytic viruses are mainly divided into two categories, one category is viruses which have specific infection or killing capacity on tumor cells, such as reovirus, newcastle disease virus and the like; the other is a virus which is artificially modified, has specific killing capacity to tumors and can replicate in the tumors, such as adenovirus, herpes simplex virus, influenza virus, human vaccinia virus and the like.
Oncolytic Viruses (OVs) selectively replicate and kill cancer cells and spread within tumors without damaging normal tissues. In addition to this direct oncolytic activity, OVs are also very effective in inducing immune responses both to themselves and to infected tumor cells. OVs comprise a wide diversity of DNA and RNA viruses that are naturally cancer selective or can be genetically engineered. OVs provide a diverse platform for immunotherapy; they act as in situ vaccines, which can be armed with immunomodulatory genes and used in combination with other immunotherapies.
Dynamic development at home and abroad: currently, 3 oncolytic virus products are approved to be marketed, T-vec (Talimogene laherparepvec, Imlygic) is the only oncolytic virus product approved by FDA to be marketed at present, is derived from HSV-1 virus, is modified to delete two genes of ICP34.5 and ICP47, the former can inhibit translation of cell proteins, and the latter can inhibit antigen presentation, and is approved to be marketed for treatment of advanced melanoma 10 months in 2015. Ragova, approved in 2004, for the treatment of various cancers, with about 75% of melanoma patients receiving raggalir therapy; the first approved oncolytic virus product H101 in 2005 was mainly used for treating primary focus, late clinical stage and recurrent head and neck tumors. At present, products of OVs entering evaluation stages such as clinical I/II and the like include Pexa-Vec for treating liver cancer and colorectal cancer, TG6002/5-FC combined treatment for glioma, LOAd703 for treating pancreatic cancer and the like.
Pexa-Vec (JX-594) is an oncolytic vaccinia virus that has been randomized phase II trials in patients with hepatocellular carcinoma (HCC). Currently, CFDA approved JX-594 for the treatment of advanced liver cancer, the large lees pharmaceutical factory has conducted a global clinical three-phase study, which will be conducted in north america, asia, australia, europe, and china. In addition, 5 months 2017, FDA in the United states awarded Reolysin, a product of ontOLytics Biotech, a rapid channel certification for intravenous oncolytic virus in metastatic melanoma patients. Toca511 is an injectable retroviral vector, and Toca FC is an under-developed extended release 5-FU drug. The FDA has granted a breakthrough therapy for the combination of Toca511 and Toca FC for the treatment of high-grade gliomas. Recruitment of phase II clinical patients is currently completed. DNAsrix is discussing the efficiency and safety assessment of DNX-2401 with Merck Cooperation for anti-PD-1 treatment in combination with pembrolizumab.
The application prospect is as follows: oncolytic viruses can overcome the barriers of tumor cells by expressing immune checkpoint inhibitors, tumor antigens, cytokines, and T cell adaptors themselves, and inhibit tumor development. Oncolytic viruses have been widely recognized as a new therapeutic for synergistic anti-cancer, and many oncolytic virus products have entered the clinical stage.
Oncolytic viruses have many characteristics that make them advantageous and distinct from current therapeutic modalities: (i) the potential for drug resistance is low (to date not seen) because OVs are usually targeted to multiple oncogenic pathways and are cytotoxic in a variety of ways; (ii) they replicate in a tumor-selective manner, are nonpathogenic, and detect only minimal systemic toxicity; (iii) due to in situ viral amplification, viral dose in tumors increases over time, whereas traditional drug pharmacokinetics decrease over time; (iv) safety functions such as medication and immune sensitivity may be built in. These characteristics lead to a high therapeutic index.
Disclosure of Invention
In order to solve the problems of the background art, the invention aims to provide a virus construct for treating liver cancer, and application and a construction method thereof. The invention also provides a novel oncolytic virus for individualized targeted therapy of liver cancer and a construction method thereof.
The invention discloses a construction method of a novel oncolytic virus for individualized targeted therapy of liver cancer, which comprises the steps of replacing a CMV promoter on adenovirus PDC316 with a liver cancer specific promoter, and connecting RGD-4C for increasing virus replication and an oncolytic gene GM-CSF caused by activating cell immunity to an adenovirus expression vector by cloning to obtain AdeAFP +RGD-4C+GMSF-Flag-GT-TAA. The invention provides a construction method of a novel oncolytic virus for individualized targeted therapy of liver cancer, and the novel virus Adv-AdeAFP+RGD-4C+GMSF-Flag-GT-TAAThe AFP promoter is adopted to realize high-selectivity propagation in liver cancer cells, so that the virus is ensured not to infect normal tissue cells; simultaneously, RGD-4C for increasing virus replication and GMCSF-flag for activating oncolytic gene caused by cell immunity are inserted, so that the cancer cells are efficiently and selectively replicated and killed without damaging normal tissues, and the cancer cells are spread in tumors. Finally, the individual liver cancer population is treated in a targeted manner by combining a molecular diagnosis technology and an oncolytic virus technology, so that the establishment of an oncolytic virus individual customized service system is realized.
In one aspect, the present application relates to a viral construct for the treatment of liver cancer comprising in operable linkage an AFP promoter, an RGD-4C gene, and a GMSF-flag gene. In one aspect, the AFP promoter comprises or consists of the nucleotide sequence of SEQ ID No. 1. In one aspect, the RGD-4C gene comprises or consists of the nucleotide sequence of SEQ ID No. 2. In one aspect, the GMSF-flag gene comprises or consists of the nucleotide sequence of SEQ ID NO. 3. In one aspect, the RGD-4C gene and GMSF-flag gene comprise or consist of the nucleotide sequence of SEQ ID No. 4. In one aspect, the viral construct may further comprise a glutamyl transpeptidase gene and a tumor-associated antigen gene.
In one aspect, the present application relates to a viral construct for the treatment of liver cancer comprising, in operative association: (a) an AFP promoter with a nucleotide sequence shown as SEQ ID NO.1, (b) an RGD-4C gene with a nucleotide sequence shown as SEQ ID NO.2, and (C) a GMSF-flag gene with a nucleotide sequence shown as SEQ ID NO. 3. In this aspect, the viral construct may further comprise (d) a glutamyl transpeptidase gene and a tumor-associated antigen gene.
"construct" as used herein refers to a nucleic acid molecule comprising one or more nucleotide sequences or nucleic acid fragments capable of performing the corresponding function. For example, the construct may comprise a promoter, a viral replication gene or genes that facilitate viral replication, and an oncolytic gene. For example, the promoter can be an AFP promoter. The viral replication gene or the gene promoting viral replication may be the RGD-4C gene. The oncolytic gene may be a GMSF-flag gene. In one aspect, the constructs of the invention may be in the form of vectors, such as viral vectors or plasmids. The form of the nucleic acid constructs can be determined by the person skilled in the art as long as they fulfill the desired function of the respective nucleic acid fragment in the target cell, for example a tumor cell. In one aspect, the constructs described herein can be viral expression vectors, such as adenoviral expression vectors.
As used herein, "operably linked" refers to the joining together or joining of two or more nucleic acid fragments into a nucleic acid construct such that each performs a corresponding function. In this context, "operatively connected" may also be abbreviated as "connected". These "nucleic acid fragments" may also be referred to as "nucleic acid components" or "components". For example, a promoter, a gene that increases viral replication, and an oncolytic gene that activates cellular immunity are "operably linked", and the promoter can drive the transcription of the latter two genes, thereby realizing the expression of the gene-encoded protein in the cell and realizing the corresponding functions. It is understood by those skilled in the art that after multiple nucleic acid fragments are operably linked, their respective functions should not be disrupted, or even not impaired. Preferably, the function of the other native nucleic acid fragments is not affected or disrupted after effective ligation. In one example, each step in the methods of the invention embodies the means by which the nucleic acid fragments or components described above are operably linked. In one example, the individual nucleic acid fragments are linked by phosphodiester linkages.
Glutamyl Transpeptidase (GT) is a liver enzyme that transports glutathione and amino acids into cells and influences glutathione metabolism. Tumor-associated antigen (TAA) may refer to antigenic material that is newly present or overexpressed during tumorigenesis, development, etc. Tumor-associated antigens may include antigens that are not specific for tumor cells, are also present on normal cells and other tissues, except in amounts that are significantly increased when cells become cancerous. The glutamyltranspeptidase gene and the tumor associated antigen gene (GT-TAA) which are effectively connected attract Antigen Presenting Cells (APC) for antigen delivery of membrane antigen expression, and act as a target recognition point of liver cancer.
In one aspect, the viral construct is an adenoviral construct. For example, the adenoviral construct may be derived from the adenoviral vector PDC 316. In one aspect, the virus described herein can be an adenovirus. The virus of the invention may comprise a viral construct of the invention.
In one aspect, the constructs described herein are nucleic acid constructs. The nucleic acid may be DNA or RNA. For example, the construct of the invention may be a DNA construct.
In one aspect, the viral construct comprises components (a), (b), and (c) and optionally (d) in a5 'to 3' orientation. In one aspect the viral construct comprises components (a), (b) and (c) and optionally (d) in the direction of transcription. In one aspect the viral construct comprises components (a), (b) and (c) and optionally (d) in the sense strand orientation.
In one aspect, a virus of the invention, such as an adenovirus, can comprise a construct of the invention.
In one aspect, the present application relates to the use of a viral construct of the invention in the manufacture of a medicament for the treatment of cancer in a patient. In one aspect, the application also relates to the use of a virus of the invention in the manufacture of a medicament for the treatment of cancer in a patient. The cancer may be liver cancer, such as hepatocellular carcinoma. The viral construct or virus may also be used in the manufacture of a medicament for inhibiting the growth of a tumor, reducing the volume of a tumor, or eliminating a tumor in a patient. The tumor may be a liver tumor.
In addition, the virus of the invention is a novel oncolytic virus for individualized targeted therapy of liver cancer.
In one aspect, the patient described herein is a human.
In one aspect, the present application relates to a method of constructing a viral construct or a virus containing the construct, comprising the steps of:
replacing the CMV promoter on the adenovirus vector with a liver cancer promoter AFP promoter,
connecting RGD-4C gene for increasing virus replication and GMSF-Flag gene for increasing cell immunity to cause tumor lysis to adenovirus expression vector with liver cancer promoter AFP promoter,
thereby constructing an oncolytic virus expression vector AdeAFP+RGD-4C+GMSF-Flag。
In one aspect, the present application relates to a method of constructing a viral construct or a virus containing the construct, comprising the steps of:
joining the RGD-4C gene which increases viral replication and the GMSF-Flag gene which increases cell immunity causing oncolysis to an adenovirus expression vector, and
replacing the CMV promoter on the adenovirus vector with a liver cancer promoter AFP promoter,
thereby constructing an oncolytic virus expression vector AdeAFP+RGD-4C+GMSF-Flag。
In one aspect, the above method further comprises linking the glutamyl transpeptidase gene and the tumor-associated antigen gene to AdeAFP+RGD-4C+GMSF-Flag+GFPThus, the Ade-PDC316-AFP-RGD-4C-GMSF-Flag-GT-TAA plasmid was constructed.
The method of constructing the constructs of the invention may be carried out in any suitable order, for example according to the written sequential order.
In one aspect, the present application relates to a method of constructing a viral construct of the invention, comprising the steps of:
construction of Ade-PDC316-CMV-GMSF-Flag plasmid: synthesizing a GMSF-Flag gene fragment with EcoRI and HindIII enzyme cutting sites, using EcoRI/HindIII to cut the GMSF-Flag gene fragment and an Ade-PDC316 vector, and constructing a plasmid Ade-PDC316-CMV-GMSF-Flag after connection;
constructing Ade-PDC316-CMV-RGD-4C-GMSF-Flag plasmid: designing forward amplification primers containing EcoRI-RGD-4C-GMSF-Flag and Hind III-GMSF-Flag reverse amplification primers, amplifying and introducing RGD-4C target genes, using EcoRI/Hind III enzyme digestion RGD-4C-GMSF-Flag gene fragments and an Ade-PDC316-CMV-GMSF-Flag vector after amplification, and constructing plasmids Ade-PDC316-CMV-RGD-4C-GMSF-Flag after connection; and
constructing Ade-PDC316-AFP-RGD-4C-GMSF-Flag plasmid: synthesizing a human AFP promoter with Xba I and EcoRI enzyme cutting sites, cutting an AFP gene fragment and an Ade-PDC316-CMV-RGD-4C-GMSF-Flag vector by Xba I/EcoRI enzyme, replacing the CMV promoter with the human AFP promoter, and constructing a plasmid Ade after connectionAFP+RGD-4C+GMSF-Flag。
In one aspect, the methods of the present application further comprise linking the glutamyl transpeptidase gene and the tumor associated antigen gene to AdeAFP+RGD-4C+GMSF-Flag+GFPThus, the Ade-PDC316-AFP-RGD-4C-GMSF-Flag-GT-TAA plasmid was constructed.
In one aspect, the method of constructing the viral constructs of the present invention may further comprise the step of constructing in parallel various corresponding vectors comprising the GFP gene. Such steps are to facilitate the detection of the expression of various embedded genes.
For example, the method of constructing the viral construct of the present invention may further comprise the steps of:
constructing Ade-PDC316-CMV-GFP plasmid: designing a forward amplification primer containing HindIII-GFP and a reverse amplification primer containing SalI-GFP, amplifying a GFP target gene with HindIII/SalI double enzyme cutting sites from PSMPuW-GFP, and connecting an Ade-PDC316-CMV vector by using HindIII/SalI double enzyme cutting to obtain a plasmid Ade-PDC 316-CMV-GFP;
construction of Ade-PDC316-CMV-GMSF-Flag-GFP plasmid: synthesizing a GMSF-Flag gene fragment with EcoRI and HindIII enzyme cutting sites, cutting the GMSF-Flag gene fragment and an Ade-PDC316-CMV-GFP vector by using EcoRI/HindIII enzyme, and constructing a plasmid Ade-PDC316-CMV-GMSF-Flag-GFP after connection;
constructing Ade-PDC316-CMV-RGD-4C-GMSF-Flag-GFP plasmid: designing forward amplification primers containing EcoRI-RGD-4C-GMSF-Flag and Hind III-GMSF-Flag reverse amplification primers, amplifying and introducing RGD-4C target genes, using EcoRI/Hind III enzyme digestion RGD-4C-GMSF-Flag gene fragments and Ade-PDC316-CMV-GMSF-Flag-GFP vectors after amplification, and constructing plasmids Ade-PDC316-CMV-RGD-4C-GMSF-Flag-GFP after connection; and/or
Constructing Ade-PDC316-AFP-RGD-4C-GMSF-Flag-GFP plasmid: synthesizing a human AFP promoter with Xba I and EcoRI enzyme cutting sites, cutting an AFP gene fragment and an Ade-PDC316-CMV-RGD-4C-GMSF-Flag-GFP vector by Xba I/EcoRI enzyme cutting, replacing the CMV promoter with the human AFP promoter, and constructing a plasmid Ade after connectionAFP +RGD-4C+GMSF-Flag+GFP。
In one aspect, the method can further comprise constructing the plasmid AdeAFP+RGD-4C+GMSF-Flag+GFPThen, the glutamyl transpeptidase gene and the tumor-associated antigen gene were ligated to AdeAFP+RGD-4C+GMSF-Flag+GFPThus, the Ade-PDC316-AFP-RGD-4C-GMSF-Flag-GT-TAA plasmid was constructed.
The application also discloses a construction method of the oncolytic virus for individualized targeted therapy of liver cancer, and the oncolytic virus comprises the virus construct.
In one aspect, the invention discloses a construction method of a novel oncolytic virus for individualized targeted therapy of liver cancer, which comprises the following steps:
optional step (1): constructing Ade-PDC316-CMV-GFP plasmid: designing a forward amplification primer containing HindIII-GFP and a reverse amplification primer containing SalI-GFP, amplifying a GFP target gene with HindIII/SalI double enzyme cutting sites from PSMPuW-GFP, and connecting an Ade-PDC316-CMV vector by using HindIII/SalI double enzyme cutting to obtain a plasmid Ade-PDC 316-CMV-GFP;
step (2): construction of Ade-PDC316-CMV-GMSF-Flag plasmid: synthesizing a GMSF-Flag gene fragment with EcoRI and HindIII enzyme cutting sites, using EcoRI/HindIII to cut the GMSF-Flag gene fragment and an Ade-PDC316 vector, and constructing a plasmid Ade-PDC316-CMV-GMSF-Flag after connection;
optional step (3): construction of Ade-PDC316-CMV-GMSF-Flag-GFP plasmid: synthesizing a GMSF-Flag gene fragment with EcoRI and HindIII enzyme cutting sites, cutting the GMSF-Flag gene fragment and an Ade-PDC316-CMV-GFP vector by using EcoRI/HindIII enzyme, and constructing a plasmid Ade-PDC316-CMV-GMSF-Flag-GFP after connection;
and (4): constructing Ade-PDC316-CMV-RGD-4C-GMSF-Flag plasmid: designing forward amplification primers containing EcoRI-RGD-4C-GMSF-Flag and Hind III-GMSF-Flag reverse amplification primers, amplifying and introducing RGD-4C target genes, using EcoRI/Hind III enzyme digestion RGD-4C-GMSF-Flag gene fragments and an Ade-PDC316-CMV-GMSF-Flag vector after amplification, and constructing plasmids Ade-PDC316-CMV-RGD-4C-GMSF-Flag after connection;
optional step (5): constructing Ade-PDC316-CMV-RGD-4C-GMSF-Flag-GFP plasmid: designing forward amplification primers containing EcoRI-RGD-4C-GMSF-Flag and Hind III-GMSF-Flag reverse amplification primers, amplifying and introducing RGD-4C target genes, using EcoRI/Hind III enzyme digestion RGD-4C-GMSF-Flag gene fragments and Ade-PDC316-CMV-GMSF-Flag-GFP vectors after amplification, and constructing plasmids Ade-PDC316-CMV-RGD-4C-GMSF-Flag-GFP after connection;
and (6): constructing Ade-PDC316-AFP-RGD-4C-GMSF-Flag plasmid: synthesizing a human AFP promoter with Xba I and EcoRI enzyme cutting sites, cutting an AFP gene fragment and an Ade-PDC316-CMV-RGD-4C-GMSF-Flag vector by Xba I/EcoRI enzyme, replacing the CMV promoter with the human AFP promoter, and constructing a plasmid Ade after connectionAFP+RGD-4C+GMSF-Flag；
Optional step (7): constructing Ade-PDC316-AFP-RGD-4C-GMSF-Flag-GFP plasmid: synthesizing a human AFP promoter with XbaI and EcoRI enzyme cutting sites, cutting an AFP gene fragment and an Ade-PDC316-CMV-RGD-4C-GMSF-Flag-GFP vector by XbaI/EcoRI enzyme cutting, replacing the CMV promoter with the human AFP promoter, and constructing a plasmid Ade after T4 DNA ligase connectionAFP+RGD-4C+GMSF-Flag+GFP(ii) a And
optional step (8): glutamyl transpeptidase gene and tumor-associated anti-tumor agentsLigation of Progenes to AdeAFP +RGD-4C+GMSF-Flag+GFPThus, the Ade-PDC316-AFP-RGD-4C-GMSF-Flag-GT-TAA plasmid was constructed.
In one aspect, the method of the invention comprises individualizing and customizing Ade-PDC316-AFP-RGD-4C-GMSF-Flag-GT-TAA plasmid, and later, finding out corresponding specific antigen by combining molecular diagnosis to individualize. In one aspect, ligation may be performed using T4 DNA ligase. The ligation in steps (7) and (8) may use T4 DNA ligase.
In the above technical scheme, the sequences of the HindIII-GFP forward amplification primer and the SalI-GFP reverse amplification primer are as follows:
HindⅢ-GFP-F-p：CCCAAGCTTATGGTGAGCAAGGGCGAGGAGC
SalⅠ-GFP-R-p:ACGC GTCGACTTACTTGTACAGCTCGTCCATG。
in the technical scheme, the sequences of the EcoRI-RGD-4C-GMSF-Flag forward amplification primer and the HindIII-GMSF-Flag reverse amplification primer are as follows:
EcoRⅠ-RGD-GMSF-Flag-F-p:CCGGAATTCTGTGACTGCCGCGGAGACTGTTTCTGCATGTGGCTGCAGAGCCTGCTGCTG
HindⅢ-RGD-GMSF-Flag-R-p:CCCAAGCTTCTTGTCGTCGTCGTCCTTGTAGTCCTCCTGCACGGGCTCCCAGCAGTCGAAG。
in the above technical scheme, in each step of the method of the present invention, all newly constructed plasmids or vectors are obtained by selecting single colonies through transformation, extracting plasmid clones, and sequencing.
In the technical scheme, the liver cancer specific promoter is an AFP promoter.
In the technical scheme, the construction method is a construction method of a novel oncolytic virus for individualized targeted therapy of liver cancer.
In the technical scheme, GT-TAA is introduced by primer design and amplification according to different liver cancer types.
Compared with the prior art, the invention has the beneficial effects that:
replacing CMV promoter on adenovirus vector with liver cancer promoter, and increasing RGD-4C gene for virus replication and cell immunity to cause tumor lysisThe gene fragment GMSF-Flag is connected to an adenovirus expression vector with a liver cancer promoter to construct a novel oncolytic virus expression vector AdeAFP+RGD-4C+GMSF-Flag。
Almost all liver cancer cells can express AFP, so the AFP promoter can ensure AdeAFP+RGD-4C+GMSF-FlagSelectively propagate in liver cancer cells. The virus does not infect normal liver cells and other normal tissues, is used in a targeted mode, has the characteristics of high efficiency/strong specificity and the like, can enhance the replication capacity of the virus by inserting an RGD-4C expression sequence, and can increase the cell immunoregulation effect by introducing GMSF-Flag of colony stimulating factors, thereby ensuring that damaged liver cancer cells are thoroughly and cleanly removed by immune cells of an organism.
Drawings
FIG. 1 shows recombinant adenovirus Ade of the present inventionAFP+RGD-4C+GMSF-FlagSchematic representation of (a).
FIG. 2 shows AdeAFP+RGD-4C+GMSF-FlagBar graph of the ability to selectively inhibit the survival of hepatoma cells HepG 2.
FIG. 3 is AdeAFP+RGD-4C+GMSF-FlagBar graph of the ability to selectively inhibit the clonogenic potential of hepatoma cells HepG 2.
FIG. 4 is AdeAFP+RGD-4C+GMSF-FlagBar graph of the ability to selectively inhibit migration of hepatoma cell HepG 2.
FIG. 5 is AdeAFP+RGD-4C+GMSF-FlagBar graph of the ability to selectively inhibit the invasion of hepatoma cell HepG 2.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the following description further explains how the invention is implemented by combining the attached drawings and the specific implementation method.
Examples
Example 1 construction of viral constructs
The restriction enzymes HindIII/SalI/EcoRI/XbaI of the invention were purchased from New England Biolabs; PCR amplification reagents purchased from TARAKA BAOri physician technology (Beijing) Co., Ltd; endotoxin-free plasmid extraction kits DP117, DP118 were purchased from Tiangen (TIANGN) Biochemical technology (Beijing) Ltd; pBHGlox _ E1,3Cre recombinant adenovirus skeleton vector and pDC316 adenovirus vector are purchased from Changsha Youbao organism; GMSF-Flag and AFP fragments and all amplification primers were synthesized in Sangon Biotech (Shanghai). DNA recovery kits were purchased from Promega.
The invention discloses a construction method of a novel oncolytic virus for individualized targeted therapy of liver cancer, which comprises the following steps:
the method comprises the following steps: constructing Ade-PDC316-CMV-GFP plasmid: in order to facilitate the detection of the expression conditions of various embedded genes, various vectors containing GFP genes are constructed in parallel, a forward amplification primer containing HindIII-GFP and a reverse amplification primer containing SalI-GFP are designed, a GFP target gene with HindIII/SalI double digestion sites is amplified from PSMPuW-GFP (stored in laboratories), and a HindIII/SalI double digestion Ade-PDC316-CMV vector is used for connection to obtain a new plasmid Ade-PDC 316-CMV-GFP.
Step two: construction of Ade-PDC316-CMV-GMSF-Flag plasmid: synthesizing a GMSF-Flag gene fragment with EcoRI and HindIII enzyme cutting sites (in a production synthesis way), cutting the GMSF-Flag gene fragment and an Ade-PDC316 vector by using EcoRI/HindIII enzyme, and constructing a new plasmid Ade-PDC316-CMV-GMSF-Flag after connection;
step three: construction of Ade-PDC316-CMV-GMSF-Flag-GFP plasmid: synthesizing a GMSF-Flag gene fragment with EcoRI and HindIII enzyme cutting sites, cutting the GMSF-Flag gene fragment and an Ade-PDC316-CMV-GFP vector by using EcoRI/HindIII enzyme, and constructing a new plasmid Ade-PDC316-CMV-GMSF-Flag-GFP after connection;
step four: constructing Ade-PDC316-CMV-RGD-4C-GMSF-Flag plasmid: designing a forward amplification primer containing EcoRI-RGD-4C-GMSF-Flag and a Hind III-GMSF-Flag reverse amplification primer, amplifying and introducing a new RGD-4C target gene, using EcoRI/Hind III to enzyme-cut RGD-4C-GMSF-Flag gene fragment and an Ade-PDC316-CMV-GMSF-Flag vector after amplification, and constructing a new plasmid Ade-PDC316-CMV-RGD-4C-GMSF-Flag after connection;
step five: constructing Ade-PDC316-CMV-RGD-4C-GMSF-Flag-GFP plasmid: designing a forward amplification primer containing EcoRI-RGD-4C-GMSF-Flag and a Hind III-GMSF-Flag reverse amplification primer, amplifying and introducing a new RGD-4C target gene, using EcoRI/Hind III to enzyme-cut RGD-4C-GMSF-Flag gene fragment and an Ade-PDC316-CMV-GMSF-Flag-GFP vector after amplification, and constructing a new plasmid Ade-PDC316-CMV-RGD-4C-GMSF-Flag-GFP after connection;
step six: constructing Ade-PDC316-AFP-RGD-4C-GMSF-Flag plasmid: synthesizing a human AFP promoter with Xba I and EcoRI enzyme cutting sites, cutting an AFP gene fragment and an Ade-PDC316-CMV-RGD-4C-GMSF-Flag vector by Xba I/EcoRI enzyme cutting, replacing a CMV promoter by the human AFP promoter, and constructing a new plasmid Ade after T4 DNA ligase connectionAFP+RGD-4C+GMSF-Flag；
Step seven: constructing Ade-PDC316-AFP-RGD-4C-GMSF-Flag-GFP plasmid: synthesizing a human AFP promoter with XbaI and EcoRI enzyme cutting sites, cutting an AFP gene fragment and an Ade-PDC316-CMV-RGD-4C-GMSF-Flag-GFP vector by XbaI/EcoRI enzyme cutting, replacing the CMV promoter with the human AFP promoter, and constructing a new plasmid Ade after T4 DNA ligase connectionAFP+RGD-4C+GMSF-Flag+GFP；
Step eight: Ade-PDC316-AFP-RGD-4C-GMSF-Flag-GT-TAA plasmid is customized individually, and corresponding specific antigen is found out by combining molecular diagnosis in the later period to be customized individually. For example, linking a glutamyl transpeptidase gene and a tumor-associated antigen gene to AdeAFP+RGD-4C+GMSF-Flag+GFPThus, the Ade-PDC316-AFP-RGD-4C-GMSF-Flag-GT-TAA plasmid was constructed. Finally, the GT-TAA is introduced by primer design and amplification according to different liver cancer types.
As shown in Table 1, the sequence of the HindIII-GFP forward amplification primer and SalI-GFP reverse amplification primer obtained in step one is as follows:
HindⅢ-GFP-F-p：CCCAAGCTTATGGTGAGCAAGGGCGAGGAGC
SalⅠ-GFP-R-p:ACGC GTCGACTTACTTGTACAGCTCGTCCATG
in the RGD-4C-GMSF-Flag target gene in the fourth step of the technical scheme,
the sequences of EcoRI-RGD-4C-GMSF-Flag forward amplification primers, and Hind III-GMSF-Flag reverse amplification primers are as follows:
EcoRⅠ-RGD-GMSF-Flag-F-p:CCGGAATTCTGTGACTGCCGCGGAGACTGTTTCTGCATGTGGCTGCAGAGCCTGCTGCTG
HindⅢ-RGD-GMSF-Flag-R-p:CCCAAGCTTCTTGTCGTCGTCGTCCTTGTAGTCCTCCTGCACGGGCTCCCAGCAGTCGAAG
TABLE 1 primers used for the construction of plasmid Ade-PDC316-AFP-RGD-4C-GMSF-Flag-GFP-GT-TAA
Figure BDA0002599150900000131
Note: the genomic sequence is indicated in bold, the restriction sites used in plasmid construction are underlined, and the unlabeled bases are the protecting bases for the cleavage sites.
In the invention, all newly constructed plasmids in the steps from the first step to the ninth step are obtained by transforming and picking single colonies, extracting plasmid clone and sequencing.
The invention also discloses a novel oncolytic virus for individualized targeted therapy of liver cancer, which adopts the following construction method: replacing CMV promoter on adenovirus vector with liver cancer promoter, connecting RGD-4C gene for increasing virus replication and gene fragment GMSF-Flag for increasing cell immunity induced oncolytic to adenovirus expression vector with liver cancer promoter to construct novel oncolytic virus expression vector AdeAFP+RGD-4C+GMSF-Flag. Wherein the liver cancer specific promoter is an AFP promoter; as shown in fig. 1.
In the invention, the construction method is the construction method in the construction method of the novel oncolytic virus for individualized targeted therapy of liver cancer.
Example 2 verification of oncolytic Effect
In the present invention, a novel oncolytic virus AdeAFP+RGD-4C+GMSF-FlagHas selective inhibition effect on liver cancer cells, does not infect normal liver cells, and has low infection rate in other tumor cells.
MTT assay
MTT is a good method to reflect the viability of cells. The present invention uses MTT to detect (0, 0.001, 0.01, 0.1, 1 Ade) at different viral titersAFP+RGD-4C+GMSF-FlagThe effect on the survival ability of liver cancer cells HepG2 and normal cells HL7702 shows that the novel virus AdeAFP+RGD-4C+GMSF-FlagCan remarkably inhibit the survival ability of liver cancer cell HepG2, has no influence on the survival ability of normal liver cell HL7702 (see figure 2)
2. Plate clone formation experiment
Clonality is a unique property of malignant cells. The plate clone formation experiment can well reflect the clone formation capability of tumor cells. Ade was detected using a plate cloning assayAFP+RGD-4C+GMSF-FlagThe influence on the clone forming capability of liver cancer cells HepG2 and normal liver cells HL7702, and the discovery of the novel oncolytic virus AdeAFP +RGD-4C+GMSF-FlagCan obviously inhibit the clonogenic capacity of hepatoma cell HepG2, and has no influence on the clonogenic capacity of normal hepatoma cell HL7702 (see figure 3).
Transwell cell migration experiment
The ability to migrate is a characteristic of malignant tumors, reflecting the metastatic ability of tumors. Transwell cell migration assays were able to detect this migration ability. Ade is detected by using Transwell cell migration experimentAFP+RGD-4C+GMSF-FlagThe influence on the migration capacity of liver cancer cells HepG2 and normal liver cells HL7702, and the discovery of the novel oncolytic virus AdeAFP +RGD-4C+GMSF-FlagCan obviously inhibit the migration ability of the liver cancer cell HepG2, and has no influence on the migration ability of the normal liver cell HL7702 (see figure 4).
Transwell cell invasion assay
Invasion is a unique characteristic of malignant tumors and the Transwell cell invasion assay can respond to this invasive capability. Ade was detected using a Transwell cell invasion assayAFP+RGD-4C+GMSF-FlagThe influence on the invasion capacity of liver cancer cells HepG2 and normal liver cells HL7702, and the discovery shows that the novel oncolytic virus AdeAFP+RGD-4C+GMSF-FlagCan obviously inhibit the invasion capacity of liver cancer cell HepG2, and has no influence on the invasion capacity of normal liver cell HL7702 (see figure 4).
The invention adopts MTT detection, plate clone formation experiment and TranThe swell cell migration experiment and the Transwell cell invasion experiment show that the novel virus Ade provided by the inventionAFP+RGD-4C+GMSF-FlagHas the capability of selectively inhibiting the survival, clone formation, migration and invasion of liver cancer cells HepG2, and shows that AdeAFP+RGD-4C+GMSF-FlagCan selectively kill liver cancer cells.
AFP promoter sequence (SEQ ID NO.1) in the present invention:
Figure BDA0002599150900000151
the sequence of RGD-4C (SEQ ID NO.2) in the invention:
1 TGTGACTGCC GCGGAGACTG TTTCTGC
sequence of GMSF-Flag of the present invention (SEQ ID NO. 3):
Figure BDA0002599150900000152
Figure BDA0002599150900000161
the sequence of RGD-4C-GMSF-Flag (SEQ ID NO.4) in the invention:
Figure BDA0002599150900000162
sequence of GFP in the invention (SEQ ID NO.5):
Figure BDA0002599150900000163
Figure BDA0002599150900000171
finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Figure BDA0002599150900000181
Figure BDA0002599150900000191
Figure BDA0002599150900000201
Sequence listing
<110> Bingding (Beijing) International cell medicine technology Ltd
<120> virus construct for treating liver cancer and application and construction method thereof
<130> YHPC12005056
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 250
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
attctgtagt ttgaggagaa tatttgttat atttgcaaaa taaaataagt ttgcaagttt 60
tttttttctg ccccaaagag ctctgtgtcc ttgaacataa aatacaaata accgctctgc 120
tgttaattat tggcaaatgt cccattttca acctaaggaa ataccataaa gtaacagata 180
taccaacaaa aggttactag ttaacaggca ttgcctgaaa agagtataaa agaatttcag 240
catgattttc 250
<210> 2
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgtgactgcc gcggagactg tttctgc 27
<210> 3
<211> 456
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgtggctgc agagcctgct gctgctgggc accgtggcct gcagcatcag cgcccccgcc 60
agaagcccca gccccagcac ccagccttgg gagcacgtga acgccatcca ggaggccaga 120
agactgctga acctgagcag agacaccgcc gccgagatga acgagaccgt ggaggtcatc 180
agcgagatgt tcgacctgca ggagcccacc tgcctgcaga ccagactgga gctgtacaag 240
cagggcctga gaggcagcct gaccaagctg aagggccccc tgaccatgat ggccagccac 300
tacaagcagc actgcccccc cacccccgag accagctgcg ccacccagat catcaccttc 360
gagagcttca aggagaacct gaaggacttc ctgctggtca tccccttcga ctgctgggag 420
cccgtgcagg aggactacaa ggacgacgac gacaag 456
<210> 4
<211> 483
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tgtgactgcc gcggagactg tttctgcatg tggctgcaga gcctgctgct gctgggcacc 60
gtggcctgca gcatcagcgc ccccgccaga agccccagcc ccagcaccca gccttgggag 120
cacgtgaacg ccatccagga ggccagaaga ctgctgaacc tgagcagaga caccgccgcc 180
gagatgaacg agaccgtgga ggtcatcagc gagatgttcg acctgcagga gcccacctgc 240
ctgcagacca gactggagct gtacaagcag ggcctgagag gcagcctgac caagctgaag 300
ggccccctga ccatgatggc cagccactac aagcagcact gcccccccac ccccgagacc 360
agctgcgcca cccagatcat caccttcgag agcttcaagg agaacctgaa ggacttcctg 420
ctggtcatcc ccttcgactg ctgggagccc gtgcaggagg actacaagga cgacgacgac 480
aag 483
<210> 5
<211> 720
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

Claims (10)

1. A viral construct for the treatment of liver cancer comprising in operative association:
(a) AFP promoter with nucleotide sequence shown as SEQ ID NO.1,
(b) RGD-4C gene with nucleotide sequence shown as SEQ ID NO.2, and
(c) GMSF-flag gene with nucleotide sequence shown in SEQ ID NO. 3.
2. The viral construct of claim 1, which is an adenoviral construct.
3. The viral construct of claim 1, further comprising (d) a glutamyl transpeptidase gene and a tumor-associated antigen gene.
4. The viral construct according to any one of claims 1 to 3, comprising in 5 'to 3' direction the components (a), (b) and (c) and optionally (d).
5. Use of the viral construct of any one of claims 1-4 in the manufacture of a medicament for treating cancer in a patient.
6. The use of claim 5, wherein the cancer is liver cancer, such as hepatocellular carcinoma.
7. A method of constructing the viral construct of any one of claims 1 to 4, comprising the steps of:
joining the RGD-4C gene which increases viral replication and the GMSF-Flag gene which increases cell immunity causing oncolysis to an adenovirus expression vector, and
replacing the CMV promoter on the adenovirus vector with a liver cancer promoter AFP promoter,
thereby constructing an oncolytic virus expression vector AdeAFP+RGD-4C+GMSF-Flag。
8. A method of constructing the viral construct of any one of claims 1 to 4, comprising the steps of:
optionally (1) constructing Ade-PDC316-CMV-GFP plasmid: designing a forward amplification primer containing HindIII-GFP and a reverse amplification primer containing SalI-GFP, amplifying a GFP target gene with HindIII/SalI double enzyme cutting sites from PSMPuW-GFP, and connecting an Ade-PDC316-CMV vector by using HindIII/SalI double enzyme cutting to obtain a plasmid Ade-PDC 316-CMV-GFP;
(2) construction of Ade-PDC316-CMV-GMSF-Flag plasmid: synthesizing a GMSF-Flag gene fragment with EcoRI and HindIII enzyme cutting sites, using EcoRI/HindIII to cut the GMSF-Flag gene fragment and an Ade-PDC316 vector, and constructing a plasmid Ade-PDC316-CMV-GMSF-Flag after connection;
optionally (3) constructing Ade-PDC316-CMV-GMSF-Flag-GFP plasmid: synthesizing a GMSF-Flag gene fragment with EcoRI and HindIII enzyme cutting sites, cutting the GMSF-Flag gene fragment and an Ade-PDC316-CMV-GFP vector by using EcoRI/HindIII enzyme, and constructing a plasmid Ade-PDC316-CMV-GMSF-Flag-GFP after connection;
(4) constructing Ade-PDC316-CMV-RGD-4C-GMSF-Flag plasmid: designing forward amplification primers containing EcoRI-RGD-4C-GMSF-Flag and Hind III-GMSF-Flag reverse amplification primers, amplifying and introducing RGD-4C target genes, using EcoRI/Hind III enzyme digestion RGD-4C-GMSF-Flag gene fragments and an Ade-PDC316-CMV-GMSF-Flag vector after amplification, and constructing plasmids Ade-PDC316-CMV-RGD-4C-GMSF-Flag after connection;
optionally (5) constructing Ade-PDC316-CMV-RGD-4C-GMSF-Flag-GFP plasmid: designing forward amplification primers containing EcoRI-RGD-4C-GMSF-Flag and Hind III-GMSF-Flag reverse amplification primers, amplifying and introducing RGD-4C target genes, using EcoRI/Hind III enzyme digestion RGD-4C-GMSF-Flag gene fragments and Ade-PDC316-CMV-GMSF-Flag-GFP vectors after amplification, and constructing plasmids Ade-PDC316-CMV-RGD-4C-GMSF-Flag-GFP after connection;
(6) constructing Ade-PDC316-AFP-RGD-4C-GMSF-Flag plasmid: synthesizing a human AFP promoter with XbaI and EcoRI enzyme cutting sites, cutting the AFP gene fragment and Ade-PDC316-CMV-RGD-4C-GMSF-Flag vector by XbaI/EcoRI enzyme, replacing CMV promoter by the human AFP promoter, preferably using the human AFP promoterConstruction of plasmid Ade following ligation of T4 DNA ligaseAFP+RGD-4C+GMSF-Flag；
Optionally (7) constructing Ade-PDC316-AFP-RGD-4C-GMSF-Flag-GFP plasmid: synthesizing a human AFP promoter with XbaI and EcoRI enzyme cutting sites, cutting an AFP gene fragment and an Ade-PDC316-CMV-RGD-4C-GMSF-Flag-GFP vector by XbaI/EcoRI enzyme cutting, replacing the CMV promoter by the human AFP promoter, preferably connecting by using T4 DNA ligase, and constructing a plasmid AdeAFP+RGD-4C+GMSF-Flag+GFP(ii) a And
optionally (8) linking the glutamyltranspeptidase gene and the tumor-associated antigen gene to AdeAFP+RGD-4C+GMSF-Flag+GFPThus, the Ade-PDC316-AFP-RGD-4C-GMSF-Flag-GT-TAA plasmid was constructed.
9. The method of claim 8, wherein in step (1), the HindIII-GFP forward amplification primer and SalI-GFP reverse amplification primer sequences are as follows:
HindⅢ-GFP-F-p：CCCAAGCTTATGGTGAGCAAGGGCGAGGAGC
SalⅠ-GFP-R-p:ACGCGTCGACTTACTTGTACAGCTCGTCCATG。
10. the method of claim 8, wherein in step (4), the EcoRI-RGD-4C-GMSF-Flag forward amplification primers, and HindIII-GMSF-Flag reverse amplification primers have the following sequences:
EcoRⅠ-RGD-GMSF-Flag-F-p:
CCGGAATTCTGTGACTGCCGCGGAGACTGTTTCTGC
ATGTGGCTGCAGAGCCTGCTGCTG
HindⅢ-RGD-GMSF-Flag-R-p:
CCCAAGCTTCTTGTCGTCGTCGTCCTTGT
AGTCCTCCTGCACGGGCTCCCAGCAGTCGAAG。
CN202010718678.9A 2020-07-23 2020-07-23 Virus construct for treating liver cancer and application and construction method thereof Pending CN111909962A (en)

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