KR102471898B1 - Tumor-targeting trans-splicing ribozyme expressing immune checkpoint inhibitor and use thereof - Google Patents

Abstract

The present invention relates to cancer specific trans-splicing ribozymes and uses thereof. The trans-splicing ribozyme does not act on normal tissue, is specifically expressed in cancer tissue, has high safety, and has excellent expression efficiency at the post-transcriptional level, and the 3' exon of the ribozyme contains one or more target genes is connected, so when introduced into the body, it expresses cancer treatment genes and immune checkpoint inhibitors together, so it can be usefully used to treat cancer.

Description

Cancer-specific trans-splicing ribozymes expressing immune checkpoint inhibitors and their uses {TUMOR-TARGETING TRANS-SPLICING RIBOZYME EXPRESSING IMMUNE CHECKPOINT INHIBITOR AND USE THEREOF}

The present invention relates to a cancer-specific trans-splicing ribozyme that expresses an immune checkpoint inhibitor in cancer cells and uses thereof.

As medical technology advances, cancer treatment technology continues to develop. In particular, as the therapeutic effect of immuno-anticancer therapy recently developed using the human body's immune system has been proven, a paradigm shift from anti-cancer therapy using conventional chemotherapeutic agents and targeted therapy agents to immuno-anticancer therapy using immunotherapeutic agents is in progress.

Conventional anticancer treatment is tumor resection through surgery. At this time, radiation therapy and chemotherapy are performed in parallel for the purpose of reducing the size of the tumor before resection or killing remaining cancer cells after resection and preventing recurrence. Radiation therapy and chemotherapy, which are referred to as first-generation anticancer drugs (since the 1970s), induce death of cancer cells by interfering with the process in which cancer cells divide and amplify indefinitely. However, radiation therapy and chemotherapy have side effects that induce the death of normal cells as well as cancer cells.

In the 2000s, targeted anticancer drugs that selectively attack cancer cells separately from normal cells were developed, and were referred to as second-generation anticancer drugs that can significantly reduce side effects of the first-generation anticancer drugs. Targeted anticancer drugs that act only on specific target proteins show therapeutic effects by selectively inhibiting cancer cells by acting only on proteins that cause cancer. Therefore, since the induced protein or the protein exhibiting the therapeutic effect is different depending on the type of cancer, an anticancer agent suitable for the type of target protein must be used. Another limitation of targeted anticancer drugs is that cancer cells have a mechanism for acquiring resistance to targeted anticancer drugs. That is, since cancer cells acquire the ability to evade anticancer drugs by mutating themselves so that they do not become targets of the target anticancer drugs, the target anticancer drugs may not be recognized as cancer cells.

Immune anticancer drugs, a third-generation anticancer drug, activate the body's immune system to increase autoimmunity, allowing immune cells to attack and eliminate cancer cells. Immune cells whose ability to attack cancer cells is enhanced by immune anti-cancer drugs remember the cancer cells they attacked first and continue to attack them unless the cancer cells completely change their functions and properties.

Immune anticancer agents can be largely classified into immune checkpoint inhibitors, immune cell therapy, therapeutic antibodies, and anticancer vaccines. Immune checkpoint inhibitors are drugs that attack cancer cells by activating T cells by blocking the activity of immune checkpoint proteins involved in T cell suppression. use antibodies.

However, the response rate of cancer patients to immunocancer drugs remains at 15 to 45%, and the response rate varies depending on the type of cancer and patient, and affects not only cancer but also various organs in the body such as the skin and gastrointestinal system, thyroid and adrenal glands. As a result, side effects are increasingly being reported.

Immuno-Oncology Development Status, BRIC View 2019-T05

The present invention is intended to solve the above problems, and includes a transsplicing ribozyme containing two or more different cancer therapeutic genes including an immune checkpoint inhibitor gene, a vector capable of expressing the ribozyme, and a gene delivery system. It aims to provide

In addition, since the trans-splicing ribozyme can actively act in cancer cells by targeting a cancer-specific gene, an object of the present invention is to provide a cancer treatment use of the ribozyme, vector, or gene delivery system. .

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

In order to solve the above problems, the present invention is a trans-splicing ribozyme targeting a cancer-specific gene, wherein the ribozyme includes a target gene linked to a 3' exon, and the target gene is an immune checkpoint inhibitor gene. It provides a trans-splicing ribozyme, characterized in that it is a gene for the treatment of two or more cancers comprising.

As an embodiment of the present invention, the trans-splicing ribozyme may have a structure of 5' - trans-splicing ribozyme - cancer treatment gene - immune checkpoint inhibitor gene - 3'.

In another embodiment of the present invention, one of the two or more cancer therapeutic genes is a gene encoding an immune checkpoint inhibitor, and the other is a gene for cancer treatment distinct from the gene encoding a drug susceptibility gene, an apoptosis gene, and a cell proliferation inhibition gene. It may be one selected from the group consisting of genes, cytotoxic genes, tumor suppressor genes, antigenic genes, cytokine genes, and anti-angiogenic genes.

As another embodiment of the present invention, the drug susceptibility gene may be HSVtk (Herpes Simplex Virus thymidine kinase), and may consist of or include the nucleotide sequence of SEQ ID NO: 5.

In another embodiment of the present invention, the cancer-specific gene is TERT (Telomerase Reverse Transcriptase) mRNA, AFP (alphafetoprotein) mRNA, CEA (carcinoembryonic antigen) mRNA, PSA (Prostate-specific antigen) mRNA, CKAP2 (Cytoskeleton-associated protein 2) It may be one selected from the group consisting of mRNA and mutant rat sarcoma (RAS) mRNA.

According to one embodiment of the present invention, the trans-splicing ribozyme may target the TERT gene, and may consist of or include the nucleotide sequence of SEQ ID NO: 4.

In another embodiment of the present invention, the immune checkpoint inhibitor is CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA or A2aR may be an inhibitor of

As another embodiment of the present invention, the trans-splicing ribozyme may further include a sequence complementary to part or all of micro RNA-122a (miR-122a) at the 3' end, and SEQ ID NO: 6 It may include a nucleotide sequence, and may include a sequence in which the sequence is repeated 2 to 10 times.

In another embodiment of the present invention, in the transsplicing ribozyme, the immune checkpoint inhibitor gene and the gene for cancer treatment other than this gene can be linked to a gene encoding a self-cleaving peptide.

As another embodiment of the present invention, the self-cleaving peptide may be P2A and may be encoded by the nucleotide sequence of SEQ ID NO: 7.

In addition, the present invention provides an expression vector capable of expressing the trans-splicing ribozyme.

In one embodiment of the present invention, the expression vector may further include a promoter operably linked to the ribozyme gene, and the promoter may be a tissue-specific promoter.

In addition, the present invention provides a gene delivery system comprising the expression vector and cells transformed with the expression vector.

In addition, the present invention provides a pharmaceutical composition for treating cancer comprising at least one selected from the group consisting of the transsplicing ribozyme, the expression vector, and the gene delivery system as an active ingredient.

In addition, the present invention provides a cancer treatment method comprising administering to a subject at least one selected from the group consisting of the transsplicing ribozyme, the expression vector, and the gene delivery system.

In addition, the present invention provides the use of the trans-splicing ribozyme, the expression vector, and/or the gene delivery system for preparing an anti-cancer drug.

As an embodiment of the present invention, the pharmaceutical composition and the anticancer agent may be administered orally or in the form of an injection intravenously, intraarterially, intravenously, intravenously, and/or subcutaneously, and in the cancer treatment method, Administration may be administered by the above route.

In another embodiment of the present invention, the cancer is liver cancer, glioblastoma, bile duct cancer, lung cancer, pancreatic cancer, melanoma, bone cancer, breast cancer, colon cancer, stomach cancer, prostate cancer, leukemia, uterine cancer, ovarian cancer, lymphoma, and brain cancer. It may be one or more cancers selected from the group consisting of:

As another embodiment of the present invention, the cancer may be immune checkpoint inhibitor resistant cancer.

The trans-splicing ribozyme according to the present invention does not act on normal tissues and is specifically expressed in cancer tissues, so it has high safety and excellent expression efficiency at the post-transcriptional level. In addition, since one or more target genes are linked to the 3' exon of the ribozyme of the present invention, when introduced into the body, it expresses a cancer treatment gene and an immune checkpoint inhibitor to increase the action and activity of immune cells, together with the expressed cancer treatment gene. It exhibits a synergistic effect on anticancer efficacy and can be usefully used for cancer treatment.

1 is a diagram schematically showing the basic configuration and anticancer mechanism of CRT-122T/ICI of the present invention. CRT-122T/ICI is a TERT-targeting trans-gene with a complementary sequence (mir-T) to miR-122a at the 3' end that expresses a cancer therapeutic gene (HSVtk) and an immune checkpoint inhibitor (ICI) in a cancer-specific manner. It is a vector capable of expressing a splicing ribozyme.
Figure 2 is a diagram schematically showing the construction of CRT-122T/ICI expressing PD1scFv (scFv to PD1) or PDL1scFv (scFv to PDL1).
Figure 3 is a result confirming the equivalence of RZ-001 by confirming the cell death inducing activity of various RZ-001+ in liver cancer cell lines. RZ-001+ is a viral vector capable of expressing vectors including CRT-122T/ICI. RZ-001 expresses cancer-specific gene (HSVtk) for cancer treatment and can express hTERT-targeted trans-splicing ribozyme having a complementary sequence (mir-T) to miR-122a at the 3' end. It is a viral vector (patent registration number; 10-2252423).
Figure 4 is a result confirming the equivalence of RZ-001 by confirming the cell death inducing activity of various RZ-001+ in brain tumor cell lines.
5 is a result confirming the equivalence of RZ-001 by confirming the cell death inducing activity of various RZ-001+ in lung cancer cell lines and melanoma cell lines.
6 shows the result of confirming the expression level of the immune checkpoint inhibitor after preparing a stabilized cell line expressing scFvPD1(N) or scFvPD1(I), which is an immune checkpoint inhibitor.
7 shows the results of reacting cell lysates expressing human PD1 (hPD1) or mouse PD1 (mPD1) with scFvPD1 recovered from a stabilized cell line expressing scFvPD1.
8 is a result of confirming the expression of mPD1 after preparing a stabilized cell line expressing mouse PD1 (mPD1) from Hepa1-6 cells, a mouse liver cancer cell line.
9 shows the result of confirming the degree of virus infection after treating Hepa1-6 cells and Hepa1-6 cells (Hepa1-6/mPD1) expressing mouse PD1 (mPD1) with mRZ-001+, respectively. mRZ-001+ is a mouse TERT-targeting trans-gene with a complementary sequence (mir-T) to miR-122a at the 3' end that expresses cancer-specific genes for cancer treatment (HSVtk) and immune checkpoint inhibitors (ICI). It is a viral vector capable of expressing a splicing ribozyme.
10 is a result of confirming cell viability after treatment of Hepa1-6, Hepa1-6/mPD1 and Hepa1c1c7 cells with mRZ-001+, respectively.
11 shows the result of confirming the expression level of immune checkpoint inhibitors after treatment of Hepa1-6, Hepa1-6/mPD1 and Hepa1c1c7 cells with mRZ-001+, respectively.
12 shows the result of confirming the level of apoptosis by flow cytometry after treating Hepa1-6 cells with mRZ-001+.
13 shows the result of confirming the level of apoptosis by flow cytometry after treating Hepa1-6/mPDL1 cells with mRZ-001+.
14 shows the results of measuring the expression levels of target genes (HSVtk and scFv) in Hep3b and SNU398 cells after treatment with RZ-001+.
FIG. 15 is a result of confirming whether trans-splicing occurred to target TERT mRNA by a ribozyme expressed in a liver cancer cell line treated with RZ-001+ and the location of the trans-splicing.
16 shows the results of Hep3b and SNU398 cells treated with RZ-001+ and PD1/PDL1 blockade bioassay performed.
17 shows the result of confirming the expression of PD-L1 in each cell of SNU398, a human liver cancer cell line, and U87MG, a human brain tumor cell line.
18 shows the results of confirming the expression level of immune checkpoint inhibitors in U87MG cells treated with RZ-001+ at a concentration of 10MOI or 20MOI.
19 shows the results of PD1/PDL1 blockade bioassay performed by treating U87MG cells with increasing amounts of RZ-001+. As a control, PD1/PDL1 blockade bioassay by Atezolizumab (anti-PDL1) was performed.
20 is a graph showing inhibition of tumor growth and weight reduction according to RZ-001 administration, RZ-001+_At administration, or RZ-001 and At combined administration (RZ-001/At) in a PBMC-humanized liver cancer model and the results of each administered drug This is the result of confirming liver toxicity.
21 is an MRI photograph of each drug administration group in an orthotopic brain tumor syngeneic model.
22 is a result confirmed by comparing the degree of tumor size reduction according to mRZ-001 administration and mRZ-001+ administration in an orthotopic brain tumor syngeneic model.
23 is a result of confirming serum ALT and AST levels of the mRZ-001 and mRZ-001+ administration groups in an orthotopic brain tumor syngeneic model.
24 is a schematic diagram of an experiment for confirming the effect of RZ-001+ in a Xenograft Orthotopic brain tumor model.
25 is IVIS imaging images of mice administered with RZ-001 or RZ-001+ to the Xenograft Orthotopic brain tumor model according to the experimental progress.
26 is a result confirming the change in body weight according to the course of the experiment in mice administered with RZ-001 or RZ-001+ in the Xenograft Orthotopic brain tumor model.
27 is a result confirming the anticancer effect of RZ-001 or RZ-001+ in a Xenograft Orthotopic brain tumor model.

In a previous study, the present inventors suppressed the proliferation and growth of cancer using a transsplicing ribozyme targeting a cancer-specific gene, specifically TERT mRNA, and used the ribozyme to target a gene, particularly a gene for cancer treatment. A transsplicing ribozyme capable of treating cancer by inducing apoptosis of cancer cells and a vector expressing the same have been prepared.

In previous studies, the transsplicing ribozyme expression vector was named CRT-122T, and its basic structure includes the following structure.

[5'-promoter-TERT targeting ribozyme-target gene (HSVtk)-miR-122T-3']

A viral vector capable of expressing vectors with SD/SA and WPRE sequences added in CRT-122T was specified as RZ-001 (KR 10-2252423), and RZ-001 acts on various carcinomas to induce cell death, and tumor It can be applied to the treatment of cancer by inhibiting the growth of

In order to develop a transsplicing ribozyme that can treat cancer more effectively, the present inventors, in addition to previous studies, expanded the target gene expressed by the ribozyme into two types, and included an immune checkpoint inhibitor gene in the target gene. , developed CRT-122T/ICI as a vector capable of expressing the ribozyme. The basic structure of the CRT-122T/ICI of the present invention is as follows (FIG. 1).

[5'-promoter-TERT targeting ribozyme-target gene-self-cleaving peptide gene-immune checkpoint inhibitor gene-miR-122T-3']

Meanwhile, according to the immune checkpoint inhibitor encoded by CRT-122T/ICI in this specification, the target immune checkpoint protein or immune checkpoint inhibitor is indicated at the end based on “/”. For example, a vector expressing Atezolizumab is indicated as CRT-122T/At.

Through specific experiments, the present inventors showed that CRT-122T/ICI had a cell death inducing activity similar to that of CRT-122T in liver cancer cell lines, brain tumor cell lines, lung cancer cell lines, and melanoma cell lines (Example 2).

Furthermore, the present inventors constructed an adenovirus (hereinafter referred to as "RZ-001+") expressing CRT-122T/ICI and confirmed its action in various cell lines through specific experiments.

Meanwhile, in the present specification, RZ-001+ is labeled as RZ-001+_ (target immune checkpoint protein or immune checkpoint inhibitor) according to the type of loaded CRT-122T/ICI. For example, an adenovirus containing CRT-122T/At is denoted as RZ-001+_At.

Specifically, after infecting cancer cells with RZ-001+, the survival rate of the cancer cells was measured to confirm the degree of apoptosis according to RZ-001+ infection, and the introduction of CRT-122T/ICI according to the RZ-001+ infection It was confirmed whether an antibody capable of binding to the immune checkpoint protein and blocking its function was expressed, and then it was confirmed whether the antibody could be smoothly secreted and acted upon. As a result, it was confirmed that cell death of cancer cells increased with RZ-001+ infection, the expression of the antibody increased, and the expressed antibody was sufficiently secreted (Examples 5 and 6).

Then, in order to confirm whether the anticancer activity of RZ-001+ confirmed in cell experiments can be equally expressed in vivo, the present inventors prepared various cancer animal models, administered RZ-001+, and measured the size and weight of tumors. In particular, in order to evaluate the efficacy as a drug for treating brain tumors, we tried to confirm the possibility of RZ-001+ as a brain tumor treatment by constructing an orthotopic brain tumor model that simulates the microenvironment in the brain such as the BBB. As a result, both the liver cancer animal model and the brain tumor animal model were reduced in tumor size and weight according to RZ-001+ treatment. In particular, in the humanized liver cancer animal model, the RZ-001+_At administration group reduced the tumor size to a similar level compared to the RZ-001 and Atezolizumab combination administration group, but showed lower liver toxicity than the combination administration group, with no side effects. It was confirmed that it can be provided as no or little anticancer agent (Example 7). Furthermore, in the orthotopic brain tumor syngeneic model, the mRZ-001+_I (mCRT-122T_scFvPD1(I))-administered group showed more effective anticancer activity than the mRZ-001-administered group. In addition, the effective anti-cancer activity of RZ-001+ was confirmed in a Xenograft Orthotopic brain tumor model in which human-derived brain tumor cells were transplanted. Trans-splicing ribozymes that can be used for cancer treatment are provided.

Each component of the trans-splicing ribozyme of the present invention and its basic structure are as follows.

[5' - Ribozyme targeting cancer specific gene - Target gene - Self-cleavage peptide gene - Immune checkpoint inhibitor gene - miR-122T - 3']

The trans-splicing ribozyme can complementarily bind to the mRNA of a cancer-specific gene in a target cell, cleave the gene, and express a transcript below the target gene.

3' 순서대로 암 특이적 유전자를 표적하는 리보자임, 목적 유전자, 자가 절단 펩타이드 유전자, 면역관문억제제 유전자, 및 miR-122T를 포함하며, 각 구성은 그 기능을 유지하는 범위 내에서 직접적 또는 간접적 방법으로 작동 가능하게 연결된 것일 수 있고, 상기 트랜스 스플라이싱 리보자임은 특히 목적 유전자의 기능을 향상시키기 위하여 각 구성 사이에 조절 인자를 추가로 포함할 수 있다. The trans-splicing ribozyme of the present invention is 5'

It includes a ribozyme targeting a cancer-specific gene in 3' order, a target gene, a self-cleavage peptide gene, an immune checkpoint inhibitor gene, and miR-122T, and each component is a direct or indirect method within the range of maintaining its function It may be operably linked to, and the trans-splicing ribozyme may further include a regulatory factor between each component in order to specifically improve the function of the target gene.

The term "ribozyme" used in the present invention is a molecule composed of an RNA molecule that acts like an enzyme or a protein containing the RNA molecule, and is also called an RNA enzyme or catalytic RNA. An RNA molecule with a clear tertiary structure that performs chemical reactions and has catalytic or autocatalytic properties. Some ribozymes inhibit their activity by cleaving their own or other RNA molecules, and other ribozymes are known to catalyze the activity of ribosome aminotransferases. Such ribozymes may include hammerhead ribozymes, VS ribozymes, and hairpin ribozymes.

The ribozyme according to the present invention inhibits the activity of cancer-specific genes through the trans-splicing reaction of Group I introns, thereby exhibiting a selective anti-cancer effect, and is expressed in a conjugated form with a cancer therapeutic gene to treat cancer. genes can be activated. Therefore, any type of material may be used as long as it exhibits characteristics capable of inactivating cancer-specific genes and activating cancer therapeutic genes.

The ribozyme according to the present invention may preferably be a ribozyme targeting hTERT mRNA described above, targeting cancer cells in which hTERT is overexpressed, specifically cleaving hTERT mRNA to inhibit expression, and It can play a role in specific expression.

As used herein, the term “trans-splicing” means linking RNAs from different genes to each other. Preferably, an hTERT target trans-splicing group I ribozyme that has been verified to have the ability to trans-splice by recognizing cancer-specific hTERT mRNA may be used.

The term "target gene" used in the present invention refers to a gene whose expression is induced by being linked to mRNA of a cancer-specific gene by the ribozyme.

The target gene according to the present invention may preferably be a gene for cancer treatment or a reporter gene, and most preferably a gene for cancer treatment.

As used herein, the term "anti-cancer therapeutic gene" refers to a polynucleotide sequence encoding a polypeptide that exhibits a therapeutic effect when expressed in cancer cells. The cancer therapeutic gene may be expressed in a conjugated form with the ribozyme or expressed independently to exhibit anticancer activity. These cancer therapeutic genes are preferably selected from the group consisting of drug susceptibility genes, apoptosis genes, cytostatic genes, cytotoxic genes, tumor suppressor genes, antigenic genes, cytokine genes, and anti-angiogenic genes. It may be one or more, and most preferably, it may be a drug susceptibility gene.

In the present invention, the cancer treatment gene may be used alone or two or more genes may be used in combination.

The drug-sensitizing gene according to the present invention is a gene encoding an enzyme that converts a non-toxic prodrug into a toxic substance, and is also called a suicide gene because cells into which the gene is introduced die. . That is, when a precursor that is not toxic to normal cells is systemically administered, the precursor is converted into a toxic metabolite only in cancer cells, thereby changing the sensitivity to the drug, thereby destroying cancer cells. These drug susceptibility genes are preferably HSVtk (Herpes simplex virus-thymidine kinase) genes using ganciclovir as a precursor, or E. coli using 5-fluorocytosine (5-FC) as a precursor. It may be a cytosine deaminase (CD) gene, and most preferably a HSVtk gene.

A proapoptotic gene according to the present invention refers to a nucleotide sequence that induces programmed cell death when expressed. Apoptosis genes known to those skilled in the art, encoding p53, adenovirus E3-11.6K (derived from Ad2 and Ad5) or adenovirus E3-10.5K (derived from Ad), adenovirus E4 gene, p53 pathway gene and caspase genes may be included.

A cytostatic gene according to the present invention refers to a nucleotide sequence that is expressed in cells and stops the cell cycle during the cell cycle. Examples include p21, retinoblastoma gene, E2F-Rb fusion protein gene, genes encoding cyclin-dependent kinase inhibitors (e.g., p16, p15, p18 and p19), growth arrest specific homeobox , GAX) gene, etc., but is not limited thereto.

A cytotoxic gene according to the present invention refers to a nucleotide sequence that is expressed in cells and exhibits a toxic effect. Examples include, but are not limited to, nucleotide sequences encoding Pseudomonas exotoxin, ricin toxin, diphtheria toxin, and the like.

A tumor suppressor gene according to the present invention refers to a nucleotide sequence capable of suppressing a tumor phenotype or inducing apoptosis by being expressed in a target cell. Representatively, tumor necrosis factor-α (TNF-α), p53 gene, APC gene, DPC-4/Smad4 gene, BRCA-1 gene, BRCA-2 gene, WT-1 gene, retinoblastoma gene, MMAC- 1 gene, adenomatous polyposis coil protein, missing colon tumor (DCC) gene, MMSC-2 gene, NF-1 gene, nasopharyngeal tumor suppressor gene located on chromosome 3p21.3, MTS1 gene , CDK4 gene, NF-1 gene, NF-2 gene, or VHL gene.

An antigenic gene according to the present invention refers to a nucleotide sequence that is expressed in a target cell and produces a cell surface antigenic protein that can be recognized by the immune system. Examples of antigenic genes known to those skilled in the art may include carcinoembryonic antigen (CEA) and p53.

A cytokine gene according to the present invention refers to a nucleotide sequence that is expressed in a cell to produce a cytokine. Representatively, GMCSF, interleukins (IL-1, IL-2, IL-4, IL-12, IL-10, IL-19, IL-20), interferon α, β, γ (interferon α-2b) and interferon α Fusions such as -2α-1 may be included.

An anti-angiogenic gene according to the present invention refers to a nucleotide sequence that is expressed and releases the anti-angiogenic factor out of the cell. Examples include angiostatin, vascular endothelial growth factor (VEGF) inhibitor, endostatin, and the like.

As used herein, the term “HSVtk (Herpes simplex virus-thymidine kinase)” refers to a thymidine kinase derived from herpes simplex virus. This enzyme is a representative example of a drug susceptibility gene that converts a non-toxic prodrug into a toxic substance, causing the transfected cell to die. In the present invention, the HSVtk gene is expressed in a form conjugated to the ribozyme according to the present invention and can be used as a cancer therapeutic gene exhibiting anticancer activity. 이러한 HSVtk 유전자는 바람직하게는 진뱅크(genbank) 등록번호 AAP13943, P03176, AAA45811, P04407, Q9QNF7, KIBET3, P17402, P06478, P06479, AAB30917, P08333, BAB84107, AAP13885, AAL73990, AAG40842, BAB11942, NP_044624, NP_044492, It may be described in CAB06747 or the like.

As used herein, the term "reporter gene" is a gene used to monitor the introduction of a recombinant vector or the expression efficiency of a ribozyme according to an embodiment of the present invention, which can be monitored without damaging infected cells or tissues. Genes may be used without limitation. Preferably, luciferase, green fluorescent protein (GFP), modified green fluorescent protein (mGFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), Modified red fluorescent protein (mRFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescence protein (EYFP), indigo fluorescent protein (CFP) or enhanced indigo fluorescent protein (ECFP).

By inserting a reporter gene into the target gene, the expression level of the cancer cell-specific ribozyme can be observed. In particular, the ribozyme of the present invention contains a promoter and a microRNA target site, so it is not expressed in normal cells and is specific to cancer cells. can manifest. It is obvious to those skilled in the art that it can be applied to diagnose whether cancer has occurred in a specific tissue using this.

In the present specification, a base sequence complementary to part or all of miR-122 is referred to as miR-122T (microRNA-122 target site). In the present invention, as long as miR-122T can complement miR-122 to form dsRNA, there may be differences in specific sequences. miR-122T may include a nucleotide sequence complementary to part or all of miR-122 one or more times, for example, 1 to 10 times, preferably 1 to 5 times, more preferably 1 time. It may be included repeatedly up to three times. miR-122 is normally expressed in normal hepatocytes, but its expression level is reduced in hepatoma cells. Using this, it is possible to develop a therapeutic agent with increased sensitivity and specificity for liver cancer cells. In the present invention, by linking a nucleic acid sequence recognizing miR-122 to a ribozyme to which a target gene is linked, expression of a liver cancer cell-specific ribozyme can be achieved. made it possible.

The term "cancer-specific gene" used in the present invention refers to a gene that is specifically expressed or markedly overexpressed only in cancer cells. The cancer-specific gene may add a feature that allows the ribozyme according to the present invention to act in a cancer-specific manner. These cancer-specific genes are preferably TERT (Telomerase reverse transcriptase) mRNA, AFP (alphafetoprotein) mRNA, CEA (carcinoembryonic antigen) mRNA, PSA (Prostate-specific antigen) mRNA, CKAP2 (Cytoskeleton-associated protein 2) mRNA, or It may be a mutant rat sarcoma (RAS) mRNA, more preferably a telomerase reverse transcriptase (TERT) mRNA, and most preferably a human telomerase reverse transcriptase (hTERT) mRNA sequence. The trans-splicing ribozyme of the present invention induces trans-splicing targeting the cancer-specific gene to express the target gene linked to the ribozyme, resulting in cell death in glioblastoma cell lines, melanoma cell lines, liver cancer cell lines, and lung cancer cell lines. The induction effect was confirmed.

The term used in the present invention, “TERT (Telomerase reverse transcriptase)” is one of the most important enzymes that regulate the immortality and proliferation ability of cancer cells, and forms telomere structures on chromosomes to form chromosome ends. It means an enzyme that inhibits cell aging through its role in protecting cells. In normal cells, each time the cell divides, the length of the telomeres decreases little by little, and eventually the genetic material is lost and the cell dies. However, in cancer cells, this enzyme continuously elongates telomeres, so the cells do not die, and it is known as a serious obstacle to cancer treatment by directly contributing to the immortality of cancer cells. In the present invention, hTERT mRNA may be used as a cancer-specific gene, but is not limited thereto.

As used herein, the term “gene delivery system” refers to a system capable of increasing expression efficiency by increasing the transfer efficiency of a desired gene and/or nucleic acid sequence into a cell, and is a virus-mediated system. and non-viral systems.

Virus-mediated systems use viral vectors such as retroviral vectors and adenovirus vectors, and have relatively higher efficiency of intracellular gene transfer than non-viral systems because they use the unique intracellular penetration mechanism of viruses that infect human cells. It is known. In addition, after entering the cell, the non-viral vector has a problem in that the endosome fuses with the lysosome and then the genes are degraded in the endolysosome, but the viral vector does not pass through the lysosome and transfers the gene into the nucleus by a mechanism It has the advantage of high gene delivery efficiency because the loss of is small.

Viral vectors that can be used in the present invention may be vectors derived from retroviruses, adenoviruses, adeno-associated viruses, and the like, as described above for recombinant vectors. These viral vectors can be assembled into viral particles and then introduced into cells by a transduction method such as infection.

In one embodiment of the present invention, a recombinant adenovirus containing the above-described recombinant vector as an example of a gene transfer vehicle was designed. That is, the recombinant adenovirus performs a function of delivering a recombinant vector expressing a trans-splicing ribozyme specific to a cancer-specific gene to a target cell (eg, cancer cell), and the recombinant delivered into the cell Vectors are expressed by the intracellular transcription system. The expressed trans-splicing ribozyme can insert a target gene linked to the ribozyme into the transcript of a cancer-specific gene present in large numbers in cancer cells.

In the present specification, RZ-001 means an adenovirus expressing CRT-122T, and RZ-001+ means an adenovirus expressing CRT-122T/ICI.

The non-viral system is a method using a cationic lipid delivery system or a cationic polymer carrier or the like as a delivery medium for nucleic acids and/or genes, or using an electroporation method.

Cationic lipid carriers use the positive charge of nanometer-sized liposomes or lipid nanoparticles mainly composed of cationic lipids to form complexes with negatively charged genes, expression vectors or nucleic acids containing genes, and then form the complexes by phagocytosis. method of delivery into cells. Complexes delivered into cells are first delivered from endosomes to lysosomes and then released into the cytosol to be expressed. Cationic polymer carriers deliver genes in a similar way to cationic lipid carriers, except that polymers are used instead of lipids. Representative cationic polymers include polyethyleneimine, poly-L-lysine, and chitosan. (chitosan), etc.

Therefore, a complex formed by combining the recombinant vector of the present invention with a cationic lipid carrier or a cationic polymer carrier can be used as a gene carrier.

In the present invention, the gene delivery system includes the recombinant vector described above, and both virus-mediated systems and non-viral systems may be used, but it is preferable to use a virus-mediated system.

As used herein, the term "vector" is an expression vector capable of expressing a target gene in a suitable host cell, and refers to a gene construct containing regulatory elements operably linked to express a gene insert contained in the vector.

As used herein, the term “operably linked” refers to functional linkage between a nucleic acid expression control sequence that performs a general function and a nucleic acid sequence encoding a gene of interest. In the ribozyme or vector of the present invention, each component is considered to be operably linked to each other unless specified as being directly linked.

For example, operably linking a ribozyme coding sequence to a promoter brings expression of the ribozyme coding sequence under the influence or control of the promoter. Two nucleic acid sequences (a ribozyme coding sequence and a promoter region sequence at the 5' end of the sequence) are operably linked when the action of a promoter is induced and the ribozyme coding sequence is transcribed, and the linkage between the two sequences changes the frame. If a frameshift mutation is not induced and the expression control sequence does not inhibit the expression of the ribozyme, it can be considered to be operably linked. Operable linkage with the recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cutting and linking can use enzymes generally known in the art.

The vector according to the present invention includes a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as a promoter, operator, initiation codon, stop codon, polyadenylation signal, and enhancer, and can be prepared in various ways according to the purpose. have. In addition, the vector of the present invention may further contain a regulatory factor capable of increasing the expression level of the trans-splicing ribozyme in cells. Non-limiting examples of regulatory factors for increased expression of ribozymes include splicing donor/splicing confer sequence (SD/SA) and WPRE. The vector's promoter may be constitutive or inducible. In addition, the expression vector may include a selectable marker for selecting host cells containing the vector, and may include an origin of replication in the case of a replicable expression vector. Vectors can replicate autonomously or integrate into host DNA.

The vector according to the present invention may preferably be a plasmid vector, a cosmid vector or a viral vector, and most preferably a viral vector. The viral vector is preferably a retrovirus, for example, human immunodeficiency virus (HIV), mouse leukemia virus (MLV), avian sarcoma / leukemia virus (Avian sarcoma / leucosis virus (ASLV), spleen necrosis virus (SNV), Rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), adenovirus, adeno-related virus (Adeno-associated virus, AAV), or a vector derived from a herpes simplex virus (Herpes simplex virus, HSV), etc., but is not limited thereto. The recombinant vector according to the present invention may most preferably be a recombinant adenoviral vector.

As used herein, the term "promoter" is involved in the binding of RNA polymerase to initiate transcription as a portion of DNA. In general, it is located adjacent to and upstream of the target gene, and is a binding site for RNA polymerase or a transcription factor, a protein that induces RNA polymerase, and can induce the enzyme or protein to be located at the correct transcription start site. can That is, it is located at the 5' site of the gene to be transcribed in the sense strand and induces RNA polymerase to bind to the corresponding position directly or through a transcription factor to initiate mRNA synthesis for the target gene. A specific gene sequence have

On the other hand, the trans-splicing ribozyme of the present invention has cell death inducing activity in various carcinomas and has no or very low liver toxicity, so it can be used for cancer treatment.

The term "cancer" used in the present invention refers to a problem in the control function of normal division, differentiation, and death of cells, which abnormally proliferates and infiltrates surrounding tissues and organs to form lumps and destroy existing structures. denotes a deformed state.

The cancer according to the present invention may preferably be liver cancer, glioblastoma, biliary tract cancer, lung cancer, pancreatic cancer, melanoma, bone cancer, breast cancer, colon cancer, stomach cancer, prostate cancer, leukemia, uterine cancer, ovarian cancer, lymphoma, or brain cancer, , more preferably liver cancer, lung cancer, melanoma, glioblastoma, and/or cholangiocarcinoma, and most preferably liver cancer and/or brain cancer.

In addition, the cancer according to the present invention may preferably have a copy number (expression level) of miR-122 expressed in cancer tissue that is less than 100 times the copy number of the ribozyme expressed in cancer tissue by the pharmaceutical composition. .

On the other hand, the present inventors compared the expression level of hTERT target ribozyme with miR-122T capable of inducing cell death in a previous study and the expression level of miR-122 in cells, and the ratio of miR-122 to ribozyme was It was confirmed that the higher the expression of the ribozyme, the lower the cell death inducing effect. Therefore, the amount of adenovirus expressing the ribozyme can be determined by inferring the amount of ribozyme to exhibit the anticancer effect according to the expression level of miR-122 in the cancer tissue. Specifically, if the minimum copy number of miR-122 is about 100 times or more than the ribozyme copy number, the function (expression) of the ribozyme having a miR-122 target site is weakened. It can be seen that high anticancer efficacy can be obtained when the number of copies of the ribozyme expressed in cancer tissue by the pharmaceutical composition according to is less than 100 times.

Also, the cancer according to the present invention may preferably be a cancer in which miR-122 is not substantially expressed in cancer tissue. The above “cancer in which miR-122 is not substantially expressed in cancer tissue” means that although miR-122 is expressed in cancer tissue, it is expressed in cancer tissue to the extent that it does not substantially affect the function of the ribozyme having a miR-122 target site. It means cancer with a low copy number of miR-122.

Through previous studies, the present inventors have found that the anticancer efficacy of the ribozyme according to the present invention in colorectal cancer, glioblastoma, melanoma, cervical cancer, lung cancer, osteosarcoma, breast cancer and cholangiocarcinoma cell lines in which miR-122 is not substantially expressed in cancer tissues confirmed.

In the present invention, the immune checkpoint inhibitor (ICI) performs the binding inhibitory function between PD-L1 and PD-1 for maintaining the immune function of T cells, and inhibits the activity of T-cells infiltrating into the tumor. Since it inhibits PD-1 or PDL-1, which inhibits it, it can increase the anticancer effect by maximizing the activity of T-cell. In a specific experiment, the present invention included the expression sequence of the immune checkpoint inhibitor of SEQ ID NOs: 1 to 3 in the ribozyme of the present invention to induce expression in cells and confirm its function, but the immune checkpoint protein was targeted for T-cell activity. It is not limited as long as it is an immune checkpoint inhibitor.

The term "prevention" used in the present invention refers to any action that suppresses or delays the onset of cancer by administering the combination or pharmaceutical composition according to the present invention.

The term "treatment" used in the present invention refers to all activities that improve cancer or beneficially change its symptoms by administration of the combination or pharmaceutical composition according to the present invention.

The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent. Examples of pharmaceutically acceptable carriers, excipients and diluents that can be used in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, and alginate. , gelatin, calcium phosphate, calcium silicate, calcium carbonate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally depending on the desired method, but is preferably administered parenterally.

According to one embodiment of the present invention, the pharmaceutical composition according to the present invention can be directly administered intravenously, intraarterially, into cancer tissue or subcutaneously, or administered as an injection. The injection according to the present invention may be in a form dispersed in a sterile medium so that it can be used as it is when administered to a patient, or may be administered after dispersing in an appropriate concentration by adding distilled water for injection. In addition, when prepared as an injection, it may be mixed with buffers, preservatives, analgesics, solubilizers, tonicity agents, stabilizers, etc., and may be prepared in unit dosage ampoules or multiple dosage forms.

The dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the patient, the severity of the disease, the drug type, the administration route and time, but can be appropriately selected by those skilled in the art. Meanwhile, the pharmaceutical composition according to the present invention may be used alone or in combination with auxiliary treatment methods such as surgical treatment.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, since various changes can be made to the embodiments, the scope of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents or substitutes to the embodiments are included within the scope of rights.

Terms used in the examples are used only for descriptive purposes and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't

In addition, in the description with reference to the accompanying drawings, the same reference numerals are given to the same components regardless of reference numerals, and overlapping descriptions thereof will be omitted. In describing the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description will be omitted.

Example 1: Production of immune checkpoint inhibitor expression trans-splicing ribozymes

A previously prepared trans-splicing ribozyme modified recombinant vector was designed. Specifically, it contains the CMV promoter, targets the site including uridine +21 of hTERT mRNA, has an anti-sense of 326 nucleotides in length, and has miR-122T, which is a miR122 target site, In the trans-splicing ribozyme expression construct containing HSV-tk as a therapeutic gene, P2A, a nucleic acid sequence encoding a peptide cleavage site, and an immune checkpoint inhibitor expression sequence were additionally introduced at the end of HSV-tk.

As immune checkpoint inhibitors, scFvPD1 (single chain variable fragment PD1) and scFvPDL1 prepared by applying PD1 antibody and PDL1 antibody sequences known in the art were used. In the present invention, scFvPD1 used two antibodies with differences in amino acid sequence, and the genes encoding each of the two antibodies are shown in SEQ ID NOs: 1 and 2 (hereinafter referred to as scFvPD1 (I) and scFvPD1 (N), respectively). box). In addition, the gene sequence encoding scFvPDL1 is shown in SEQ ID NO: 3 (hereinafter referred to as scFvPDL1 (A)).

A FLAG Tag sequence is introduced at the end of the immune checkpoint inhibitor, and 3 copies of miR-122T are inserted at the 3' end of the construct to express the ribozyme by miR-122. this could be regulated.

On the other hand, a recombinant vector (mCRT-122T/ immune checkpoint inhibitors) were also prepared.

The designed trans-splicing ribozyme expression vector was named mCRT-122T/ICI, and its vector structure is shown in FIG. 2 .

Meanwhile, in the experiment below, the activities of mCRT-122T and mCRT-122T/ICI prepared in previous studies are compared and confirmed, and the structure of mCRT-122T is as follows.

5' - CMV promoter - mTERT ribozyme - HSVtk - miR-122T (3X) - 3'

Meanwhile, the adenovirus expressing the CRT-122T/ICI vector is named RZ-001+, and the adenovirus expressing the mCRT-122T/ICI vector is named mRZ-001+, respectively, and the adenovirus expressing the CRT-122T vector is named mRZ-001+. It was named RZ-001.

Example 2. Confirmation of equivalence to RZ-001 through RZ-001+ cell death induction experiment

2-1. Confirmation of equivalence in human liver cancer cell lines

In order to compare the equivalence of RZ-001 prepared in a previous study, cell death induction experiments were performed in human liver cancer cell lines.

Specifically, SNU398 and Hep3b were dispensed at 1x10 ⁴ cells/well/100ul in a 96-well plate, and the used medium was removed the next day, and then RZ-001 and RZ-001+ were incubated in FBS 2% medium at different multiplicities of infection (Multiplicity of Infection, MOI) (0.01-10 MOI). 24 hours after virus treatment, 100uM GCV was treated three times at 2-day intervals. Finally, on the next day after treatment with GCV, each well was treated with 10ul EZ-Cytox regent (DOGEN, EZ-1000), and after incubation at 37°C, absorbance was measured at Abs 450nm.

As a result, it was confirmed that apoptosis of liver cancer cells increased as the concentration of RZ-001 or RZ-001+ treatment increased, indicating that there was no problem with the cell death induction activity by hTERT targeting ribozyme following the addition of the immune checkpoint inhibitor gene. could In addition, at the in vitro cell line level, in the absence of the immune system, it was found that RZ-001+ had equal activity by RZ-001 and hTERT targeting ribozyme (FIG. 3).

2-2. Confirmation of equivalence in human brain tumor cell lines

The human brain tumor cell line U87MG was dispensed in a 96-well plate at 1x10 ⁴ cells/well/100ul, and the next day after removing the used medium, RZ-001 and RZ-001+ were treated at various concentrations in FBS 2% medium (0.01 to 10 MOI). ). 24 hours after virus treatment, 100uM GCV was treated three times at 2-day intervals. Finally, on the next day after treatment with GCV, each well was treated with 10ul EZ-Cytox regent (DOGEN, EZ-1000), and after incubation at 37°C, absorbance was measured at Abs 450nm.

Mock was used as an adenovirus control that does not express a transgene because it does not contain a transgene, and was used as a negative control to confirm that there is no non-specific induction of cell death by adenovirus infection. In addition, CT was CMV-HSVtk, which was used as a positive control to confirm cell death induction caused by GCV phosphorylation by HSVtk.

From the absorbance measurement results, it was confirmed that cell death was induced in RZ-001 and all RZ-001+ treated groups. From the above, it can be seen that all RZ-001+ exhibited cell death inducing activity in brain tumor cells, and in particular, all RZ-001+ exhibited the same effect as RZ-001 at 0.5 MOI or higher (FIG. 4).

2-3. Confirmation of equivalence in lung cancer cell lines and melanoma cell lines

The apoptotic effect of RZ-001+ was investigated in the human lung cancer cell line A549 cell line and the human melanoma cell lines A375P and A375SM. Each cell line was dispensed in a 96-well plate at 1x10 ⁴ cells/well/100ul, and the next day, the used medium was exchanged with FBS 2% medium, and RZ-001+ was treated at different concentrations. From the day after virus treatment, GCV at 100uM concentration was treated three times at 2-day intervals, and 24 hours after the last GCV treatment, each well was treated with 10ul EZ-Cytox reagent (DOGEN, EZ-1000), cultured at 37°C, and Abs at 450nm. Absorbance was measured.

As a result, RZ-001+ induces apoptosis of melanoma cells and lung cancer cells, and at an MOI of 1 or higher, RZ-001+ excluding RZ-001+_PD1(I) has a similar level of apoptosis-inducing activity to RZ-001. It was found that it represents (FIG. 5).

Example 3: Production of immune checkpoint inhibitor expression stabilization cell line

3-1. Production of stable cell lines

Before confirming the effect of the vector constructed in Example 1, a stable cell line expressing an immune checkpoint inhibitor was prepared as follows.

After dispensing 2x10 ⁵ cells of 293A cells in a 35 mm culture dish, they were cultured in a 37°C 5% CO ₂ incubator for 24 hours. Then, 1 μg of each immune checkpoint inhibitor expression vector and 100 μl of Opti-MEM were mixed in a 1.5 ml tube, and 5 μl of Lipofectamine 2000 and 100 μl of serum-free medium were put in another 1.5 ml tube, mixed, and left at room temperature for 5 minutes. did Then, the contents of the two tubes were mixed and stored at room temperature for 20 minutes to form a liposome-type complex. After 20 minutes, the tube was centrifuged for 10 seconds, sprinkled on each cell for transfection, and 4 hours later, the medium was replaced with a new medium. After culturing for 24 hours in a 37°C 5% CO ₂ incubator, the cells were washed with 1X PBS, treated with trypsin to detach the cells, and then transferred to a 100 mm culture dish and cultured. Every 2 to 3 days, the medium was replaced with a medium containing the antibiotic geneticin (G418) at a concentration of 5 μg/ml. After selecting cell clones and growing each, expression of immune checkpoint inhibitors was confirmed.

Proteins were isolated from the cell supernatant, Western blotting was performed, RNA was extracted from the cells, and the expression level of the immune checkpoint inhibitor was confirmed by RT-PCR.

3-2. Immune checkpoint inhibitor expression and affinity confirmation

After culturing the stabilized cell line prepared in 3-1 above, Western blotting was performed by isolating proteins from the cell supernatant, RNA was extracted from the cells, and expression of the immune checkpoint inhibitor was confirmed by RT-PCR.

As a result, it was confirmed that all immune checkpoint inhibitors introduced into 293A cells were expressed well (FIG. 6A), and as a result of checking the mRNA level, it was confirmed that they were well expressed (FIG. 6B).

3-3. Confirmation of affinity of expressed scFvPD1

Cells expressing human PD1 (hPD1) or mouse PD1 (mPD1) were cultured to prepare a cell lysate, which was attached to a 96-well plate. Then, the antibody affinity was confirmed by ELISA method by reacting with scFvPD1 (I) recovered from the stabilized cell line culture medium of 2-1. As a control, 293A cell culture medium was used. As a result, it was confirmed that the absorbance increased as the concentration of the cell lysate increased, indicating that scFvPD1(I) functions well as an antibody (FIG. 7).

3-4. Construction of mouse PDL1 expression stabilization cell line

In order to confirm the combined effect of co-expression of cancer therapeutic genes and immune checkpoint inhibitors, a stable cell line expressing mouse PDL1 (mPDL1) was prepared in the same manner as in Example 2-1. Briefly, mPDL1/pCMV6 was introduced into Hepa1-6 cell line, a mouse-derived liver cancer cell line, and cell clones were selected for 3 weeks by treatment with geneticin. As a result of confirming the expression level of mPDL1 by culturing the selected cell clone, it was confirmed that mPDL1 was expressed at a high level compared to other mouse liver cancer cell lines (Hepa1-6, Hepa1c1c7) (FIG. 8).

Hereinafter, the Hepa1-6 stabilized cell line into which mPDL1 has been introduced is referred to as Hepa1-6/mPDL1.

Example 4. Confirmation of effect of RZ-001+ expression vector in Hepa1-6 stabilization cell line

4-1. infection test

Hepa1-6 or Hepa1-6/mPDL1 cell lines were treated with mRZ-001+ prepared in Example 1 at a concentration of 10 MOI.

The vectors used are:

mCRT-122T (CMV promoter + mTERT ribozyme + HSVtk + miR-122T (3X)),

mCRT-122T/scFvPD1(I) (CMV promoter + mTERT ribozyme + HSVtk + scFvPD1(I) + miR-122T (3X)),

After 24 hours of virus treatment, genomic DNA was extracted from the cells, and the degree of virus infection was confirmed by performing RT-PCR targeting E4.

As a result, it was confirmed that the average Ct values of E4 appeared at a similar level in each experimental group (FIG. 9).

4-2. Check cell viability

Hepa1-6, Hepa1-6/mPDL1, and Hepa1c1c7 cells were respectively seeded in 96-well, 1×10 ⁴ , and then treated with adenovirus containing the mRZ-001+ expression vector prepared in Example 1 at different MOIs. 24 hours after adenovirus treatment, GCV (ganciclovir) was diluted in the cell culture medium to a final concentration of 100 μM, and then added to each well. GCV was treated a total of three times at 2-day intervals, and 24 hours after the last GCV treatment, MTS assay reagent was added and absorbance was measured at a wavelength of 450 nm to confirm cell viability.

As a result of confirmation, it was found that cell death increased in the experimental group treated with the ribozyme expression vector compared to the control group (EGFP). It was confirmed that the level of apoptosis was remarkably high in scFvPD1 (I)) (FIG. 10).

4-3. Comparison of expression of PD1 or PDL1 antibodies

Hepa1-6, Hepa1-6/mPDL1, and Hepa1c1c7 cells were treated with adenovirus containing the immune checkpoint inhibitor expression vector at 50 MOI, and after 24 hours, proteins were isolated from the cells to determine the expression level of the checkpoint inhibitor. The expected size of the protein is about 28 kDa including the signal peptide.

As a result of confirmation, immune checkpoint inhibitors were expressed in all three cell lines (FIG. 11).

4-4. apoptotic efficacy

Hepa1-6 and Hepa1-6/mPDL1 cells were respectively seeded in 2x10 ⁵ cells in a 6-well plate, and adenovirus containing the mRZ-001+ expression vector was treated at an MOI of 5. 24 hours after virus treatment, GCV was diluted in cell culture medium to a final concentration of 100 μM, and then added to each well. After further culturing for 24 hours, each well was treated with propidium iodide (PI), and the degree of apoptosis was analyzed by flow cytometry.

As a result of the analysis, both Hepa1-6 and Hepa1-6/mPDL1 cells showed little change in the level of early apoptosis by GCV treatment in the mCRT-122T vector-treated group, but significantly increased early apoptosis after GCV treatment in the immune checkpoint inhibitor-expressing vector-treated group. It was found to increase (Figs. 12 and 13).

Example 5. Introduction of RZ-001+ expression vector in human liver cancer cell line

5-1. Confirmation of immune checkpoint inhibitor secretion following introduction of RZ-001+

Hep3b and SNU398 cells, which are human liver cancer cell lines, were seeded at 1x10 ⁶ cells in a 60π culture dish, and the next day, the medium used was exchanged with 2% FBS medium, and then treated with 30 MOI of adenovirus containing the RZ-001+ expression vector. After 48 hours of virus treatment, the medium was placed in a 15ml tube, centrifuged at 1,500 rpm for 5 minutes, and centrifuged to remove cell debris. Each sample was transferred to a 10k centricon and concentrated at 3,000 rpm for 15-30 minutes so that the total volume of the sample was 500ul. Concentrated samples were prepared using 5x sample buffer and denaturation was induced at 100 ° C for 5 min. The prepared samples were loaded in 40ul each on 12% SDS-PAGE. Each cell was harvested by washing with PBS, treated with RIPA buffer at 4 ° C. for 20 minutes, and then centrifuged at 13,000 rpm for 10 minutes to obtain a supernatant, which was transferred to a new tube and quantified by BCA quantification. Samples were prepared so that the quantified protein was 30 μg/well and loaded on SDS-PAGE in the same manner as above. After PAGE separation, it was transferred to PVDF and blocked in 5% skim milk in PBS-T for 30 minutes, and anti-FLAG was diluted 1:1,000 and reacted O/N at 4°C. The next day, after washing with PBS-T, the secondary antibody anti-mouse/HRP was diluted 1:2,000, reacted for 1 hour, and then the expression level was detected with ECL solution.

As a result, it was confirmed that scFv was secreted from the liver cancer cell line treated with RZ-001+. In particular, the highest level of scFv secretion was confirmed in RZ-001+_PDL1 (At). From the above, it can be seen that the cells into which the RZ-001+ expression vector was introduced actively secrete scFv, and that RZ-001+ can be applied to cancer cells (FIG. 14).

5-2. Confirmation of trans-splicing reaction following introduction of RZ-001+

In Example 5-1, it was confirmed whether RZ-001+ effectively transsplices the target TERT mRNA in adenovirus-treated liver cancer cell lines. Cells treated with 30 MOI virus in Example 5-1 were quantified by preparing RNA using TRIzol after 48 hours of virus treatment. Using RT kit (Genet bio #SR3000), 3 ug RNA and 1uL RT primer were mixed and reacted (50℃ 60min, 70℃ 10min). PCR was performed using the primer premix (Bionia, #k-2611) shown in Table 1 below, and 40 cycles were performed under the conditions of 95°C 30 sec, 59°C 30 sec, and 72°C 30 sec. Subsequently, the amplification product was subjected to electrophoresis on a 2% agarose gel to confirm a band of the target size. In the target size band, cloning was performed using a TA vector through gel elution and sequencing was performed.

RT primer (HSV-tk) 5' - agttagcctcccccatctc - 3' hTERT 5′-GGAATTCGCAGCGCTGCGTCCTGCT-3′ HSVtk 5′-GTGAGGACCGTCTATATAAACCCGCAGTAG-3′

As a result, in the liver cancer cell line sample treated with RZ-001+, a band of product size that can be produced when trans-splicing has occurred was confirmed, and the product nucleotide sequence of the band was trans-spliced at the target site of the target gene. It was confirmed that plicing occurred. From the above, it can be seen that RZ-001+ can induce a trans-splicing reaction by accurately reacting to the target gene and target site in the introduced liver cancer cells (FIG. 15).

5-3. Confirmation of bioactivity of secreted immune checkpoint inhibitors

Subsequently, a PD1/PDL1 blockade bioassay (Promega, #J1250) was performed to confirm whether the immune checkpoint inhibitor secreted by the RZ-001+-introduced liver cancer cell line could effectively bind to the immune checkpoint protein and block the signal. The PD1/PDL1 blockade bioassay is a PD1/PDL1 interaction when PD-1 on the surface of PD-1 effector cells binds to PDL1 present on APC cells or cancer cells in the absence of PD-1 or PDL-1 immunotherapeutic agents. Although the luciferase signal is not detected by suppressing this TCR-mediated luminescence, in the presence of an immuno-anticancer agent, the combination of PDL1 and PD1 is hindered by the combination of the immuno-anticancer agent, which activates the TCR and activates the NFAT signal to increase the expression of the luciferase gene. It is to use a system in which the luciferase signal is increased by inducing. Experiments were performed according to the manufacturer's recommended protocol. Specifically, PDL1 aAPC/CHO-K1 cells were released and prepared on a white plate. The next day, the medium of the cells was removed, 40ul of the concentrated sample of Example 4-1 was loaded on the plate, and PD-1 effector cells were released on the plate loaded with the concentrated sample, and the reaction was induced at 37° C. for 6 hours. Subsequently, bio-Glo reagent was added and incubated for 5-10 minutes at room temperature, and fluorescence was measured using a luminometer.

As a result of measuring the activity of the immuno-anticancer agent secreted using the culture medium of the liver cancer cell line sample treated with RZ-001+, an increase in bioactivity was confirmed in all cells treated with RZ-001+, and from the above, RZ-001+ It can be seen that immune checkpoint inhibitors are produced and secreted within cells by viral infection. In particular, in response to the results of Example 5-1, the bioactivity increased the most in the case of RZ-001 + _At, so the most immuno-anticancer agent was produced and secreted, and it can be seen that the secreted immuno-anticancer agent has excellent activity. (FIG. 16).

Example 6. Introduction of RZ-001+ Expression Vector in Human Brain Tumor Cell Lines

6-1. Confirmation of PD-L1 expression in RZ-001+ introduced cell lines

The human liver cancer cell line SNU398 and the human brain tumor cell line U87MG were dispensed in a 12 well plate at 5x10 ⁴ cells/well/1mL and cultured for 2 days. Cells are lysed using RIPA buffer to extract total protein. The extracted total protein was quantified by the BCA quantification method, and all samples were prepared to have the same amount of protein. The expression level of the target protein was measured.

As a result, by confirming the expression of PD-L1 protein in both liver cancer cell line SNU398 and brain tumor cell line U87MG, the effectiveness of RZ-001+_PDL1 expressing immune checkpoint inhibitors in liver cancer and brain cancer can be suggested. (FIG. 17).

6-2. Confirmation of immune checkpoint inhibitor secretion following introduction of RZ-001+

In the same manner as in Example 5-1, scFv secretion was confirmed from the adenovirus-infected brain tumor cell line containing the RZ-001+ expression vector. The experimental method was performed in the same manner as in Example 5-1 with only the virus treatment concentrations of 10 MOI and 20 MOI.

As a result, in U87MG, a Glioblastoma cell line, scFv secretion was confirmed in cells following the introduction of the RZ-001+ expression vector, and in particular, the highest amount of scFv secretion was confirmed in cells introduced with the RZ-001+_At expression vector ( Figure 18).

6-3. Confirmation of bioactivity of secreted immune checkpoint inhibitors

Next, the supernatant of the cells treated with the virus containing the RZ-001+_PDL1(At) expression vector was isolated and a PD1/PDL1 blockade bioassay (Promega, #J1250) was performed in the same manner as in Example 5-3. As a control, commercially available atezolizumab was used.

As a result, it was confirmed that the luciferase activity increased as the concentrated sample obtained from the cells treated with the virus at a high MOI was treated (FIG. 19).

Example 7. in vivo Confirmation of anticancer effect of RZ-001+ in

7-1. Confirmation of the effect of RZ-001+ in PBMC-humanized liver cancer model

Human PBMC 5 x 10 ⁶ cells/head was injected into a mouse xenograft subcutaneous model (mouse xenograft subcutaneous model: , 6-week-old male NOG) mouse to construct a PBMC humanized mouse (PBMC-humice), and the mouse was treated for 7 to 10 days. After checking the body weight and condition, subcutaneous injection of 5 x 10 ⁶ cells /50 μl of SNU-398 cells was performed, followed by breeding for 2 weeks to construct a liver cancer tumor model. Subsequently, tumor growth and weight were measured, group separation was performed, and drug administration was performed for each group. Additionally, AST/ALT levels were measured to confirm liver toxicity of the drug.

Each group was divided into a control group, Atezolizumab (At) administration group, RZ-001 administration group, RZ-001+_At administration group, and RZ-001 and Atezolizumab combination administration group. Drug administration was 1 x 10 ⁹ VP/head, 48 hour intervals. Two doses were administered directly into the tumor (intratumoral injection), and co-administration of Atezolizumab was administered intravenously (intravenous injection) three times at 2-day intervals from 2 days after virus administration at a dose of 5 mg/kg.

As a result, it was confirmed that the tumor size or weight decreased when RZ-001+_At was administered compared to the single administration of RZ-001 and Atezolizumab. . On the other hand, as a result of liver toxicity measurement, the RZ-001+_At administration group showed a decrease in AST/ALT level compared to the Atzolizumab alone or combination administration group, and showed a similar level compared to Ad-Mock, indicating that liver toxicity was significantly suppressed ( Figure 20).

7-2. Confirmation of the effect of RZ-001+ in a syngeneic orthotopic brain tumor model

After anesthetizing a 5-week-old male C57BL/6 mouse with immunoreactivity, a 1cm midline incision was made on the scalp using a stereotactic tool, and mouse-derived brain cancer cell lines were placed at depths of 1mm anterior, 2.3mm lateral, and 3mm based on the bregma. (GL261) An orthotopic brain tumor model was constructed by transplanting 1 x 10 ⁵ cells/head. After 7 days, tumor growth was measured, group separation was performed, and drug administration was performed for each group.

Administration of the drug was carried out according to the following dosage and usage.

For mRZ-001 and mRZ-001+, 3ⅹ10 ⁹ VP/5uL is directly administered intratumorally once.

GCV is administered at 50 mg/kg in a 100 ul liquid volume, and once daily from the next day after the end of virus administration, a total of 10 administrations.

Experimental animal groups were divided into mRZ-001 administration group and mRZ-001+ administration group, and were divided into PBS administration group as a control group.

In order to measure the size of the tumor, MRI was performed every 3 days, including the day after drug administration, and 5 slices were selected based on the tumor implantation location, and the region of interest of the tumor over time was selected using the ImageJ system. ROI) analysis was performed (FIG. 21). Specifically, a Biospec 47/40 USR (Bruker, Ettlingen, Germany) horizontal bore magnet was used for MRI imaging. During imaging, animals were anesthetized, and during image acquisition, respiratory rate, heart rate, and body temperature were observed using an animal monitoring-gating system, and a warm bed was used to maintain body temperature. Images to confirm tumor growth and growth inhibition were taken using the RARE sequence of 15 consecutive axial slices, and the related conditions are as follows.

repetition time (TR) = 2200 ms

echo time (TE) = 40 ms

slice thickness = 0.75 mm

matrix = 192 x 192

flip angle (FA) = 90

field of view (FOV) = 18 x 18 mm ²

average = 4

echo train length (ETL) = 8

The size of the tumor was set as Day 1 for the next day based on mRZ-001, and was the result of a total of 10 treatments until Day 10 along with GCV treatment, and the degree of tumor size reduction was confirmed compared to the PBS-administered group. As a result, the size of the tumor decreased according to the virus infection, and the administration of mRZ-001+ reduced the size of the tumor more significantly than the administration of mRZ-001 (FIG. 22).

On the other hand, the mRZ-001+ administration group showed lower AST and ALT levels than the control group, indicating that the in vivo toxicity of the treated samples was remarkably low (FIG. 23).

7-3. Confirmation of the effect of RZ-001+ in a xenograft orthotopic brain tumor model

Following Example 7-3, the anticancer effect of RZ-001+ was confirmed in an animal model transplanted with human-derived brain tumor cells. Accordingly, a 5-week-old male BALB/C nude mouse was transplanted into a mouse in the same manner as in Example 7-3 using U87MG-Luci, a human-derived brain tumor cell stably expressing Luciferase, to prepare a Xenograft Orthotopic mouse brain tumor model. After 7 days, tumor growth was measured through IVIS imaging, and group separation was performed, and drug administration was performed for each group. (FIG. 24).

Administration of the drug was carried out according to the following dosage and usage.

For RZ-001 and RZ-001+_AT, 1x10 ¹⁰ VP/Head is directly administered into the tumor once.

GCV is administered at 50 mg/kg once a day, a total of 10 times, from the next day after the end of virus administration.

After virus administration, IVIS imaging was performed at 3-day intervals to track the growth of luciferase-expressing tumor cells. After 19 days of virus administration, the mice were sacrificed and necropsied on the next day (Day 20) after IVIS imaging. (FIG. 25). As the mice in each drug-administered group maintained a steady body weight during the experimental period, it could be seen that there was no or very low toxicity of RZ-001+ (FIG. 26). On the other hand, both RZ-001 and RZ-001+ were excellent in anticancer effect, and in particular, it was confirmed that the anticancer activity of RZ-001+_At was superior to that of RZ-001. It can be seen that there is (FIG. 27).

As described above, although the embodiments have been described with limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, the described techniques may be performed in an order different from the method described, and/or components of the described system, structure, device, circuit, etc. may be combined or combined in a different form than the method described, or other components may be used. Or even if it is replaced or substituted by equivalents, appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents of the claims are within the scope of the following claims.

<110> RZNOMICS INC. <120> TUMOR-TARGETING TRANS-SPLICING RIBOZYME EXPRESSING IMMUNE CHECKPOINT INHIBITOR AND USE THEREOF <130> APC-2021-0824 <150> KR 10-2021-0010416 <151> 2021-01-25 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 744 <212> DNA <213> artificial sequence <220> <223> gene encoding scFv PD1 (I) <400> 1 caggtacaac ttgttcagtc cggagctgaa gtgaaaaagc cgggcgcgtc tgttaaggta 60 agctgcaagg cttcaggcta tactttcacg gcgcagtaca tgcattgggt cagacaagct 120 ccaggccaag gtcttgaatg gatggggatc atcaacccga gtgggggtga aacaggctat 180 gctcaaaagt tccagggtcg agtcaccatg actcgggata cctccacgtc taccgtttac 240 atggagctga gcagtttgag gagcgaagat actgccgtat actattgtgc caaggaaggt 300 gttgcggacg gttatgggct cgttgatgta tgggggcaag gcacgatggt taccgtctca 360 tctggtggag gaggttctgg gggtgggaggc tcaggaggag gggggtcagg tggcggagga 420 tccgaaatcg tgttgaccca gagtcctgca acactgagtc tgtccccagg ggagagagcc 480 accttgtcct gtagagcgag ccagtctgta agctcttatc tggcttggta tcagcaaaaa 540 cctgggcagg cgccgcgcct cctcatctat gacgcgagca agagggcaac agggatacca 600 gcgagattct ctgggtcagg atcagggaca gacttcacac tcacgatcag ctctttggaa 660 ccagaagatt ttgcagtcta ctattgtgat caacgaaaca actggcctct cacgttcgga 720 ggagggacta aagtagaaat taaa 744 <210> 2 <211> 720 <212> DNA <213> artificial sequence <220> <223> gene encoding scFv PD1 (N) <400> 2 caggtccaac ttgtcgagag cggcggtgga gtggtacagc ctgggcgatc cttgcggctt 60 gattgcaagg cgagcggaat aaccttcagc aacagtggga tgcactgggt aagacaagcg 120 ccaggcaagg ggctcgagtg ggtcgctgtc atctggtatg acggaagtaa acgatattac 180 gccgatagtg taaagggaag gtttacgatc agtagggata actctaaaaa tacgctcttt 240 cttcaaatga acagtcttcg agcagaagat acagcggtgt attattgtgc tactaatgac 300 gattattggg gccagggtac tctcgttacg gtaagctctg gtggaggagg aagtggtggc 360 ggaggtagtg gaggtggcgg ctccgggggt ggaggatccg agatagtact cacacaaagt 420 cctgctacgc tttcactctc ccctggagag agagctactc tctcatgccg agcctcccag 480 agtgtgagtt catatttggc gtggtaccag cagaagcccg gccaagcccc ccgattgctc 540 atatatgacg ccagtaatcg cgcgactggt atacctgccc ggtttagcgg aagtggatcc 600 gggacggact ttaccctgac aatttcttca ctggagcctg aagacttcgc cgtatattat 660 tgtcaacaat cctccaattg gccaagaact tttggccaag gaacgaaagt tgagataaaa 720 720 <210> 3 <211> 735 <212> DNA <213> artificial sequence <220> <223> gene encoding scFv PDL1 (A) <400> 3 gaagttcaac tggtggagtc tggagggggt ttggtgcagc caggcggggag tttgaggctc 60 agctgcgccg cctctggatt caccttctcc gatagctgga tccattgggt caggcaagcg 120 cctggtaaag ggttggagtg ggtcgcatgg atatctcctt atggagggtc tacatattat 180 gccgactctg tcaagggaag attcacgata tccgcagaca caagtaagaa tacagcatac 240 cttcaaatga actccctgcg cgctgaagac acggcggttt attattgcgc taggcggcac 300 tggccagggg gttttgacta ttggggtcaa ggtaccttgg tcacggtttc atccggcggc 360 ggtggtagcg gtggtggagg tagcgggggt ggtggaagtg ggggtggagg ctcagacatc 420 caaatgacac aaagcccatc ctccctgagc gctagtgtgg gggaccgggt cacgataacc 480 tgccgggcta gccaagatgt gagcacagca gtcgcctggt atcagcagaa gcccgggaag 540 gccccaaaac tcctcatata ctctgcttct tttctctatt ccggtgtgcc ctctcgattc 600 tcaggcagtg ggtcaggaac cgacttcacg ctgaccatct caagtttgca gccggaagac 660 ttcgcaacgt attattgcca gcaatacctc taccatcctg ccactttcgg tcaggggacg 720 aaagtagaga ttaaa 735 <210> 4 <211> 393 <212> DNA <213> artificial sequence <220> <223> sequence encoding hTERT targeting ribozyme <400> 4 ggcaggaaaa gttatcaggc atgcacctgg tagctagtct ttaaaccaat agattgcatc 60 ggtttaaaag gcaagaccgt caaattgcgg gaaaggggtc aacagccgtt cagtaccaag 120 tctcagggga aactttgaga tggccttgca aagggtatgg taataagctg acggacatgg 180 tcctaaccac gcagccaagt cctaagtcaa cagatcttct gttgatatgg atgcagttca 240 cagactaaat gtcggtcggg gaagatgtat tcttctcata agatatagtc ggacctctcc 300 ttaatgggag ctagcggatg aagtgatgca acactggagc cgctgggaac taatttgtat 360 gcgaaagtat attgattagt tttggagtac tcg 393 <210> 5 <211> 1128 <212> DNA <213> artificial sequence <220> <223> sequence encoding HSVkt <400> 5 atggcttcgt acccctgcca tcaacacgcg tctgcgttcg accaggctgc gcgttctcgc 60 ggccatagca accgacgtac ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc 120 cgcctggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc tcacgggatg 180 gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga tatcgtctac 240 gtacccgagc cgatgactta ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc 300 tacaccacac aacaccgcct cgaccagggt gagatatcgg ccggggacgc ggcggtggta 360 atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc cgttctggct 420 cctcatatcg ggggggaggc tgggagctca catgccccgc ccccggccct caccctcatc 480 ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcgata ccttatgggc 540 agcatgaccc cccaggccgt gctggcgttc gtggccctca tcccgccgac cttgcccggc 600 acaaacatcg tgttgggggc ccttccggag gacagacaca tcgaccgcct ggccaaacgc 660 cagcgccccg gcgagcggct tgacctggct atgctggccg cgattcgccg cgtttacggg 720 ctgcttgcca atacggtgcg gtatctgcag ggcggcgggt cgtggcggga ggattgggga 780 cagctttcgg ggacggccgt gccgccccag ggtgccgagc cccagagcaa cgcgggccca 840 cgaccccata tcggggacac gttatttacc ctgtttcggg cccccgagtt gctggccccc 900 aacggcgacc tgtacaacgt gtttgcctgg gccttggacg tcttggccaa acgcctccgt 960 cccatgcacg tctttatcct ggattacgac caatcgcccg ccggctgccg ggacgccctg 1020 ctgcaactta cctccgggat ggtccagacc cacgtcacca cccccggctc cataccgacg 1080 atctgcgacc tggcgcgcac gtttgcccgg gagatggggg aggctaac 1128 <210> 6 <211> 22 <212> DNA <213> artificial sequence <220> <223> sequence encoding miR-122T <400> 6 caaacaccat tgtcacactc ca 22 <210> 7 <211> 57 <212> DNA <213> artificial sequence <220> <223> sequence encoding p2a <400> 7 gccacaaact tctctctgct aaagcaagca ggtgatgttg aagaaaaccc cgggcct 57 <210> 8 <211> 19 <212> DNA <213> artificial sequence <220> <223> RT primer (HSV-tk) of primer premix <400> 8 agttagcctc ccccatctc 19 <210> 9 <211> 25 <212> DNA <213> artificial sequence <220> <223> hTERT primer of primer premix <400> 9 ggaattcgca gcgctgcgtc ctgct 25 <210> 10 <211> 30 <212> DNA <213> artificial sequence <220> <223> HSVkt primer of primer premix <400> 10 gtgaggaccg tctatataaa cccgcagtag 30

Claims (19)

As a trans-splicing ribozyme targeting TERT (Telomerase Reverse Transcriptase) mRNA,
The ribozyme includes a target gene operably linked to the 3' exon,
The target gene is a HSVtk (Herpes Simplex Virus thymidine kinase) and immune checkpoint inhibitor gene, trans-splicing ribozyme. According to claim 1,
The trans-splicing ribozyme has a structure of 5' - trans-splicing ribozyme - cancer treatment gene - immune checkpoint inhibitor gene - 3',
The gene for cancer treatment is a trans-splicing ribozyme that is distinct from the immune checkpoint inhibitor gene. delete delete delete According to claim 1,
The immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA or A2aR, trans splicing Ribozyme. According to claim 1,
The trans-splicing ribozyme is a trans-splicing ribozyme further comprising at least one copy of a sequence complementary to part or all of micro RNA-122a (miR-122a) at the 3' end. . According to claim 1,
The two or more genes for cancer treatment are linked to a gene encoding a self-cleaving peptide, a trans-splicing ribozyme. According to claim 8,
Wherein the self-cleaving peptide is P2A, a trans-splicing ribozyme. A non-viral gene delivery system comprising the trans-splicing ribozyme of claim 1. An expression vector capable of expressing the trans-splicing ribozyme of claim 1. According to claim 11,
The expression vector further comprises a promoter operably linked to the ribozyme gene. A gene delivery system expressing the expression vector of claim 11 . A cell transformed with the expression vector of claim 11. The trans-splicing ribozyme of any one of claims 1, 2, and 6-9; The non-viral gene delivery system of claim 10; The expression vector of claim 11 or claim 12; And the gene delivery system of claim 13; A pharmaceutical composition for the treatment of cancer comprising any one selected from the group consisting of as an active ingredient. According to claim 15,
The pharmaceutical composition is a pharmaceutical composition that is administered orally or in the form of an injection intravenously, intraarterially, cancer tissue, or subcutaneous route. According to claim 15,
The cancer is at least one selected from the group consisting of liver cancer, glioblastoma, bile duct cancer, lung cancer, pancreatic cancer, melanoma, bone cancer, breast cancer, colon cancer, stomach cancer, prostate cancer, leukemia, uterine cancer, ovarian cancer, lymphoma, and brain cancer Cancer, pharmaceutical composition. According to claim 15,
Characterized in that the cancer is immune checkpoint inhibitor-resistant cancer, the pharmaceutical composition. A cancer treatment method comprising administering the composition of claim 15 to a non-human subject.

Images