Modified virus targeting abnormal ETS family transcription factor-related tumors

Abstract

Provided is a modified virus targeting abnormal ETS family transcription factor-related tumors. The virus comprises a cis-acting element capable of binding to an ETS family transcription factor and a target gene operably linked to the cis-acting element. The virus can be used for treating tumors.

Description

Modified viruses targeting tumors associated with abnormal ETS family transcription factors TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a modified virus as an oncolytic virus drug for treating tumors. BACKGROUND

[0002] Oncolytic virus therapy is an emerging cancer treatment strategy that uses viruses with oncolytic activity to selectively attack and destroy cancer cells. This treatment is based on the unique biological characteristics of viruses that selectively infect and replicate in cancer cells during their life cycle, and the mechanism of enhancing anti-tumor effects by stimulating the immune system. Once the oncolytic virus infects the cancer cells, it uses the biological mechanisms of the host cells to replicate. This usually leads to the rupture and death of cancer cells, releasing a large number of newly born virus particles into the surrounding environment, which in turn infect nearby cancer cells. In addition, the ruptured cancer cells may also attract the attention of the immune system, activating the body's immune response, thereby producing further anti-tumor effects on cancer cells.

[0003] However, current oncolytic virus therapy still has many technical problems, such as improving the tumor-specific infection and replication of viruses, enhancing the replication and oncolytic ability of viruses, etc. Therefore, it is still necessary to develop new oncolytic virus therapy to better adapt to the complex tumor environment and improve the therapeutic effect. SUMMARY

[0004] The present application provides a cis-acting element targeting ETS family transcription factors, which has high specificity and only enables the regulation of expression of a target gene in cells with abnormal ETS family transcription factors, while the target gene regulated by it is not expressed or is lowly expressed in normal cells, has high safety and small side effects, thereby realizing tumor specificity.

[0005] In addition, the present application also provides a modified virus containing the cis-acting element, which uses the killing ability of the virus on tumors, places the virus proliferation essential gene behind the cis-acting element described in the present application, activates the expression of downstream proteins when abnormal ETS family transcription factors are recognized, promotes the explosive proliferation of viruses, kills cancer cells, and achieves the purpose of oncolysis. The modified virus provided by the present application not only has a high degree of specificity in tumor cells, showing excellent killing effect on tumor cells, but also can target and kill a variety of tumor cells. It has high effectiveness and safety, and has wide application potential.

[0006] In one aspect, the present application provides a modified virus comprising a first regulatory system comprising a first cis-acting element and a first gene of interest operably linked thereto, wherein the first cis-acting element is capable of binding to an ETS family transcription factor, and the first gene of interest comprises a viral propagation-essential gene.

[0007] In certain embodiments, wherein expression and / or activity of at least one endogenous viral propagation-essential gene of the virus is down-regulated in the modified virus.

[0008] In certain embodiments, wherein the at least one endogenous viral propagation-essential gene is down-regulated by gene editing and / or gene recombination.

[0009] In certain embodiments, wherein the gene editing comprises use of antisense RNA, siRNA, shRNA, and / or CRISPR / Cas system.

[0010] In certain embodiments, wherein the virus is selected from the group consisting of adenovirus, vaccinia virus, herpes simplex virus, parvovirus, reovirus, coxsackievirus, Seneca Valley virus, Semliki Forest virus, poliovirus, measles virus, Newcastle disease virus, vesicular stomatitis virus, Venezuelan equine encephalitis virus, chicken anemia virus, Maraba virus, and echovirus.

[0011] In certain embodiments, the virus is an adenovirus, and the first gene of interest is selected from one or more of the viral propagation-essential genes consisting of: early protein 1A, early protein 1B 19K, early protein 1B 55K, encapsidation protein Iva2, DNA polymerase, terminal protein precursor pTP, encapsidation protein 52K, capsid protein precursor pIIIa, penton base, core protein pVII, core protein precursor pX, core protein precursor pVI, hexon, protease, single-stranded DNA binding protein, hexamer assembly protein 100K, protein 33K, encapsidation protein 22K, capsid protein precursor, protein U, fiber protein, regulatory protein E4 open reading frame 6 / 7, regulatory protein E4 34K, regulatory protein E4 open reading frame 4, regulatory protein E4 open reading frame 3, regulatory protein E4 open reading frame 2, and regulatory protein E4 open reading frame 1.

[0012] In certain embodiments, wherein the first gene of interest is an early protein 1A gene.

[0013] In certain embodiments, wherein the first gene of interest comprises a nucleotide sequence set forth in SEQ ID NO: 21, or encodes an amino acid sequence set forth in SEQ ID NO: 22.

[0014] In some embodiments, the first regulatory system further comprises a second gene of interest, which comprises a gene of interest capable of increasing the anti-tumor efficacy of the modified virus.

[0015] In some embodiments, the second gene of interest comprises a gene of interest that enhances antigen presentation and / or stimulates an immune response.

[0016] In some embodiments, the second gene of interest comprises a gene encoding a cytokine.

[0017] In some embodiments, the cytokine comprises an interleukin, a tumor necrosis factor, an interferon, a chemokine, a lymphokine, and / or a growth factor.

[0018] In some embodiments, the second gene of interest comprises a gene encoding a hyaluronidase and / or a gene encoding a granulocyte-macrophage colony-stimulating factor.

[0019] In some embodiments, the first gene of interest and the second gene of interest are located in the same expression cassette, and the expression of both the first gene of interest and the second gene of interest are regulated by the first cis-acting element.

[0020] In some embodiments, the first gene of interest and the second gene of interest are connected by a nucleotide sequence encoding a linker. In some embodiments, the linker comprises a 2A peptide.

[0021] In some embodiments, the second gene of interest comprises a nucleotide sequence set forth in SEQ ID NO: 46 or 48, or encodes an amino acid sequence set forth in SEQ ID NO: 47 or 49.

[0022] In some embodiments, the first regulatory system further comprises a polyadenylation signal sequence located at the 3' end of the first gene of interest and / or the second gene of interest.

[0023] In some embodiments, the first regulatory system comprises a nucleotide sequence set forth in any one of SEQ ID NO: 32 and SEQ ID NO: 44-45.

[0024] In some embodiments, the first regulatory system comprises one or more copies.

[0025] In some embodiments, the first regulatory system is integrated into the genome of the modified virus.

[0026] In some embodiments, the integration comprises using a gene editing method and / or a gene recombination method.

[0027] In certain embodiments, wherein the cis-acting element comprises a domain that binds to an ETS family transcription factor.

[0028] In certain embodiments, wherein the domain that binds to an ETS family transcription factor is capable of binding to a DNA binding domain of an ETS family transcription factor.

[0029] In certain embodiments, wherein the cis-acting element comprises a nucleotide sequence set forth in SEQ ID NO: 1 or n number of GGAA sequences, wherein n is an integer greater than or equal to 1.

[0030] In certain embodiments, wherein the cis-acting element comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 2-9.

[0031] In certain embodiments, wherein the cis-acting element comprises a nucleotide sequence set forth in SEQ ID NO: 8.

[0032] In certain embodiments, wherein the cis-acting element comprises a promoter and / or an enhancer.

[0033] In certain embodiments, wherein the cis-acting element is a promoter, the promoter comprises a domain that binds to an ETS family transcription factor and a minimal promoter.

[0034] In certain embodiments, wherein the minimal promoter is located 3' to the domain that binds to an ETS family transcription factor.

[0035] In certain embodiments, wherein the minimal promoter comprises at least one TATA box element.

[0036] In certain embodiments, wherein the minimal promoter comprises a minimal CMV promoter.

[0037] In certain embodiments, wherein the minimal promoter comprises a nucleotide sequence set forth in SEQ ID NO: 10.

[0038] In certain embodiments, wherein the cis-acting element comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 11-18.

[0039] In certain embodiments, wherein the ETS family transcription factor comprises an aberrant ETS family transcription factor.

[0040] In certain embodiments, the modified virus further comprises a second regulatory system capable of regulating expression of the first regulatory system, wherein the second regulatory system comprises:

[0041] a) a gene encoding a second transcription factor, said second transcription factor being capable of responding to a regulatory molecule; and

[0042] b) a response module comprising a second cis-acting element and operably linked thereto a regulatory sequence, wherein said second cis-acting element is capable of binding said second transcription factor, and said regulatory sequence is capable of modulating the expression level and / or activity of a gene of interest in the first regulatory system.

[0043] In certain embodiments, wherein said regulatory sequence is capable of directly or indirectly binding an expression product of a gene of interest in the first regulatory system.

[0044] In certain embodiments, wherein said expression product of a gene of interest in the first regulatory system comprises mRNA.

[0045] In certain embodiments, wherein said expression product of a gene of interest in the first regulatory system comprises a binding site for a regulatory sequence.

[0046] In certain embodiments, wherein said regulatory sequence comprises an RNA interference targeting sequence targeting a gene of interest in the first regulatory system or an expression product thereof.

[0047] In certain embodiments, wherein said regulatory sequence comprises an RNA interference targeting sequence targeting an expression product of a gene of interest in the first regulatory system, said expression product of a gene of interest in the first regulatory system comprising a target site for the RNA interference targeting sequence.

[0048] In certain embodiments, wherein said target site is located in the 3' UTR of the mRNA of said gene of interest in the first regulatory system.

[0049] In certain embodiments, wherein said regulatory sequence comprises a nucleotide sequence encoding an antisense RNA, shRNA, siRNA and / or miRNA.

[0050] In certain embodiments, wherein said regulatory sequence comprises a gene encoding an RNA binding protein, said RNA binding protein being capable of binding an expression product of a gene of interest in the first regulatory system.

[0051] In certain embodiments, wherein said expression product of a gene of interest in the first regulatory system comprises a binding site for an RNA binding protein.

[0052] In certain embodiments, wherein said binding site for an RNA binding protein is located in the 5' UTR of the mRNA of said gene of interest in the first regulatory system.

[0053] In certain embodiments, wherein said RNA binding protein comprises MCP, L7Ae and / or PP7.

[0054] In some embodiments, wherein the regulatory sequence comprises a RNA interference targeting sequence targeting a gene of interest in a first regulatory system.

[0055] In some embodiments, wherein the regulatory sequence comprises a nucleotide sequence encoding an antisense RNA, shRNA, siRNA, and / or miRNA.

[0056] In some embodiments, wherein the second cis-acting element comprises a domain capable of binding to the second transcription factor.

[0057] In some embodiments, wherein the second cis-acting element comprises an inducible promoter.

[0058] In some embodiments, wherein the regulatory molecule comprises a small molecule compound.

[0059] In some embodiments, wherein the regulatory molecule comprises Grazoprevir.

[0060] In some embodiments, wherein the second transcription factor comprises a domain capable of responding to the regulatory molecule.

[0061] In some embodiments, wherein the second transcription factor comprises a DNA binding domain, a domain capable of responding to the regulatory molecule, and a transcriptional activation domain.

[0062] In some embodiments, wherein the second transcription factor comprises a DNA binding domain of GAL4, a NS3 enzyme or fragment thereof, and a VP64 transcriptional activation domain.

[0063] In some embodiments, wherein the second transcription factor comprises an amino acid sequence set forth in SEQ ID NO: 54, or is encoded by a nucleotide sequence set forth in SEQ ID NO: 53.

[0064] In some embodiments, wherein the second cis-acting element comprises a GAL4 binding sequence.

[0065] In some embodiments, wherein the GAL4 binding sequence comprises a nucleotide sequence set forth in SEQ ID NO: 55.

[0066] In some embodiments, it comprises an amino acid sequence set forth in SEQ ID NO: 52 or SEQ ID NO: 62.

[0067] In another aspect, the present application provides an expression cassette comprising, in a 5’ to 3’ direction, a cis-acting element capable of binding to a ETS family transcription factor and a gene of interest operably linked to the cis-acting element, wherein the gene of interest comprises a viral propagation essential gene.

[0068] In another aspect, the present application provides an isolated nucleic acid molecule comprising, in the 5' to 3' direction, a nucleotide sequence encoding a cis-acting element capable of binding to an ETS family transcription factor and a nucleotide sequence of a gene of interest operably linked to the cis-acting element, wherein the gene of interest is an essential gene for viral replication or a gene encoding a reporter protein.

[0069] In certain embodiments, wherein the cis-acting element comprises the nucleotide sequence set forth in SEQ ID NO: 1.

[0070] In certain embodiments, wherein the cis-acting element comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 2-9.

[0071] In certain embodiments, wherein the cis-acting element comprises the nucleotide sequence set forth in SEQ ID NO: 8.

[0072] In certain embodiments, wherein the nucleotide sequence of the gene of interest comprises the nucleotide sequence set forth in SEQ ID NO: 21 or a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 22.

[0073] In another aspect, the present application provides an isolated nucleic acid molecule encoding the modified virus described herein.

[0074] In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule described herein.

[0075] In certain embodiments, wherein the vector comprises a viral vector.

[0076] In another aspect, the present application provides a cell comprising the modified virus described herein, the isolated nucleic acid molecule described herein and / or the vector described herein.

[0077] In certain embodiments, which is capable of producing the modified virus described herein.

[0078] In another aspect, the present application provides a pharmaceutical composition comprising the modified virus described herein, the expression cassette described herein 50, the isolated nucleic acid molecule described herein and / or the vector described herein and / or the cell described herein, and optionally a pharmaceutically acceptable carrier.

[0079] In another aspect, the present application provides use of the modified virus described herein, the expression cassette described herein, the isolated nucleic acid molecule described herein, the vector described herein and / or the cell described herein in the manufacture of a medicament, wherein the medicament is for treating a tumor.

[0080] In another aspect, the present application provides use of the modified virus described herein, the expression cassette described herein, the isolated nucleic acid molecule described herein, the vector described herein, the cell described herein and / or the pharmaceutical composition described herein in treating a tumor.

[0081] In another aspect, the present application provides a method of treating a tumor, comprising administering to a subject in need thereof an effective amount of the modified virus described herein, the expression cassette described herein, the isolated nucleic acid molecule described herein, the vector described herein, the cell described herein and / or the pharmaceutical composition described herein.

[0082] In certain embodiments, wherein the tumor comprises a tumor associated with an ETS family abnormal transcription factor.

[0083] In certain embodiments, wherein the tumor is selected from one or more of the group consisting of Ewing sarcoma, lung cancer, breast cancer, prostate cancer, melanoma, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer and brain tumor.

[0084] In certain embodiments, wherein the tumor comprises lung adenocarcinoma.

[0085] In certain embodiments, wherein the tumor comprises drug resistant breast cancer.

[0086] In certain embodiments, wherein the tumor comprises doxorubicin resistant breast cancer.

[0087] Other aspects and advantages of the present application can be readily ascertained by one skilled in the art from the following detailed description. Only the preferred embodiments of the application are shown and described in the following detailed description. As will be realized by those skilled in the art, the application is capable of modifications in various obvious aspects, all without departing from the application as recited in the claims. Accordingly, the drawings and descriptions should be regarded as illustrative in nature and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0088] The specific features of the application as claimed are set forth in the appended claims. The features and advantages of the application claimed can be better understood from the detailed description set forth below when read in conjunction with the accompanying drawings. Brief Description of Drawings is as follows:

[0089] FIG. 1A-FIG. 1H show the effect of the cis-acting elements described herein on ETS family transcription factor members.

[0090] FIG. 2A-2B show the results of the functional verification of the cis-acting element described in the present application. FIG. 2A shows the response effect of the cis-acting element described in the present application in different cells, and FIG. 2B shows that the cis-acting element described in the present application responds to the fusion protein or high expression of ETS family transcription factor to control the replication of adenovirus. FIG. 2C shows the killing effect of A549 cells by the cis-acting element described in the present application operably linked to different therapeutic genes evaluated by crystal violet staining. FIG. 2D shows the organ tissue specificity detection results of the cis-acting element described in the present application in vivo in mice.

[0091] FIG. 3 shows the flow of preparing the modified virus described in the present application.

[0092] FIG. 4 shows the microscopic results of the oncolytic adenovirus described in the present application after transfection into HEK293 cells.

[0093] FIG. 5A-5E show the in vitro functional verification results of the modified virus described in the present application. AD T-ETS and AD ΔE1A The killing effect on positive cancer cell lines (A549, A673, MCF-7 / ADR) and negative cancer cell lines (RD, MCF-7, HeLa) under fluorescence microscope (FIG. 5A), CCK8 apoptosis detection (FIG. 5B), crystal violet staining (FIG. 5C), qPCR detection of virus proliferation effect (FIG. 5D), and mixed cell-specific killing test (FIG. 5E).

[0094] FIG. 6A-6M show the in vivo functional verification results of the modified virus described in the present application. FIG. 6A shows the schematic diagram of in vivo functional verification; FIG. 6B and FIG. 6C show the tumor size change after treatment of A549 tumor-bearing mice and the tumor weight after treatment; FIG. 6D shows the body weight change of A549 tumor-bearing mice after treatment and the virus load of each tissue and organ, and the control (control) is a mouse without injection of any virus; FIG. 6E and FIG. 6F show the tumor size change after treatment of RD tumor-bearing mice and the tumor weight after treatment; FIG. 6G shows the experimental flow of administering the modified virus described in the present application by tracheal administration; FIG. 6H shows the bioluminescence signal detection results of A549 tumor-bearing mice after tracheal administration of AD ΔE1A , AD T-ETS ; FIG. 61 shows the H&E staining results of lung sections of A549 tumor-bearing mice after tracheal administration of AD ΔE1A , AD T-ETS ; FIG. 6J shows the body weight change and virus load of each tissue and organ of A549 tumor-bearing mice after tracheal administration of AD ΔE1A , AD T-ETS ; FIG. 6K shows the PBS, AD ΔE1A , ADT-ETS Body weight changes of C57BL / 6J mice; Fig. L shows the viral load of various tissue organs of C57BL / 6J mice treated with PBS, AD ΔE1A , AD T-ETS , AD ΔE1A , AD T-ETS H&E staining results of lung sections of C57BL / 6J mice treated with PBS, AD

[0095] Fig. 7A-7E show the killing effect of the modified virus described herein on various tumors. Fig. 7A shows the results of the killing effect of AD T-ETS on various tumor cells evaluated by crystal violet staining; Fig. 7B shows the results of the killing effect of AD T-ETS on various tumor cells evaluated by CCK8 assay; Fig. 7C shows the results of the detection of viral proliferation using qPCR; Fig. 7D shows the tumor volume changes of MCF-7 / ADR cancer cell bearing mice after treatment; Fig. 7E shows the tumor size changes and tumor mass after treatment of MCF-7 / ADR cancer cell bearing mice.

[0096] Fig. 8A-8E show that long-term administration of the modified virus inhibits lung tumor growth. Fig. 8A shows a schematic diagram of the long-term administration regimen of the modified virus. Fig. 8B and Fig. 8C show the bioluminescence signal for monitoring tumor size during the entire experiment. Fig. 8D shows the survival curve of mice in each group during the entire experiment. Fig. 8E shows lung tissue sections (H&E staining) of mice in each group on D60. Data is shown as the mean ± SEM of n=5 mice per group.

[0097] Fig. 9A-9E show the administration dose exploration of the modified virus. Fig. 9A shows a schematic diagram of the experimental design. Fig. 9B shows the luminescence signal measured to indicate the final tumor volume; Fig. 9C shows H&E staining of lung tissue to quantify residual metastasis; Fig. 9D shows qRT-PCR analysis of adenovirus E4 gene expression in various tissues to indicate whether AD T-ETS replicates. Fig. 9E shows the body weight changes of mice in each group. Fig. 9B, 9C and 9E group data is represented as mean ± SEM (n=5 mice per group), and D group is represented as mean ± SD (n=3 independent experiments).

[0098] Fig. 10 shows the expression amount of the modified virus co-expressing cytokines or hyaluronidase after transfecting cells.

[0099] Fig. 11 shows that the cis-acting element of the present application remains silent in normal proliferating tissues. Fig. 11A shows that after 5 days of establishing the excision wound healing model of C57 mice, 5 μg of PETS* or P hSurv P. Intradermal injection of plasmid DNA containing mCherry expression units into scar tissue. hCMV-mCherry Used as a positive control, P hCMVmin-mCherry Used as a negative control. Microscopic analysis of DAPI-stained skin tissue (scale bar: 200 μm) 48 h post-transfection shows representative images from n = 3 mice. Figure 11B shows P... ETS*- P hSurv- or P hTERT- Lentiviral particles specifically driving adenovirus replication gene (E1A) expression were used to infect retinal pigment epithelial cells (ARPE-19). After 48 hours, replication-deficient adenovirus (ADΔE1A, 5 MOI) containing constitutive EGFP expression units was added. Cell viability was assessed by crystal violet staining after another 48 hours. ARPE-19 cells infected with PhCMV-mCherry lentiviral particles served as a negative control, reflecting the absence of cell lysis. Data are presented as mean ± standard deviation; n = 3 separate experiments.

[0100] Figures 12A-12E show the construction and functional verification results of the second regulatory system. Figure 12A: Schematic diagram of the construction of the second regulatory system; Figure 12B: The effect of the second regulatory system on reducing leakage of the first regulatory system under Grazoprevir regulation, with Grazoprevir concentration on the horizontal axis; Figure 12C: The effect of the second regulatory system on shutting down the first regulatory system after activation under Grazoprevir regulation, with Grazoprevir concentration on the horizontal axis; Figure 12D: Observation under a fluorescence microscope, the effect of the second regulatory system on shutting down the first regulatory system under Grazoprevir regulation, with green fluorescence representing GFP; Figure 12E: The second regulatory system shuts down viral proliferation caused by the activation of the first regulatory system under Grazoprevir regulation, with green fluorescence representing ADE1A.

[0101] Figures 13A-13D show the construction and functional verification results of the second regulatory system. Figure 13A: The L7Ae-C / DBOX system is a schematic diagram of the second regulatory system; Figure 13B: Using SEAP as a reporter gene, the leakage prevention effect of the second regulatory system and the effect of shutting down the first regulatory system were tested; Figure 13C: Using mCherry as a reporter gene, the leakage prevention effect of the second regulatory system and the effect of shutting down the first regulatory system were tested; Figure 13D: Using E1A as the target gene, the effect of shutting down the first regulatory system and activating viral replication was tested. Detailed Implementation

[0102] The following detailed description of the application is presented in order to better demonstrate the nature and use of the present application, and to put it into practice in its intended use. Other advantages will be realized and further objects will be realized after a study of the description of the preferred embodiments presented herein.

[0103] Definitions of Terms

[0104] In the present application, the term "virus" can include live virus, inactivated virus, virus particle, viral inclusion bodies, virion, virus like particle, virus components, and mixtures thereof, and can also include viral genome. In the present application, the term "modified virus" generally refers to a virus that has been altered compared to the parental strain of the virus. For example, the modified virus can be an artificially engineered virus. A typical modified virus has one or more truncations, mutations, insertions, or deletions in the viral genome. The modified virus can have one or more endogenous viral genes modified and / or one or more intergenic regions modified. The modified virus can have one or more heterologous nucleotide sequences inserted into the viral genome. For example, the modified virus can have one or more heterologous nucleotide sequences in the form of a gene expression cassette to express a heterologous gene. For example, the modified virus can have a heterologous cis-acting element. For example, in the modified virus, the cis-acting element can regulate an endogenous viral gene and / or a heterologous nucleic acid sequence.

[0105] In the present application, the term "oncolytic virus" generally refers to a virus that is capable of selectively replicating in tumor cells and slowing the growth of tumor cells or inducing or over-proliferating cell death in vitro or in vivo, with no or minimal effect on normal cells.

[0106] In the present application, the term "ETS family transcription factor" can be used interchangeably with "ETS transcription factor family" or "ETS transcription factor", also known as "E-twenty-six-specific sequence" or "E26 transforming sequence". ETS family transcription factors include more than 30 members, which play important regulatory roles in many physiological and pathological processes by modulating cell proliferation, differentiation, apoptosis and cell-cell interactions. In the present application, the term "ETS family transcription factor" can include a class of transcription factors, for example one or more of the ETS family transcription factors. Structurally, ETS family transcription factors are characterized by a conserved DNA-binding domain: ETS domain, which can bind to specific DNA sequences in the promoters and / or enhancers through the ETS domain to regulate the expression of target genes. ETS family transcription factors can include 12 subfamilies: ETS, ERG, PEA3, ETV2, TCF, ERF, PDEF, ELF, ESE, TEL, SPI and ELG. The ETS family transcription factors described in the present application can include: ELF4 (MEF), ERG, FLI1, ELF3 (ESE1 / ESX), ELF5 (ESE2), EHF, ETS1, ETS2, ETV4 (PEA3 / E1AF), ETV5 (ERM), ETV1 (ER81), ETV2 (ER71), SPI1 (PU.1), SPIB, SPIC, ELK1 and / or ELK3 (NET / SAP2). The gene names and UniProt IDs of some members of the ETS family transcription factors can be shown in the following table:

[0107] In the present application, the ETS family transcription factor can include any natural ETS family transcription factor of any vertebrate origin, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise specified. The ETS family transcription factor can include "full-length", unprocessed ETS family transcription factor as well as any form of ETS family transcription factor derived from processing in cells; can also include naturally occurring variants of ETS family transcription factors, such as splice variants or allelic variants. For example, the ETS family transcription factor includes a chimeric protein comprising the ETS family transcription factor.

[0108] In the present application, each of the terms "chimeric protein", "fusion protein" or "fusion polypeptide" also means a protein whose amino acid sequence represents a fusion product of sub-sequences of amino acid sequences from at least two different proteins. The fusion protein is usually not produced by direct manipulation of the amino acid sequence, but rather is expressed from a "chimeric" gene encoding the chimeric amino acid sequence. In the present application, the chimeric protein comprising an ETS family transcription factor can be a gene expression resulting from gene rearrangement or gene fusion. The chimeric protein comprising an ETS family transcription factor can include, but is not limited to, TMPRSS2-ERG, EWS-ERG, FUS-ERG, EWS-FLI1, EWS-FEV, TMPRSS2-ETV4, EWS-ETV4, TMPRSS2-ETV5, TMPRSS2-ETV1 and / or EWS-ETV1.

[0109] In the present application, the term "gene of interest" generally refers to an exogenous DNA or cDNA contained in a nucleic acid molecule, a vector, a host cell or a kit, which can encode a gene product, which can be a polypeptide, a protein or a polynucleotide per se and has a function or activity. In the present application, unless otherwise specified, the gene of interest generally refers to a downstream gene operably linked to the cis-acting element of the present application, i.e. the gene of interest generally refers to a gene whose expression is regulated by the cis-acting element of the present application.

[0110] In the present application, the term "cis-acting element" generally refers to a regulatory element of gene expression that can affect the expression of a gene. Typically, a cis-acting element is a nucleotide sequence that can be located at or near the 5' end, 3' end, both ends, and / or inside of the sequence of the gene it regulates. A cis-acting element usually does not encode any protein, but only provides a binding site to affect the expression of a gene through interaction with a trans-acting factor. A "trans-acting factor" generally refers to various molecules that can play different regulatory roles (activation or inhibition) on gene expression by directly binding to or indirectly acting on nucleic acid molecules such as DNA, RNA, etc. Generally, a trans-acting factor refers to a specific protein, i.e., a transcription factor, that can bind to a gene sequence. A cis-acting element can regulate the expression of a gene by binding to a transcription factor, which in the present application can also be referred to as the "response" of the cis-acting element to the transcription factor. The binding site of the transcription factor is included in the cis-acting element. The regulation of a gene by a cis-acting element can include positive regulation or negative regulation. For example, when the regulation of a gene by a cis-acting element is positive regulation, the activity and / or expression of the gene it regulates is activated and / or up-regulated when the cis-acting element binds to its corresponding transcription factor. For example, when the regulation of a gene by a cis-acting element is negative regulation, the activity and / or expression of the gene it regulates is silenced and / or down-regulated when the cis-acting element binds to its corresponding transcription factor. The types of cis-acting elements can include, but are not limited to, promoters, enhancers, and silencers.

[0111] In the present application, the term "transcription factor" generally refers to a class of protein molecules that can bind to specific sequences on DNA and regulate the process of gene transcription. Transcription factors affect the binding of RNA polymerase to the promoter region of a gene by binding to specific sequences on DNA, thereby regulating the transcriptional activity of the gene. In the present application, the term "DNA binding domain" generally refers to the part of the molecular structure that binds to specific sequences of DNA. These domains usually include specific amino acid sequences or structural motifs that can form stable interactions with specific sequences in DNA. The DNA binding domain can include the DNA binding domain of a transcription factor, which usually occurs in the promoter region of a gene and can promote or inhibit the transcription of the gene. In the present application, the terms "transcriptional activator" and "transcriptional activation domain" can be used interchangeably and generally refer to a regulatory element or functional region that enhances or activates the transcription of a target gene. For example, a transcription factor can include a DNA binding domain and a transcriptional activation domain.

[0112] In the present application, the term "operably linked" generally refers to the arrangement of two or more nucleic acid sequence elements that are physically connected and have a functional relationship with each other. For example, operable linkage can include placing a regulatory sequence necessary for expression of a coding sequence in the proper position relative to the coding sequence so as to effect expression of the coding sequence. For example, operable linkage can mean the arrangement of a gene sequence of interest and a sequence of a cis-acting element. For example, a gene of interest and a cis-acting element are operably linked if the cis-acting element sequence is capable of affecting transcription or expression of the gene of interest. In certain embodiments, "operably linked" can also mean that a gene of interest is linked into a vector such that the gene regulatory sequences within the vector perform their intended function of regulating transcription and translation of the gene of interest.

[0113] In the present application, the term "binding" generally refers to a physical or chemical interaction between two or more biological molecules or compounds. Binding includes ionic, non-ionic, hydrogen bonding, van der Waals, hydrophobic interactions, and the like. Binding can be direct or indirect, indirect being due to or through the influence of another biological molecule or compound. Direct binding refers to an interaction that occurs without the influence of another biological molecule or compound, and without other substantial chemical intermediates. Binding can include specific binding.

[0114] In the present application, the term "promoter" generally refers to a necessary nucleotide sequence for driving the transcription of a downstream (3' end) or upstream (5' end) nucleotide sequence. The promoter is usually located near the gene it transcribes. In the present application, the promoter can include a functional fragment of the promoter. For example, a functional fragment of the promoter can be composed of fewer polynucleotides than the promoter, but still retains the ability to drive transcription. In the present application, the term "inducible promoter" generally refers to a promoter that can be activated or induced under specific conditions, which can regulate the expression of a gene linked thereto under a specific environment or physiological state. These promoters are usually affected by external signals or regulatory elements, and when these signals or factors are present, the promoter will drive the transcriptional activity of the gene.

[0115] In the present application, the term "minimal promoter" generally refers to the smallest functional fragment that has the ability to drive transcription. For example, a promoter can refer to a nucleotide sequence comprising a minimal promoter plus regulatory elements capable of controlling the expression of a coding sequence or a functional RNA. A minimal promoter can comprise a promoter's essential TATA box element, to which RNA polymerase II (pol II), TATA-binding protein (TBP), and / or TBP-associated factors (TAFs) can bind to initiate transcription. A minimal promoter can comprise at least one TATA box element. In the present application, the term "TATA box" generally refers to a segment of a promoter that resembles the 5'-TATAAA-3' nucleotide sequence. The TATA box is usually located 25-35 nucleotides upstream of the transcription start site of a gene of interest.

[0116] A minimal promoter can be derived from a viral promoter, such as the SV40 early or late promoter, the cytomegalovirus (CMV) immediate early promoter, or the Rous sarcoma virus (RSV) early promoter; or from a eukaryotic cell promoter, such as the beta-actin promoter, the GADPH promoter, the TK-1 (thymidine kinase) promoter, the HSP (heat shock protein) promoter, the UbB or UbC promoter, the PGK, the Efl-alpha promoter, or any eukaryotic promoter containing a TATA box element. In the present application, the term "minimal CMV promoter" generally refers to the shortest sequence derived from the cytomegalovirus immediate early promoter that has the ability to drive transcription. For example, a minimal CMV promoter can comprise the nucleotide sequence set forth in SEQ ID NO: 10.

[0117] In the present application, the term "enhancer" generally refers to a regulatory DNA sequence that can be bound by proteins (activators) to stimulate or enhance the transcription of a gene or several genes. Enhancers are usually cis-acting, but can be located upstream or downstream of the start site of a gene or genes they regulate. In addition, enhancers can be forward or backward oriented, and do not need to be located near the transcription start site to affect transcription. Enhancers can also be located in introns.

[0118] In the present application, the term "propagation" generally refers to an increase in number. For example, viral propagation can include viral replication. Viral replication can include any virus-mediated biological pathway required to complete transcription and / or translation of a viral genome and / or viral packaging and / or formation of functional virions and / or budding of nascent virions from a target host cell membrane. A "viral propagation essential gene" generally refers to a viral gene that is required to be expressed for a virus to propagate in vivo or in tissue culture. For example, if a viral propagation essential gene is missing or its function is impaired, the virus will be unable to complete the propagation process. In the present application, viral propagation essential genes include genes that encode viral propagation essential proteins. When a protein name is mentioned when describing a viral propagation essential gene, it generally refers to the gene that encodes it.

[0119] In the present application, the terms "5'" and "3'" are conventional expressions used to describe nucleotide sequence features, relating to the position of genetic elements and / or the direction of events such as RNA polymerase transcription or ribosome translation that proceed in a 5' to 3' direction (5' to 3'). Synonyms are upstream (5') and downstream (3'). Typically, DNA sequences, gene maps, vector maps, and RNA sequences are drawn with the 5' to 3' direction from left to right, or alternatively, the 5' to 3' direction is indicated with an arrowhead symbol, where the arrowhead points in the 3' direction. So when following this conventional usage, 5' (upstream) refers to placing a genetic element to the left hand side, while 3' (downstream) refers to placing a genetic element to the right hand side.

[0120] In the present application, the term "gene editing" generally refers to a genetic engineering that can modify a nucleotide sequence, including insertion, deletion, modification, or substitution of DNA in the genome of an organism. In the present application, gene editing can be performed using enzymes, such as nucleases that have been engineered to target specific DNA sequences, where they can introduce a cut in a DNA strand, enabling removal of existing DNA and insertion of a substituted DNA. Gene editing can be performed by the CRISPR / Cas system.

[0121] In the present application, the term "genetic recombination" generally refers to an exchange of genetic material between multiple chromosomes and / or between different regions of the same chromosome. Genetic recombination can be mediated by homology; that is, homologous regions of chromosomes are lined up in preparation for exchange, and can require some degree of sequence identity.

[0122] In the present application, the term "immune response" or "immunological response" generally includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity. In addition, the term immune response includes immune responses that are indirectly affected by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells, such as macrophages. Immune cells involved in immune responses include lymphocytes, such as B cells and T cells (e.g., CD4 + + T cells, Thl and Th2 cells); antigen presenting cells (e.g., professional antigen presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, etc., and non-professional antigen presenting cells such as endothelial cells, fibroblasts, and epithelial cells); myeloid cells such as macrophages, eosinophils, mast cells, basophils, and neutrophils.

[0123] In the present application, the term "cytokine" generally refers to a protein released by one cell that has an intercellular modulator effect on another cell. For example, the cytokine can include a cytokine that can be used for tumor therapy. Examples of "cytokine" include, but are not limited to, interleukins, tumor necrosis factors, interferons, chemokines, lymphokines, and / or growth factors.

[0124] In the present application, the term "polyadenylation signal sequence" or "polyA signal sequence" generally refers to a nucleotide sequence used to induce cleavage and polyadenylation of the primary transcript of a particular nucleotide sequence segment.

[0125] In the present application, the term "vector" generally refers to a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector. In the present application, the term "viral vector" generally refers to a nucleic acid vehicle that has been constructed based on a viral genome and is capable of carrying foreign nucleotide sequences. Generally, viral vectors are capable of self-replication and / or expression of genes (endogenous and exogenous) they contain in an appropriate host cell. Viral vectors can comprise the genome of an intact wild-type virus, or a mutated or modified viral genome.

[0126] ​In the present application, the term "treatment" generally refers to slowing or improving the progression, severity, and / or duration of a proliferative disorder, or improving one or more symptoms (e.g., one or more discernible symptoms) of a proliferative disorder, as a result of administering one or more therapies (e.g., one or more therapeutic agents such as the modified viruses of the present application). In the present application, the term "treatment" can also refer to improving at least one measurable physical parameter of a proliferative disorder such as tumor growth, not necessarily discernible by the patient. The term "treatment" in the present application can also refer to inhibiting the progression of a proliferative disorder, either physically by, for example, stabilization of a discernible symptom, physiologically by, for example, stabilization of a physical parameter, or both. In certain instances, the term "treatment" can refer to reducing or stabilizing tumor size or cancer cell count.

[0127] In the present application, the term "pharmaceutical composition" generally refers to a preparation that is in a form that is effective for the biological activity of the active ingredient and that contains no additional ingredients that are unacceptable to the subject to whom the preparation is administered. The pharmaceutical composition can also include one or more pharmaceutically acceptable carriers. Acceptable ingredients of a pharmaceutical composition are preferably non-toxic to the recipient at the dosages and concentrations employed. Pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0128] In the present application, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the present application is contemplated.

[0129] In the present application, the term "tumor" or "tumor cell" generally refers to or describes a physiological condition characterized by unregulated cell growth. For example, a tumor can refer to neoplastic cell growth and proliferation. For example, a tumor described herein can include a cancer.

[0130] In the present application, the term "cancer" generally refers to a new growth of tissue that results from abnormal cell growth, which is unregulated in response to various carcinogenic factors, leading to clonal abnormal proliferation of a cell in a local tissue of the body, resulting in a neoplasm, because the neoplasm is often a space-occupying mass that protrudes, it is also called a neoplasm. The term "cancer" includes primary malignancies (e.g., those malignancies whose cells do not migrate to sites other than the primary tumor site in the body of an individual) and secondary malignancies (e.g., those malignancies resulting from metastasis, tumor cells migrating to a second site different from the primary tumor site).

[0131] In the present application, the term "tumor associated with an abnormality in an ETS family transcription factor" generally refers to a tumor associated with an abnormal expression of an ETS family transcription factor, or a tumor associated with a cell that expresses an abnormal ETS family transcription factor. In the present application, "abnormal" in relation to an ETS family transcription factor can include an altered number, an altered gene copy number, an altered expression level, an altered biological activity, and / or the formation of a chimeric protein of an ETS family transcription factor in a cell (e.g., a tumor cell or a cell of a subject) relative to a normal cell of the subject, a cell of a normal healthy subject, and / or a normal healthy cell. For example, the abnormal ETS family transcription factor can include an increased copy number of an ETS family transcription factor in a cell. For example, the abnormal ETS family transcription factor can include an overexpression of an ETS family transcription factor in a cell. For example, the abnormal ETS family transcription factor can include a loss of tumor suppressor function of an ETS family transcription factor. For example, the abnormal ETS family transcription factor can include a chimeric protein comprising an ETS family transcription factor expressed by a fusion gene resulting from a chromosomal translocation. For example, the abnormal ETS family transcription factor can include a case where an ETS family transcription factor is over-accumulated in a cell due to abnormal degradation. Methods for detecting the expression of an ETS family transcription factor in a tumor are known in the art and include Western blot, immunohistochemical assay.

[0132] In the present application, the term "expression level" generally refers to a protein, RNA, or mRNA level of a particular relevant gene. Any method known in the art can be employed to determine the expression level of a particular relevant gene (e.g., a human ETS family transcription factor). In the present application, the "expression" generally refers to a process of transforming information encoded by a gene into a structure present in and operating in a cell. For example, it can include reverse transcription and amplification analysis (e.g., PCR, reverse transcription RT-PCR, or quantitative qPCR), hybridization analysis, Northern blotting, dot blotting, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, Western blotting, immunohistochemistry, immunostaining, or mass spectrometry. The analysis can be performed directly on a biological sample or on a protein / nucleic acid isolated from the sample.

[0133] In the present application, the term "activity" generally refers to a functional property or nature of a substance. In relation to transcription factors, the term "activity" relates to their ability to regulate gene transcription. The transcriptional activity of a gene can be determined using routinely used assays such as reporter gene assays.

[0134] In the present application, the term "expression cassette" generally refers to a recombinantly or synthetically produced polynucleotide having a series of specified nucleic acid elements that allow for the transcription of a particular nucleic acid in a target cell. Generally, an expression cassette can include a nucleotide sequence to be transcribed and a promoter, along with other sequences.

[0135] In the present application, the terms "nucleic acid," "polynucleotide," and "oligonucleotide" generally refer to polymers of nucleotides (e.g., ribonucleotides or deoxyribonucleotides) and include naturally occurring (adenine, guanine, cytosine, uracil, and thymine), non-naturally occurring, and modified nucleic acids. The term is not limited by the length of the polymer (e.g., number of monomers). The nucleic acids can be single-stranded or double-stranded, and generally contain 5'-3' phosphodiester bonds, although in some cases nucleotide analogs can have other linkages. The monomers are often referred to as nucleotides. The term "nucleotide sequence" as used in the present application generally refers to the specific arrangement of nucleotides of a nucleic acid molecule.

[0136] In the present application, the term "isolated" generally refers to a biological component (such as a nucleic acid molecule, protein, virus, or cell) that has been purified away from other cellular components (such as other chromosomal and extra-chromosomal DNA and RNA, proteins, and cells) with which it is naturally associated. Nucleic acid molecules and proteins that have been isolated include those that have been purified by standard purification methods. The term also encompasses nucleic acid molecules and proteins that are prepared by recombinant expression in a host cell and chemically synthesized nucleic acid molecules and proteins.

[0137] In the present application, the terms "subject" or "individual" or "animal" or "patient" are used interchangeably herein to refer to a subject, such as a mammalian subject, in need of administration of a pharmaceutical composition of the present application. Animal subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, yellow-tail, dairy cattle, and the like, e.g., humans.

[0138] In the present application, the terms "identity," "homology," and grammatical variations thereof, generally refer to sequences that are "aligned." Thus, for example, two polypeptide sequences are identical if they have the same amino acid sequence, at least within the region or portion being referred to. If two polynucleotide sequences are identical, they have the same polynucleotide sequence, at least within the region or portion being referred to. Identity can be of a defined region (region or domain) of a sequence. A "region" or "region" of identity refers to the identical portion of two or more referenced entities. Thus, two protein or nucleic acid sequences are identical if they are identical within one or more sequence regions or domains. Sequences that are "aligned" often contain inserted or appended bases or amino acids (gaps) as compared to a reference sequence. The degree of identity (homology) between two sequences can be determined using computer programs and mathematical algorithms. Such algorithms that calculate percent sequence identity (homology) generally calculate the gaps and mismatches between sequences in the comparison region or domain. For example, the BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschul et al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI) has exemplary search parameters as follows: mismatch -2, gap open 5, gap extend 2.

[0139] In the present application, the term "comprising" generally refers to including the recited features but not excluding other elements.

[0140] In the present application, the term "about" generally refers to a variation within a range of 0.5-10% above or below the specified numerical value, e.g., within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified numerical value.

[0141] DETAILED DESCRIPTION

[0142] Cis-acting element

[0143] In one aspect, the present application provides a cis-acting element capable of binding to an ETS family transcription factor. The cis-acting element provided by the present application is capable of regulating the expression of a gene of interest operably linked thereto.

[0144] Current studies have shown that ETS family transcription factors are closely related to the occurrence, development and metastasis of cancer. For example, ETV4 and EHF are highly expressed in lung cancer and promote the progression and transformation of lung cancer; ETS1 high expression promotes the generation of drug-resistant breast cancer (e.g., doxorubicin-resistant breast cancer); ETS2 high expression promotes the progression of colorectal cancer; FLI1 and ERG form a fusion gene with EWSR1, leading to the occurrence of Ewing sarcoma; ETV1 and ERG form a fusion gene with TMPRSS2, promoting the occurrence of prostate cancer. Therefore, the present application contemplates constructing a cis-acting element responsive to a fusion protein (EWS-FLI1, TMPRSS2-ETV1, etc.) or high expression of ETS family transcription factors.

[0145] In certain embodiments, the cis-acting element is capable of binding to an ETS family transcription factor, and the gene activity and / or expression regulated by the cis-acting element can be activated and / or up-regulated after binding to the ETS family transcription factor. For example, the cis-acting element is capable of binding to one or more ETS family transcription factors.

[0146] In certain embodiments, the cis-acting element is capable of binding to an abnormal ETS family transcription factor.

[0147] In certain embodiments, the cis-acting element is capable of binding to an abnormal ETS family transcription factor, and the gene activity and / or expression regulated by the cis-acting element can be activated and / or up-regulated after binding to the ETS family transcription factor. In certain embodiments, the cis-acting element is capable of specifically binding to an abnormal ETS family transcription factor, so that the gene activity and / or expression regulated by the cis-acting element can be specifically activated and / or up-regulated in a cell containing an abnormal ETS family transcription factor. For example, the gene activity and / or expression regulated by the cis-acting element is activated and / or up-regulated, which can be relative to the gene activity and / or expression regulated by the cis-acting element in a cell without an abnormal ETS family transcription factor, can be relative to the gene activity and / or expression regulated by the cis-acting element in a normal cell of a subject, a cell of a normal healthy subject, and / or a normal healthy cell, or can be relative to the gene activity and / or expression without being regulated by the cis-acting element.

[0148] In certain embodiments, the gene regulated by the cis-acting element is not active and / or not expressed in normal cells of the subject, in cells of a normal healthy subject, and / or in normal healthy cells. In certain embodiments, the gene regulated by the cis-acting element is less active and / or has a lower level of expression in normal cells of the subject, in cells of a normal healthy subject, and / or in normal healthy cells. For example, the gene regulated by the cis-acting element can be less active and / or have a lower level of expression relative to the activity and / or expression of the gene regulated by the cis-acting element in a cell in which an ETS family transcription factor is present, relative to the activity and / or expression of the gene regulated by the cis-acting element in a tumor cell, or relative to the activity and / or expression of the gene in the absence of regulation by the cis-acting element.

[0149] In certain embodiments, the activity of the gene can include a functional property or property of a polynucleotide (e.g., a polynucleotide encoding the gene, a messenger RNA (mRNA) produced by transcription of the gene, etc.), an amino acid product, a protein, and / or a post-translationally modified protein corresponding to the gene and / or a functional fragment of the gene.

[0150] In certain embodiments, the level of expression of the gene can include an amount of a polynucleotide (e.g., a polynucleotide encoding the gene, a messenger RNA (mRNA) produced by transcription of the gene, etc.), an amino acid product, a protein, and / or a post-translationally modified protein corresponding to the gene and / or a functional fragment of the gene.

[0151] In certain embodiments, the cis-acting element comprises a domain that binds to an ETS family transcription factor. For example, the domain that binds to an ETS family transcription factor can be capable of binding to a DNA-binding domain of an ETS family transcription factor.

[0152] In certain embodiments, the domain that binds to an ETS family transcription factor can comprise n number of GGAA nucleotide sequence repeats, where n is an integer greater than or equal to 1. In certain embodiments, n can be an integer between 7-25. In certain embodiments, n can be 21. In certain embodiments, the domain that binds to an ETS family transcription factor can comprise n number of GGAA, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In certain embodiments, there can be a spacer sequence between the repeated GGAA. For example, there can be 1 or more nucleotides as a spacer sequence between the repeated GGAA. For example, there can be 1 or 2 G (guanine), A (adenine), C (cytosine), and / or T (thymine) as a spacer sequence between the consecutive two GGAA. In certain embodiments, there can be no spacer sequence between the repeated GGAA.

[0153] In certain embodiments, there can be no spacer sequence between the repeated GGAA. In certain embodiments, the domain that binds to an ETS family transcription factor comprises a sequence of (GGAA)n, where n is an integer greater than or equal to 1. n In certain embodiments, n can be an integer between 7-25. In certain embodiments, n can be 21. In certain embodiments, the domain that binds to an ETS family transcription factor comprises a sequence of (GGAA)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. n

[0154] In certain embodiments, the cis-acting element can be a polynucleotide sequence. In certain embodiments, the cis-acting element can comprise a nucleotide sequence set forth in SEQ ID NO: 1 (the nucleotide sequence of SEQ ID NO: 1 is (GGAA)n, where n is an integer greater than or equal to 1). In certain embodiments, the cis-acting element can comprise a nucleotide sequence set forth in any one of SEQ ID NOs: 2-9 (the nucleotide sequence of SEQ ID NO: 2 is: GGAA). In certain embodiments, the cis-acting element can comprise a nucleotide sequence set forth in SEQ ID NO: 8.

[0155] ​In some embodiments, the cis-acting element can further comprise a component other than the domain that binds to an ETS family transcription factor.

[0156] In some embodiments, the cis-acting element can be capable of driving transcription of its regulated gene. In some embodiments, the cis-acting element can be a promoter, which comprises a domain that binds to an ETS family transcription factor and a component that has a function of driving transcription. For example, the component that has a driving function can be a minimal promoter.

[0157] In some embodiments, the cis-acting element comprises a domain that binds to an ETS family transcription factor and a minimal promoter. In some embodiments, the domain that binds to an ETS family transcription factor can be directly adjacent to the minimal promoter, or separated by an intervening nucleotide. In some embodiments, the minimal promoter can be located at the 3' end of the domain that binds to an ETS family transcription factor.

[0158] In some embodiments, the minimal promoter can comprise at least one TATA box. For example, the minimal promoter can be a nucleotide sequence of at least one TATA box.

[0159] In some embodiments, the minimal promoter can be derived from a viral promoter, such as the SV40 early or late promoter or the Rous sarcoma virus (RSV) early promoter; or can be derived from a eukaryotic cell promoter, such as the beta-actin promoter, the GADPH promoter, the TK-1 (thymidine kinase) promoter, the HSP (heat shock protein) promoter, the UbB or UbC promoter, the PGK, the EF1-alpha promoter, or any eukaryotic promoter containing a TATA box. In some embodiments, the minimal promoter can be tissue-specific, such as one of the muscle cell-specific promoters minimal TnISlow promoter, minimal TnIFast promoter, or muscle creatine kinase promoter.

[0160] In some embodiments, the minimal promoter can be a minimal CMV (cytomegalovirus) promoter. In some embodiments, the minimal promoter can be a minimal human CMV promoter (P hCMVmini ). In some embodiments, the minimal promoter can comprise the amino acid sequence set forth in SEQ ID NO: 10.

[0161] In some embodiments, the cis-acting element can comprise a nucleotide sequence as set forth in any one of SEQ ID NOs: 11-18. In some embodiments, the cis-acting element can comprise a nucleotide sequence that is at least 90% (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to a nucleotide sequence as set forth in any one of SEQ ID NOs: 11-18.

[0162] In some embodiments, the cis-acting element described herein can be a tumor-specific promoter. For example, the tumor-specific promoter can have high tumor specificity and high promoter activity, and can be effectively used for gene therapy. For example, the tumor-specific promoter can be an ETS family transcription factor abnormality-associated tumor-specific promoter.

[0163] In some embodiments, the cis-acting element can increase the transcription level of a gene that it regulates. In some embodiments, the cis-acting element can be an enhancer. In some embodiments, the cis-acting element can be a tumor-specific enhancer.

[0164] Expression cassette

[0165] In another aspect, the present application also provides an expression cassette comprising a cis-acting element described herein and a gene of interest operably linked thereto, wherein the cis-acting element can regulate the expression of the gene of interest.

[0166] In some embodiments, the cis-acting element and the gene of interest can be directly adjacent to each other, or separated by an intervening nucleotide. In some embodiments, the cis-acting element and the gene of interest can be separated by an intron.

[0167] In some embodiments, the cis-acting element can be located at the 5' end of the gene of interest. In some embodiments, the cis-acting element can be located at the 3' end of the gene of interest. In some embodiments, the expression cassette comprises, in the 5' to 3' direction, a cis-acting element capable of binding to an ETS family transcription factor and a gene of interest operably linked to the cis-acting element.

[0168] In some embodiments, the relative position of the domain that binds to the ETS family transcription factor to the gene of interest can be adjusted as long as the regulation of the gene of interest can be achieved. For example, the distance between the domain that binds to the ETS family transcription factor and the gene of interest can vary from 20 base pairs upstream to 2000 base pairs upstream. For example, the distance between the domain that binds to the ETS family transcription factor and the gene of interest can vary from 20 base pairs upstream to 500 base pairs upstream.

[0169] In the present application, the expression cassette can further comprise other elements, including but not limited to transcription and translation termination signals, translation initiation signals, post-transcriptional regulatory elements, and other elements.

[0170] In the present application, the transcription termination signal can include but is not limited to a polyadenylation signal sequence, such as a polyadenylation signal sequence derived from SV40, bovine growth hormone (BGH) gene, rabbit beta globin (RBG) gene, and thymidine kinase gene (TK), or variants thereof.

[0171] In the present application, the translation initiation sequence can include a Kozak sequence, which can be located between the cis-acting element and the gene of interest.

[0172] The expression cassette described in the present application can comprise, in the 5' to 3' direction, the cis-acting element capable of binding to the ETS family transcription factor, the gene of interest, and the polyadenylation signal sequence. The expression cassette described in the present application can comprise, in the 5' to 3' direction, the cis-acting element capable of binding to the ETS family transcription factor, a Kozak sequence, the gene of interest, and the polyadenylation signal sequence.

[0173] In the present application, the gene of interest can encode a polypeptide, a protein, or a polynucleic acid, or can itself be transcribed into a polynucleic acid with functionality or activity, such as an antisense nucleic acid or an inhibitory oligonucleotide, including antisense DNA and RNA (e.g., miRNA, siRNA, and shRNA). The gene of interest described in the present application is operably linked to the cis-acting element capable of binding to the ETS family transcription factor, and the expression of the gene of interest is regulated by the ETS family transcription factor.

[0174] In some embodiments, the expression cassette can comprise a nucleotide sequence as set forth in any one of SEQ ID NOs: 23-35 and SEQ ID NOs: 44-45.

[0175] Gene of interest encodes a reporter protein

[0176] In certain embodiments, the gene of interest can encode a protein of interest, which can include a reporter protein. As used herein, the term "reporter protein" generally refers to a protein that, when expressed in a cell or organism, produces a detectable signal or imparts a measurable property that can be used as an indicator of a particular biological, biochemical, or physiological process. The presence, absence, or level of a reporter protein can be detected using standard methods known in the art, such as fluorescence microscopy, luminescence measurement, colorimetric analysis, or selection under particular culture conditions. In some embodiments, a reporter protein can include a fluorescent protein (e.g., green fluorescent protein (GFP), enhanced GFP (EGFP), mCherry, mOrange), a luminescent protein (e.g., luciferase variants such as firefly luciferase, Renilla luciferase, NanoLuc), or an enzymatic reporter (e.g., beta-galactosidase, alkaline phosphatase). Exemplary reporter proteins include, but are not limited to, secreted alkaline phosphatase (SEAP), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (eCFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), MmGFP, dsRed, mCherry, luciferase, and beta-galactosidase (lacZ).

[0177] In certain embodiments, the expression cassette comprises a cis-acting element described herein and a gene of interest operably linked thereto, wherein the gene of interest encodes a reporter protein, and the expression cassette is useful for detection or diagnostic purposes. For example, the expression cassette can be used to detect whether an ETS family transcription factor is aberrantly expressed, and / or to assess whether a tumor or tumor cell associated with an ETS family transcription factor abnormality is present in a sample. In certain embodiments, the cis-acting element is operably linked to a gene of interest that encodes a reporter protein, and by detecting the expression level of the reporter protein, the expression activity of the ETS family transcription factor can be reflected, and / or whether a tumor or tumor cell associated with an ETS family transcription factor abnormality is present in a sample can be assessed.

[0178] Gene of interest is a therapeutic gene

[0179] In certain embodiments, the gene of interest can be a therapeutic gene. The therapeutic gene can encode a therapeutic peptide, a therapeutic polypeptide, a therapeutic protein, or a therapeutic polynucleic acid. The therapeutic peptide, the therapeutic polypeptide, or the therapeutic protein can be a peptide, a polypeptide, or a protein that can be used to restore or replace the function of a defective endogenous peptide, polypeptide, or protein. In certain cases, the therapeutic protein or the therapeutic polynucleic acid can be used to alter the expression level and / or activity of one or more proteins or polynucleic acids in a host cell. In certain embodiments, the therapeutic gene can include the coding sequence for all or part of an active molecule for a different therapeutic product. For example, in oncolytic virus therapy, the therapeutic gene can be a key gene necessary for viral replication.

[0180] In certain embodiments, the expression cassette comprises a cis-acting element described herein and a gene of interest operably linked thereto, the gene of interest encoding a therapeutic protein or other therapeutic product, the expression cassette being useful for therapeutic use. For example, the expression cassette can be used to selectively express a therapeutic product in a cell or tissue in which an ETS family transcription factor is aberrantly expressed, to achieve a targeted therapeutic effect.

[0181] Gene of interest is a prophylactic gene

[0182] In certain embodiments, the gene of interest can be a prophylactic gene. The prophylactic gene can encode a prophylactic peptide, a prophylactic polypeptide, a prophylactic protein, or a prophylactic polynucleic acid. The prophylactic peptide, the prophylactic polypeptide, or the prophylactic protein can be a peptide, a polypeptide, or a protein that can be used to restore or replace the function of a defective endogenous peptide, polypeptide, or protein. In certain cases, the prophylactic protein or the prophylactic polynucleic acid can be used to alter the expression level and / or activity of one or more proteins or polynucleic acids in a host cell.

[0183] In certain embodiments, the expression cassette comprises a cis-acting element described herein and a gene of interest operably linked thereto, the gene of interest being a prophylactic gene, the expression cassette being useful for prophylactic use. The expression cassette can be used to selectively express a prophylactic protein or other functional molecule in a cell in which an ETS family transcription factor is aberrantly expressed, to intervene in the relevant signaling pathway before the pathological state has fully established, to delay disease progression or to reduce the risk of occurrence.

[0184] Modified virus

[0185] In another aspect, the present application provides a modified virus.

[0186] The present application provides a modified virus comprising a cis-acting element described herein and / or an expression cassette described herein.

[0187] The modified virus described herein can have a cis-acting element described herein. In certain embodiments, the cis-acting element described herein can be integrated into the genome of the modified virus.

[0188] In certain embodiments, the modified virus comprises an expression cassette described herein. In certain embodiments, the modified virus can comprise, in a 5' to 3' direction, a cis-acting element capable of binding to an ETS family transcription factor and a gene of interest operably linked to the cis-acting element.

[0189] In certain embodiments, the modified virus can have one or more copies of an expression cassette described herein. When the modified virus has two or more copies of an expression cassette described herein, the proteins of interest in the expression cassettes can be the same or different.

[0190] In certain embodiments, the cis-acting element described herein and / or the expression cassette described herein can be integrated into the genome of the modified virus. In certain embodiments, the cis-acting element described herein and / or the expression cassette described herein can be integrated into any position of the genome of the modified virus as long as the cis-acting element and / or the expression cassette can achieve their functions. For example, the cis-acting element and / or the expression cassette can be integrated into the site of a gene to be knocked out or down in the viral genome.

[0191] The present application provides a modified virus comprising a cis-acting element capable of binding to an ETS family transcription factor and a gene of interest operably linked to the cis-acting element. By virtue of the cis-acting element capable of binding to an ETS family transcription factor, the modified virus is capable of targeting tumors associated with ETS family transcription factor abnormalities. The "targeting" generally refers to the fact that the modified virus has a relatively higher replication capacity in tumor cells and / or tumor tissues with abnormal expression levels of ETS family transcription factors, while the replication efficiency is significantly reduced in normal tissues or cells with low expression levels of ETS. The "specificity" can be relative specificity, for example, by comparing the viral replication efficiency, the expression level of the gene of interest, the cell lysis activity, etc. with non-tumor cells, it reflects its enhanced effect in the targeted tissues, rather than no replication at all in other tissues.

[0192] In certain embodiments, the cis-acting element capable of binding to an ETS family transcription factor in the modified virus can comprise a cis-acting element provided herein.

[0193] The modified viruses provided herein can be used for the treatment of tumors and can be referred to as "oncolytic viruses". Oncolytic viruses generally refer to viruses that are designed or engineered to selectively infect and kill tumor cells. Oncolytic viruses can have two characteristics: first, they have a relatively low impact on normal cells during infection and replication, and tend to preferentially infect tumor cells; second, they can induce the death or destruction of tumor cells and can trigger an immune response to eliminate the tumor. For example, the modified viruses provided herein can be oncolytic viruses that target tumors associated with abnormalities in ETS family transcription factors. In certain embodiments, the oncolytic effect of the viruses is achieved by placing the expression of a viral propagation essential gene under the control of a cis-acting regulatory element that binds to an ETS family transcription factor.

[0194] The modified viruses described herein can include viral types that can function as oncolytic viruses. For example, the modified viruses can be selected from the group consisting of adenoviruses, vaccinia viruses, herpes simplex viruses, parvoviruses, reoviruses, coxsackieviruses, Seneca Valley viruses, Semliki Forest viruses, polioviruses, measles viruses, Newcastle disease viruses, vesicular stomatitis viruses, Venezuelan equine encephalitis viruses, chicken anemia viruses, Maraba viruses, and echoviruses.

[0195] In certain embodiments, the gene of interest operably linked to the cis-acting element can include a viral propagation essential gene. By taking advantage of the killing ability of the virus on tumor cells, the viral propagation essential gene is placed under the control of the cis-acting element described herein, and when an abnormal ETS family transcription factor is recognized, the downstream protein expression is activated, promoting explosive viral proliferation, killing cancer cells, and achieving the oncolytic purpose. The modified viruses provided herein not only have a high degree of specificity in tumor cells and exhibit excellent killing effects on tumor cells, but also can target and kill a variety of tumor cells.

[0196] In certain embodiments, the class of viral propagation essential genes can be one or more, wherein each viral propagation essential gene is regulated by a respective cis-acting element described herein.

[0197] For different viruses, different viral propagation essential genes can be selected.

[0198] For example, for a herpes simplex virus, the virus propagation essential gene can be selected from one or more of envelope glycoprotein L, uracil DNA glycosylase, capsid protein, primase subunit of helicase, DNA replication initiation binding helicase, myristate derivative protein, deoxyribonuclease, tegument serine / threonine protein kinase, DNA packaging terminase subunit 1, tegument protein UL16, DNA packaging protein UL17, capsid trimer subunit 2, major capsid protein, envelope protein UL20, nuclear protein UL24, DNA packaging protein UL25, capsid maturation proteinase, capsid protein, envelope glycoprotein B, single-stranded DNA binding protein, DNA polymerase catalytic subunit, nuclear egress layer protein, DNA packaging protein UL32, DNA packaging protein UL33, nuclear egress membrane protein, large capsid protein, capsid trimer subunit 1, ribonucleotide reductase subunit 1, ribonucleotide reductase subunit 2, envelope host shutoff protein, DNA polymerase processivity subunit, membrane protein UL45, tegument protein VP13 / 14, transactivation protein VP16, tegument protein VP22, envelope glycoprotein N, tegument protein UL51, helicase-primease primase subunit, envelope glycoprotein K, ICP27, nuclear protein UL55, nuclear protein UL56, transcriptional regulatory factor ICP4, regulatory protein ICP22, envelope glycoprotein D, and membrane protein US8A.

[0199] For example, for an adenovirus, the virus propagation essential gene can be selected from one or more of early protein 1A (E1A), early protein 1B 19K, early protein 1B 55K, encapsidation protein Iva2, DNA polymerase, terminal protein precursor pTP, encapsidation protein 52K, capsid protein precursor pIIIa, penton base, core protein pVII, core protein precursor pX, core protein precursor pVI, hexon, protease, single-stranded DNA binding protein, hexamer assembly protein 100K, protein 33K, encapsidation protein 22K, capsid protein precursor, protein U, fiber protein, regulatory protein E4 open reading frame 6 / 7, regulatory protein E4 34K, regulatory protein E4 open reading frame 4, regulatory protein E4 open reading frame 3, regulatory protein E4 open reading frame 2, and regulatory protein E4 open reading frame 1.

[0200] For example, for vaccinia virus, the virus propagation essential gene can be selected from one or more of the group consisting of nucleotide reductase small subunit, serine / threonine kinase, DNA binding viral core protein, polymerase large subunit, RNA polymerase subunit, DNA polymerase, sulfhydryl oxidase, putative DNA binding viral nucleoprotein, DNA binding phosphoprotein, viral core cysteine protease, RNA helicase NPH-II, putative metalloprotease, transcription elongation factor, glutathione-like protein, RNA polymerase, putative viral nucleoprotein, late transcription factor VLTF-1, DNA binding viral nucleoprotein, viral coat protein, polymerase small subunit, DNA-dependent RNA polymerase subunit rpo22, DNA-dependent RNA polymerase subunit rpo147, serine / threonine protein phosphatase, IMV heparin binding surface protein, DNA-dependent RNA polymerase, late transcription factor VLTF-4, DNA topoisomerase type I, mRNA capping enzyme large subunit, viral core protein 107, viral core protein 108, uracil-DNA glycosylase, triphosphatase, 70 kDa small subunit of early gene transcription factor VETF, DNA-dependent RNA polymerase subunit rpo18, nucleoside triphosphate hydrolase-I, mRNA capping enzyme small subunit, rifampicin target, late transcription factor VLTF-2, late transcription factor VLTF-3, disulfide bond formation pathway, core protein 4b precursor p4b, core protein 39 kDa, DNA-dependent RNA polymerase subunit rpo19, 82 kDa large subunit of early gene transcription factor VETF, 32 kDa small subunit of transcription factor VI TF-3, IMV membrane protein 128, core protein 4a precursor P4a, IMV membrane protein 131, phosphorylated IMV membrane protein, IMV membrane protein A17L, DNA helicase, viral DNA polymerase processing factor, IMV membrane protein A21L, palmitoyl protein, 45 kDa large subunit of intermediate gene transcription factor VI TF-3, DNA-dependent RNA polymerase subunit rpo132, DNA-dependent RNA polymerase rpo35, IMV protein A30L, putative ATPase, serine / threonine kinase, EEV maturation protein, palmitoylated EEV membrane glycoprotein, IMV surface protein A27L, EEV membrane phosphoglycoprotein, IEV and EEV membrane glycoprotein, EEV membrane glycoprotein, disulfide bond formation pathway protein, putative viral nucleoprotein, IMV membrane protein I2L, poxviral myristoyl protein, IMV membrane protein L1R, late 16 kDa putative membrane protein, putative viral membrane protein H2R, IMV membrane protein A21L, chemokine binding protein, epidermal growth factor-like protein, and IL-18 binding protein.

[0201] For example, for a Coxsackievirus, the viral propagation essential gene can be selected from one or more of protein Vpg, core protein 2A, protein 2B, RNA helicase 2C, protein 3A, protease 3C, reverse transcriptase 3D, coat protein Vp4, and protein Vpl.

[0202] For example, for a Measles virus, the viral propagation essential gene can be selected from one or more of nucleoprotein N, phosphoprotein P, matrix protein M, transmembrane glycoprotein F, transmembrane glycoprotein H, and RNA-dependent RNA polymerase L.

[0203] For example, for a Measles virus, the viral propagation essential gene can be selected from one or more of nucleoprotein N, phosphoprotein P, matrix protein M, transmembrane glycoprotein F, transmembrane glycoprotein H, and RNA-dependent RNA polymerase L.

[0204] For example, for a Measles virus, the viral propagation essential gene can be selected from one or more of nucleoprotein N, phosphoprotein P, matrix protein M, transmembrane glycoprotein F, transmembrane glycoprotein H, and RNA-dependent RNA polymerase L.

[0205] For example, for a Measles virus, the viral propagation essential gene can be selected from one or more of nucleoprotein N, phosphoprotein P, matrix protein M, transmembrane glycoprotein F, transmembrane glycoprotein H, and RNA-dependent RNA polymerase L.

[0206] For example, for an Influenza virus, the viral propagation essential gene can be selected from one or more of hemagglutinin, neuraminidase, nucleoprotein, membrane protein Ml, membrane protein M2, polymerase PA, polymerase PB1-F2, and polymerase PB2.

[0207] In certain embodiments, the modified virus can be an Adenovirus, and the gene of interest to which the cis-acting element is operably linked is early protein 1A.

[0208] In certain embodiments, in order to achieve that the modified virus is capable of specifically proliferating in cells with abnormal ETS family transcription factor, the expression and / or activity of one or more originally (i.e., "endogenous") viral proliferation essential genes in the genome of the modified virus is down-regulated, for example, so that the modified virus does not have the ability to self-replicate in cells or tissues with normal expression of ETS family transcription factor, and only self-replicates and proliferates in cells or tissues with abnormal ETS family transcription factor. For example, the down-regulated viral proliferation essential gene does not have the cis-acting element described in the present application upstream. For example, the down-regulated viral proliferation essential gene is the same gene essential for the proliferation of the virus as the viral proliferation essential gene regulated by the cis-acting element, but the down-regulated viral proliferation essential gene is on the original genome before the virus is modified. In the present application, the down-regulation can include the reduction or elimination of the gene expression of one or more genes.

[0209] In certain embodiments, the down-regulation of the viral proliferation essential gene can include down-regulation using gene editing and / or gene recombination methods. For example, the gene editing can include using antisense RNA, siRNA, shRNA, and / or CRISPR / Cas system.

[0210] In certain embodiments, the cis-acting element described in the present application and the viral proliferation essential gene operably linked to the cis-acting element can be integrated into the genome of the modified virus. For example, the integration site can be a viral proliferation essential gene.

[0211] In certain embodiments, the modified virus described in the present application is an adenovirus, which comprises the cis-acting element described in the present application and the early protein 1A gene operably linked to the cis-acting element on its genome, wherein the cis-acting element and the early protein 1A gene operably linked to the cis-acting element are integrated into the early protein 1A gene site on the genome, the early protein 1A gene on the genome is unable to be expressed, so that the expression of the early protein 1A gene is regulated by the cis-acting element described in the present application.

[0212] In certain embodiments, the modified virus comprises a first regulatory system, wherein the first regulatory system comprises a first cis-acting element and a first target gene operably linked thereto, wherein the first cis-acting element comprises the cis-acting element capable of binding to the ETS family transcription factor, and the first target gene comprises the viral proliferation essential gene described in the present application. In certain embodiments, the first cis-acting element can be the cis-acting element capable of binding to the ETS family transcription factor described in the present application.

[0213] In some embodiments, the modified virus provided herein can further comprise a modification to enhance its tumor specificity, anti-tumor efficacy and / or safety. For example, the modified virus provided herein, in addition to comprising a virus propagation essential gene operably linked to a cis-acting element as described herein, can express one or more exogenous genes, knock out and / or down regulate one or more genes.

[0214] In some embodiments, the modified virus comprises a first regulatory system, wherein the first regulatory system, in addition to comprising the first cis-acting element and the first gene of interest as described above, the first regulatory system further comprises a second gene of interest, which comprises a gene of interest capable of enhancing the anti-tumor efficacy of the modified virus.

[0215] The "enhancing anti-tumor efficacy" generally refers to that, compared to a corresponding virus not comprising the second gene of interest, for example, relative to a virus comprising only the first cis-acting element and the first gene of interest, the further introduction of the second gene of interest can enhance the lysis ability of the virus in tumor cells, or promote the clearance response of the host immune system to the tumor, thereby enhancing the overall anti-tumor activity. The "anti-tumor efficacy" can be measured by various indicators known in the art, including but not limited to: changes in tumor volume or weight, lysis rate or apoptosis rate of tumor cells, prolongation of survival time or reduction of tumor load in animal models, and other in vivo pharmacodynamic indicators, etc. The detection methods of the above indicators are known to those skilled in the art, for example, can be achieved by real-time quantitative PCR (qPCR), flow cytometry (FACS), ELISA, immunohistochemical staining, in vivo fluorescence imaging, tumor measurement, pathological scoring, survival curve analysis, etc.

[0216] In some embodiments, the second gene of interest can include a gene that enhances antigen presentation and / or stimulates immune response. In some embodiments, the second gene of interest can include a gene encoding a cytokine. For example, the cytokine can include interleukins, tumor necrosis factors, interferons, chemokines, lymphokines, and / or growth factors. For example, the gene encoding a cytokine can be from a human. For example, the cytokine can include human granulocyte-macrophage colony-stimulating factor (GM-CSF), which can have an amino acid sequence as set forth in SEQ ID NO: 47. In some embodiments, the modified virus described herein can also express a hyaluronidase to degrade extracellular matrix within a tumor to enhance the spread of the oncolytic virus to promote oncolytic effect. In some embodiments, the second gene of interest can include a gene encoding a hyaluronidase. For example, the gene encoding a hyaluronidase can include a member of the human hyaluronidase family. For example, the hyaluronidase can include human hyaluronidase PH20, which can have an amino acid sequence as set forth in SEQ ID NO: 49.

[0217] In some embodiments, the expression of the second gene of interest is also regulated by a cis-acting element capable of binding to an ETS family transcription factor.

[0218] In some embodiments, the first gene of interest and the second gene of interest are located in the same expression cassette. For example, the first gene of interest and the second gene of interest are operably linked to the same cis-acting element capable of binding to an ETS family transcription factor. In some embodiments, the first gene of interest and the second gene of interest can comprise a nucleotide sequence encoding a linker therebetween. The linker can comprise a 2A peptide. For example, the linker can include a P2A, T2A peptide, and the like. In some embodiments, the modified virus comprises, in the 5’ to 3’ direction, in order: a cis-acting element capable of binding to an ETS family transcription factor, a first gene of interest (i.e., a viral propagation-essential gene), a gene encoding a P2A peptide, and a gene encoding a second gene of interest (i.e., a gene capable of improving the anti-tumor efficacy of the modified virus). In some embodiments, the modified virus described herein comprises, in the 5’ to 3’ direction, in order: a cis-acting element capable of binding to an ETS family transcription factor, a viral propagation-essential gene, a gene encoding a P2A peptide, and a gene encoding a cytokine. For example, the cytokine is human granulocyte-macrophage colony-stimulating factor.

[0219] In some embodiments, the modified virus described herein comprises, in the 5' to 3' direction, a cis-acting element capable of binding to an ETS family transcription factor, a gene essential for virus propagation, a gene encoding a P2A peptide, and a gene encoding a hyaluronidase. For example, the hyaluronidase is human hyaluronidase PH20.

[0220] In some embodiments, the first gene of interest and the second gene of interest are located in different expression cassettes. For example, the first gene of interest and the second gene of interest are each operably linked to their respective cis-acting elements.

[0221] For example, the modified virus can further comprise a gene that down-regulates a gene that is harmful to normal cells. For example, the modified virus can further comprise a gene that expresses a tumor-specific antigen. For example, the modified virus can further express a ligand gene that specifically binds to a tumor surface receptor.

[0222] In this application, the modified virus can have an enhanced ability to target tumor cells and / or kill tumor cells. For example, the modified virus described herein can have an enhanced ability to target tumor cells and / or kill tumor cells compared to an unmodified virus. For example, the modified virus described herein can have an enhanced ability to target tumor cells and / or kill tumor cells compared to a virus containing other cis-acting elements or other tumor-specific promoters (e.g., cis-acting elements other than the cis-acting element capable of binding to an ETS family transcription factor described herein). For example, the modified virus described herein can have an enhanced ability to target tumor cells and / or kill tumor cells compared to a virus in which the essential gene for virus replication is regulated by other cis-acting elements or other tumor-specific promoters.

[0223] For example, the ability to target tumor cells or the ability to kill tumor cells can be detected and assessed by methods known in the art. For example, the ability to kill tumor cells can be manifested as stable or reduced tumor size. For example, the ability to kill tumor cells can be manifested as an inhibitory effect on tumor cell growth.

[0224] In the present application, the modified virus can have reduced toxicity. In the present application, the modified virus can have no or reduced killing ability to normal cells. For example, the modified virus of the present application can have no or reduced killing ability to normal cells compared to unmodified virus. For example, the modified virus of the present application can have no or reduced killing ability to normal cells compared to virus containing other cis-acting elements or other tumor-specific promoters. For example, the modified virus of the present application can have no or reduced killing ability to normal cells compared to virus in which the essential genes for virus replication are regulated by other cis-acting elements or other tumor-specific promoters.

[0225] For example, the reduced toxicity and / or the killing ability to normal cells can be detected and evaluated by methods known in the art. For example, toxicity can be determined in vivo in animal models. For example, the degree of toxicity can be illustrated using the body weight of the animals to which the drug is administered in animal models. For example, the degree of toxicity can be illustrated using the survival rate of the animal models.

[0226] In certain embodiments, the modified virus can comprise a nucleotide sequence as set forth in any one of SEQ ID NOs: 23-35. In certain embodiments, the modified virus can comprise a nucleotide sequence as set forth in any one of SEQ ID NOs: 44-45.

[0227] Second regulatory system

[0228] Further, the present application also provides an expression construct for regulating the expression of a gene of interest, which comprises: the expression cassette of the present application (which can be referred to as a first regulatory system) and a second regulatory system. The second regulatory system can improve the regulatory specificity of the cis-acting element in the expression cassette, which includes a cis-acting element capable of binding to an ETS family transcription factor (which can be referred to as a first cis-acting element). By introducing the second regulatory system, the expression regulation effect of the cis-acting element on the gene of interest, which includes the first gene of interest and / or the second gene of interest of the present application, can be optimized.

[0229] In certain embodiments, the second regulatory system can regulate the expression of the first regulatory system. In certain embodiments, the second regulatory system can improve the regulatory specificity of the cis-acting element. For example, the regulatory specificity can be manifested in that the gene of interest is not expressed or expressed less when the abnormal ETS family transcription factor is absent, and the gene of interest is regulated by the cis-acting element to be expressed only when the abnormal transcription factor is present.

[0230] The present application also provides a modified virus comprising the first regulatory system and the second regulatory system. In some embodiments, the second regulatory system is capable of increasing the tumor specificity of the modified virus containing the first regulatory system, so as to enhance the specificity of the killing effect of the modified virus on the tumor associated with abnormal ETS family transcription factor.

[0231] In some embodiments, the second regulatory system comprises:

[0232] a) a gene encoding a second transcription factor, which is capable of responding to a regulatory molecule; and

[0233] b) a response module comprising a second cis-acting element and a regulatory sequence operably linked thereto, wherein the second cis-acting element is capable of binding to the second transcription factor, and the regulatory sequence is capable of regulating the expression and / or activity of the gene of interest of the first regulatory system.

[0234] In some embodiments, the second transcription factor is different from the first transcription factor (e.g., an ETS family transcription factor), and the second cis-acting element is also different from the first cis-acting element (e.g., an element capable of binding to an ETS family transcription factor). In some embodiments, the second cis-acting element is incapable of binding to the first transcription factor.

[0235] In some embodiments, the second transcription factor comprises a DNA binding domain, a domain capable of responding to the regulatory molecule, and a transcription activation domain. The DNA binding domain is capable of binding to the second cis-acting element, and the transcription activation domain is capable of promoting the expression of the regulatory sequence. The domain capable of responding to the regulatory molecule and the transcription activation domain enable the function of the second transcription factor to be regulated by the regulatory molecule.

[0236] In some embodiments, the regulatory molecule is generally an exogenous molecule, and is also unrelated to the ETS family transcription factor. In some embodiments, the regulatory molecule comprises a small molecule compound.

[0237] In some embodiments, the domain capable of responding to the regulatory molecule can comprise NS3 enzyme and / or fragments thereof, and the regulatory molecule can comprise NS3 enzyme inhibitors. In some embodiments, the regulatory molecule comprises Grazoprevir (Grazo for short), and the second transcription factor comprises a domain capable of responding to Grazoprevir, such as NS3 enzyme and / or fragments thereof. In the present application, NS3 protein generally refers to Hepatitis C Virus (HCV) nonstructural protein 3 (NS3), and Grazoprevir is a Hepatitis C Virus (HCV) NS3 / 4A protease inhibitor, which is the generic name of Grazoprevir, which can block NS3 protease. Grazoprevir can be represented by CAS No. 1350514-68-9, and its molecular formula is C 38 H50N6O9S. Since small molecule Grazoprevir inhibits the self-cleavage of NS3 enzyme, the second transcription factor comprises NS3 enzyme and / or fragments thereof, so the second transcription factor can be regulated by Grazo.

[0238] In some embodiments, the DNA-binding domain in the second transcription factor is capable of binding to the second cis-acting element. In some embodiments, the second transcription factor comprises the DNA-binding domain of GAL4, and the second cis-element comprises the GAL4 binding sequence. In the present application, GAL4 generally refers to a transcription factor that plays a positive regulatory role in gene expression in yeast, particularly genes involved in galactose metabolism. It functions by binding to specific DNA sequences known as upstream activation sequences (UAS). For example, the GAL4 binding sequence can comprise the nucleotide sequence set forth in SEQ ID NO: 55.

[0239] In some embodiments, the transcription activation domain comprises a VP64 transcription activation domain. In the present application, the VP64 transcription activation domain generally refers to an artificial activation domain composed of 4 tandem Herpes simplex virus VP16 activation domains.

[0240] In some embodiments, the second transcription factor comprises the DNA-binding domain (DBD) of GAL4.

[0241] In some embodiments, the second transcription factor comprises the amino acid sequence set forth in SEQ ID NO: 54, or is encoded by the nucleotide sequence set forth in SEQ ID NO: 53.

[0242] In certain embodiments, the second cis-acting element comprises a sequence that is capable of binding to the DNA binding domain of the second transcription factor. In certain embodiments, the second cis-acting element is a promoter, which further comprises a minimal promoter. In certain embodiments, the second cis-acting element is a promoter comprising a GAL4 binding sequence.

[0243] In certain embodiments, the regulatory sequence comprises a nucleotide sequence that is capable of modulating the expression and / or activity of the first and / or second gene of interest. In certain embodiments, the regulatory sequence is capable of down-regulating or knocking down the expression and / or activity of the first and / or second gene of interest.

[0244] In certain embodiments, the regulatory sequence is capable of directly or indirectly binding to the first and / or second gene of interest, or the regulatory sequence is capable of directly or indirectly binding to an expression product of the first and / or second gene of interest. In the present application, the expression product can include various products produced during the expression of a nucleotide sequence, including but not limited to mRNA, non-coding RNA (such as shRNA, miRNA, guide RNA), and protein. For example, the expression product of the first and / or second gene of interest can include mRNA containing the RNA sequence encoding the first and / or second gene of interest.

[0245] In certain embodiments, the expression product of the first and / or second gene of interest comprises a binding site for the regulatory sequence. For example, the expression product of the first and / or second gene of interest is mRNA containing the RNA sequence encoding the first and / or second gene of interest, and the binding site for the regulatory sequence can be located in the 5' UTR or 3' UTR of the mRNA, depending on the way the regulatory sequence exerts its regulatory effect.

[0246] In certain embodiments, the regulatory sequence comprises one or more RNA interference targeting sequences. In certain embodiments, the RNA interference targeting sequence is capable of targeting the mRNA of the first and / or second gene of interest, and the RNA interference targeting sequence can target the coding region in the mRNA, or target a non-coding region (such as 5' UTR, 3' UTR). In certain embodiments, the regulatory sequence comprises an RNA interference targeting sequence, and the first regulatory system comprises a binding site for the RNA interference targeting sequence, for example, a binding site for the RNA interference targeting sequence is contained downstream of the first and / or second gene of interest.

[0247] In some embodiments, the regulatory sequence can comprise a nucleotide sequence encoding an antisense RNA, shRNA, siRNA and / or miRNA targeting the first and / or second gene of interest or expression product thereof. For example, the regulatory sequence can comprise a nucleotide sequence encoding an shRNA or siRNA targeting the first and / or second gene of interest or expression product thereof.

[0248] In some embodiments, the expression product of the regulatory sequence is capable of binding to the first and / or second gene of interest or expression product thereof. For example, the regulatory sequence can comprise a nucleotide sequence encoding a protein, the expression product of which is capable of binding to the first and / or second gene of interest or expression product thereof.

[0249] In some embodiments, the regulatory sequence comprises a gene encoding an RNA binding protein capable of binding to the expression product of the gene of interest in the first regulatory system, for example, the mRNA thereof.

[0250] In this application, RNA binding protein generally refers to a protein capable of specifically binding to a certain structure or sequence on an RNA molecule. In some embodiments, the RNA binding protein can bind to a target mRNA molecule, thereby affecting its translation, stability and / or degradation rate, thereby regulating the expression level of the gene. For example, when its binding sequence is in the 5' UTR of the mRNA, the RNA binding protein inhibits translation initiation by binding thereto.

[0251] In some embodiments, the regulatory sequence comprises a gene encoding an RNA binding protein, and the first regulatory system further comprises a binding site corresponding to the RNA binding protein. For example, between the first and / or second gene of interest and the first cis-acting element, a binding site corresponding to the RNA binding protein is included. For example, the binding site of the RNA binding protein is located in the 5' UTR of the mRNA of the gene of interest regulated by the first regulatory system.

[0252] The RNA binding protein and its corresponding RNA binding site described in this application can be known in the art. In some embodiments, the RNA binding protein includes but is not limited to MCP, L7Ae and / or PP7.

[0253] L7Ae protein is derived from archaeal or some eukaryotic ribosome, which is a natural RNA binding protein capable of recognizing and binding the kink-turn (K-turn) structure in the RNA molecule, which is usually present in C / D box small nucleolar RNA (snoRNA), and the binding site of the L7Ae can include one or more C / D box structural units, such as (C / D box)4structure. The amino acid sequence of L7Ae can be as shown in SEQ ID NO: 57, and L7Ae can be encoded by the nucleotide sequence shown in SEQ ID NO: 56. The binding site of L7Ae can be encoded by the nucleotide sequence shown in SEQ ID NO: 58. MS2 coat protein (MCP) is derived from MS2 phage, which can specifically recognize and bind MS2 stem-loop structure, which is a stable hairpin structure formed by specific RNA sequence after transcription. PP7 coat protein (PP7) is derived from PP7 phage, which can specifically bind PP7 stem-loop structure, similar to the MS2 system.

[0254] In some embodiments, the second regulatory system comprises:

[0255] a) a gene encoding a second transcription factor comprising a DNA binding domain, a domain capable of responding to the regulatory molecule, and a transcription activation domain; and

[0256] b) a response module comprising a second cis-acting element and a regulatory sequence operably linked thereto, wherein the second cis-acting element comprises a sequence capable of binding the DNA binding domain of the second transcription factor, and the regulatory sequence is capable of modulating the expression and / or activity of the gene of interest in the first regulatory system.

[0257] In some embodiments, the second regulatory system comprises:

[0258] a) a gene encoding a second transcription factor comprising a DNA binding domain of GAL4, NS3 enzyme or a fragment thereof, and a VP64 transcription activation domain (GAL4-NS3-VP64); and

[0259] b) a response module comprising a second cis-acting element and a regulatory sequence operably linked thereto, wherein the second cis-acting element is a promoter comprising a GAL4 binding sequence, and the regulatory sequence comprises a nucleotide sequence encoding an antisense RNA, shRNA, siRNA and / or miRNA of the gene of interest in the first regulatory system.

[0260] In some embodiments, the second regulatory system comprises:

[0261] a) a gene encoding a second transcription factor comprising a DNA binding domain of GAL4, NS3 enzyme or a fragment thereof, and a VP64 transcriptional activation domain (GAL4-NS3-VP64); and

[0262] b) a response module comprising a second cis-acting element and a regulatory sequence operably linked thereto, wherein the second cis-acting element is a promoter comprising a GAL4 binding sequence, and the regulatory sequence comprises a nucleotide sequence encoding an RNA binding protein, and the first regulatory system comprises a binding site for the RNA binding protein upstream of the gene of interest.

[0263] In certain embodiments, in the presence of the NS3 enzyme inhibitor (e.g., Grazoprevir), the self-cleavage of the NS3 enzyme is inhibited, and the second transcription factor (e.g., GAL4-NS3-VP64) is capable of inducing the expression of the second cis-acting (e.g., a promoter comprising a GAL4 binding sequence) to drive the expression of the second gene of interest (e.g., a nucleotide sequence encoding a shRNA targeting the first gene of interest), thereby knocking down the expression of the first gene of interest, and further, when the first gene of interest is a viral propagation essential gene, the virus is unable to propagate or the efficiency of propagation is reduced in the presence of the NS3 enzyme inhibitor.

[0264] The expression construct described herein for regulating the expression of a gene of interest comprises a first regulatory system and a second regulatory system, and the second regulatory system is capable of enabling the expression of the gene of interest in the first regulatory system to be regulated by both the ETS transcription factor and the regulatory molecule. In certain embodiments, the gene of interest regulated by the first cis-acting element (the first gene of interest and / or the second gene of interest) is capable of being expressed only when both the ETS transcription factor and the regulatory molecule are present simultaneously.

[0265] In certain embodiments, the modified virus described herein comprises the first regulatory system and the second regulatory system, the first regulatory system comprises a first cis-acting element and a first gene of interest operably linked thereto, the first cis-acting element comprises a cis-acting element described herein (i.e., a cis-acting element capable of binding to an ETS family transcription factor), and the second regulatory system is capable of enabling the expression of the first gene of interest to be regulated by both the ETS family transcription factor and the regulatory molecule. In certain embodiments, the first gene of interest comprises a viral propagation essential gene.

[0266] In some embodiments, the modified virus described herein comprises the first regulatory system comprising the first cis-acting element, and the first and second genes of interest operably linked thereto, and the second regulatory system capable of enabling the expression of the first and / or second gene of interest to be under the dual regulation of the ETS family transcription factor and the regulatory molecule. In some embodiments, the first gene of interest comprises a virus propagation essential gene, and the second gene of interest comprises a gene of interest capable of enhancing the anti-tumor efficacy of the modified virus.

[0267] In some embodiments, the modified virus comprises the amino acid sequence set forth in SEQ ID NO: 52 or SEQ ID NO: 62.

[0268] Isolated nucleic acid molecule / vector / cell

[0269] In another aspect, the present application provides an isolated nucleic acid molecule capable of encoding the cis-acting element described herein.

[0270] In another aspect, the present application provides an isolated nucleic acid molecule capable of encoding the expression cassette described herein.

[0271] In another aspect, the present application provides an isolated nucleic acid molecule capable of encoding the modified virus described herein. In some embodiments, the nucleic acid molecule “encoding the modified virus” can be one or more independent nucleic acid sequences, which can be comprised in one vector, or distributed in two or more vectors respectively. For example, in a virus packaging or expression system, the genome of the modified virus can exist in a multi-component form, and different genetic modules can be carried by different nucleic acid molecules or vectors, collectively enabling the virus assembly or functional reconstitution.

[0272] In some embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 1-9, 11-18, 23-35, 38, 44-45, 50-52, 53, and / or 59-62.

[0273] In another aspect, the present application provides a vector comprising the nucleic acid molecule described herein. In some embodiments, the “vector” can be a combination of one or more vectors, i.e. the nucleic acid molecule can be comprised in two or more vectors respectively, and the nucleic acid molecules in different vectors can work together to enable the encoding of the modified virus or the functional expression of the regulatory system. For example, in a virus packaging or regulatory construction system, the multiple vectors can carry the first regulatory system, the second regulatory system, the auxiliary protein expression element, etc. respectively, for the purpose of working together to construct or regulate the modified virus.

[0274] In certain embodiments, the vector can be a viral vector. For example, the viral vector can include a DNA viral vector, an RNA viral vector, and / or a retroviral vector. For example, the viral vector can include an integrating viral vector and / or an episomal vector. For example, the vector can include an adenoviral vector, a vaccinia viral vector, a herpes simplex viral vector, a parvoviral vector, a reoviral vector, a coxsackie viral vector, a semliki forest viral vector, a polioviral vector, a measles viral vector, a newcastle disease viral vector, a vesicular stomatitis viral vector, a venezuelan equine encephalitis viral vector, a chicken anemia viral vector, a maraba viral vector, and / or an echoviral vector. For example, the vector can include an adeno-associated viral vector and / or a lentiviral vector.

[0275] In certain embodiments, the vector can also be a polynucleotide vector, e.g., a plasmid, cosmid, or transposon.

[0276] Vectors suitable for use have been widely described and are well known in the art. Those skilled in the art will appreciate that vectors comprising the nucleic acid molecules described herein can also comprise additional sequences and elements that can be useful for replication of the vector in prokaryotic and / or eukaryotic cells. For example, the vectors described herein can include a prokaryotic replicon, i.e., a nucleotide sequence that has the ability to direct the host's own replication and maintenance in a prokaryotic host cell (e.g., a bacterial host cell). Such replicons are well known in the art. In certain instances, the vector can comprise a shuttle element that makes the vector suitable for replication and integration in both prokaryotes and eukaryotes. In addition, the vector can also include a gene that is capable of expressing a detectable marker (e.g., a drug resistance gene). The vector can also have a reporter gene, e.g., a gene that encodes a fluorescent or other detectable protein.

[0277] The present application also provides a cell comprising the isolated nucleic acid molecule and / or the vector. In some cases, the cell can be used to amplify, replicate, package and / or purify the modified virus, the isolated nucleic acid molecule or the vector. In other cases, the cell can be used to express a gene of interest contained in the modified virus, the isolated nucleic acid molecule or the vector. Accordingly, the present application also encompasses a method of expressing a gene of interest, wherein the modified virus of the present application, the isolated nucleic acid molecule of the present application or the vector of the present application is introduced into a cell, e.g., a tumor cell. One skilled in the art will understand the conditions necessary to introduce the modified virus, the isolated nucleic acid molecule or the vector into a cell, as well as conditions that support or facilitate expression of the gene of interest in the cell. In addition, the method can be an in vivo or in vitro method. The cell can include prokaryotic and eukaryotic cells. In some cases, the cell can be a mammalian cell. For example, when the cell is used to package the viral vector, the cell can also be transfected with one or more plasmids or infected with one or more viruses that provide the necessary helper molecules for packaging. In other cases, the cell can stably express one or more helper molecules from the genome. One skilled in the art is able to select an appropriate host cell for amplification, replication, packaging and / or purification of the vector of the present application. Exemplary mammalian cells can include, but are not limited to, HEK-293 cells.

[0278] The present application also provides a method of expressing a gene of interest, the method comprising introducing the isolated nucleic acid molecule, the vector and / or the modified virus into a host cell and allowing the gene of interest to be expressed in the host cell.

[0279] The present application also provides a method of making the modified virus of the present application. In some embodiments, the method can further comprise the step of isolating the modified virus.

[0280] Pharmaceutical compositions and kits

[0281] The present application also provides a diagnostic or pharmaceutical composition comprising the modified virus of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application and / or the cell of the present application.

[0282] The modified viruses described herein, the isolated nucleic acid molecules described herein, the vectors described herein, and / or the cells and other compositions, agents, drugs, biologies (proteins) described herein can be incorporated into the diagnostic and pharmaceutical compositions, e.g., pharmaceutically acceptable carriers, diluents and adjuvants. The carriers, diluents and adjuvants can include buffers, such as phosphate, citrate or other organic acids; antioxidants, such as ascorbic acid; low molecular weight polypeptides (e.g., less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants, e.g., Tween, Pluronics or polyethylene glycol (PEG). In certain instances, the physiologically acceptable carrier is an aqueous pH buffered solution.

[0283] In instances where the composition comprises a nucleic acid molecule or a nucleic acid molecule carrier (e.g., a plasmid), the nucleic acid molecule or nucleic acid molecule carrier can be present as “naked DNA” or prepared in a delivery vehicle, such as a microparticle or nanoparticle, including a liposome, a micelle, a lipid particle, a ceramic / inorganic particle, and a virus-like particle.

[0284] The present application also provides a kit comprising the modified viruses described herein, the isolated nucleic acid molecules described herein, the vectors described herein, and / or the host cells described herein. In another aspect, the present application also provides use of the cis-acting elements described herein, the expression cassettes described herein, the modified viruses described herein, the isolated nucleic acid molecules described herein, the vectors described herein, the cells described herein in the manufacture of a kit.

[0285] The kits will also typically include a label or package insert providing instructions for use of the components therein, including descriptions of the components and instructions for using the components in in vitro, in vivo, or ex vivo procedures.

[0286] The term “packaging material” refers to the physical structure housing the components of the kit. The packaging material can maintain the components in sterile condition, can be made of material commonly used for such purposes, such as paper, foil, glass, plastic, fϊlm, ampoules, vials, tubes, etc.

[0287] The label or package insert can include information concerning one or more components of the kit, such as identification, dosage, clinical pharmacology, pharmacokinetics, and pharmacodynamics of the active ingredients including mechanism of action. The label or package insert can include information identifying the manufacturer, batch number, place of manufacture, and date of manufacture, expiration date. The label or package insert can include information identifying the manufacturer, batch number, place of manufacture, and date of manufacture. The label or package insert can include information concerning the disease for which the kit components can be used. The label or package insert can include instructions for the clinician or subject for using one or more kit components in a method, use, or treatment regimen or treatment method. The instructions can include formulation dosage, frequency, or duration, and directions for practicing any of the methods, uses, treatment regimens, or prophylactic or therapeutic methods described herein.

[0288] The label or package insert can include information concerning any benefit that the components can provide, such as a prophylactic or therapeutic benefit. The label or package insert can include information concerning potential adverse side effects, complications, or reactions, such as warning information for the subject or clinician concerning situations in which use of a particular composition can not be appropriate. Adverse side effects or complications can also occur when the subject is already taking, will take, or is taking one or more other drugs that can not be compatible with the composition, or when the subject is already undergoing, will undergo, or is undergoing another treatment regimen or treatment method that can not be compatible with the composition, and thus the instructions should include information concerning such incompatibilities.

[0289] Detection and / or diagnostic methods

[0290] In another aspect, when the cis-acting element described herein is used to initiate expression of a reporter protein, the present application also provides use of the cis-acting element described herein, the expression cassette described herein, the modified virus described herein, the isolated nucleic acid molecule described herein, the vector described herein, the cell described herein, the pharmaceutical composition described herein, and / or the kit described herein in detection and / or diagnosis, for example, for detecting whether an ETS family transcription factor is abnormally expressed, and / or for assessing whether a tumor or tumor cell associated with abnormal expression of an ETS family transcription factor is present in a sample.

[0291] In another aspect, the present application also provides a method for detecting the expression activity of an ETS family transcription factor, comprising the steps of:

[0292] a) introducing into a cell to be tested an expression cassette comprising a cis-acting element described herein and a gene of interest encoding a reporter protein, wherein the cis-acting element is operably linked to the gene of interest;

[0293] b) culturing the cell so that the reporter protein in the expression cassette is expressed;

[0294] c) detecting the expression level of the reporter protein; and

[0295] d) assessing the expression activity of ETS family transcription factor in the cell based on the expression level of the reporter protein.

[0296] In another aspect, the present application also provides a method for assessing whether a tumor or tumor cell associated with abnormal expression of ETS family transcription factor exists in a sample, comprising the following steps:

[0297] a) introducing a virus or expression cassette comprising an ETS response element and a gene of interest into the sample or cells therein to be tested;

[0298] b) allowing the virus or expression cassette to express in the sample;

[0299] c) detecting the product of the gene of interest (e.g. a reporter protein or virus replication level);

[0300] d) determining whether a tumor or tumor cell with abnormal expression of ETS family transcription factor exists in the sample based on the expression level of the product.

[0301] In certain embodiments, the use is a diagnostic use. In certain embodiments, the kit can be used for diagnosis. For example, for diagnosing a tumor associated with abnormal expression of ETS family transcription factor. The diagnostic method comprises detecting the expression level of a reporter protein initiated by the cis-acting element in a sample of a subject, and comparing with a reference value to determine whether the subject has the associated tumor. The reference value can be the expression level of the normal tissue of the subject, or the average expression level of the normal tissue of the normal population. When the expression of the reporter protein in the sample is higher than the reference value, it can indicate that the subject or a specific tissue thereof has abnormal expression of ETS family transcription factor, and thus has the risk of the associated tumor.

[0302] In certain embodiments, the detection or diagnosis process is performed in vitro, for example by analyzing an ex vivo sample of the subject.

[0303] Prevention and / or treatment of diseases

[0304] In another aspect, the present application provides use of the cis-acting element described herein, the expression cassette described herein, the modified virus described herein, the isolated nucleic acid molecule described herein, the vector described herein, or the cell described herein in the preparation of a medicament for the prevention and / or treatment of a disease.

[0305] In another aspect, the present application provides a use of the cis-acting element described herein, the expression cassette described herein, the modified virus described herein, the isolated nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the pharmaceutical composition described herein in the treatment of a disease.

[0306] In another aspect, the present application provides a method of treating a disease, comprising administering to a subject in need thereof an effective amount of the cis-acting element described herein, the expression cassette described herein, the modified virus described herein, the isolated nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the pharmaceutical composition described herein.

[0307] In certain embodiments, the present application also provides a method of treatment, comprising administering to a subject a medicament (e.g., a modified virus) comprising a first regulatory system described herein and a second regulatory system described herein. In certain embodiments, the method of treatment further comprises a step of administering a regulatory molecule capable of modulating the activity of the second transcription factor in the second regulatory system, thereby achieving control of the expression of the gene of interest in the first regulatory system. The administration of the medicament and the regulatory molecule can be simultaneous, or administered separately. For example, the administration of the regulatory molecule can be simultaneous with the administration of the modified virus, or can be administered within a specific time window after the administration of the virus, to achieve a time-sequential control effect. For example, the regulatory molecule can be administered during the duration of the pharmacodynamic effect of the modified virus, thereby improving the tumor targeting of the modified virus and reducing the impact on normal tissues.

[0308] The cis-acting element described herein, the expression cassette described herein, the modified virus described herein, the isolated nucleic acid molecule described herein, the vector described herein, the cell described herein, the pharmaceutical composition described herein, and / or the medicament for preventing and / or treating a disease described herein can be administered in a therapeutically effective amount. For example, a therapeutically effective amount can include an amount effective, when administered to a subject, to prevent or ameliorate the symptoms of, or the progression of, one or more diseases or conditions. For example, a therapeutically effective amount can include an amount of a binding compound sufficient to result in an improvement in symptoms, e.g., an amount that treats, cures, prevents or ameliorates the relevant medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition. For example, a therapeutically effective dose refers only to the active ingredient itself in the case of administering an active ingredient alone. For example, in the case of combination therapy, a therapeutically effective dose would refer to the combined amounts of active ingredients that would act synergistically when administered together, whether by co-administration, sequential administration, or separate administration.

[0309] The cis-acting element described herein, the expression cassette described herein, the modified virus described herein, the isolated nucleic acid molecule described herein, the vector described herein, the cell described herein, the pharmaceutical composition described herein, and / or the medicament for preventing and / or treating a disease described herein can be delivered to a subject in need thereof by any route known in the art. Pharmaceutical carriers and formulations or compositions are also well known in the art. For example, the route of administration can include intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral, respiratory, or mucosal. The modified virus described herein, the pharmaceutical composition described herein, and / or the medicament for preventing and / or treating a disease described herein can be suitable for administration via a variety of routes of administration. For example, for a lung cancer patient, the modified virus described herein, the pharmaceutical composition described herein, and / or the medicament for preventing and / or treating a disease described herein can be administered via trachea.

[0310] The cis-acting element described herein, the expression cassette described herein, the modified virus described herein, the isolated nucleic acid molecule described herein, the vector described herein, the cell described herein, the pharmaceutical composition described herein, and / or the medicament for preventing and / or treating a disease described herein can be administered to a subject in need thereof in combination with at least one agent. For example, the agent can include an anticancer agent, an agonist, an antagonist, a chemotherapeutic agent, and a radiation agent.

[0311] In the present application, the prevention and / or treatment includes not only prevention and / or treatment of a disease, but also generally includes preventing the onset of a disease, slowing or reversing the progression of a disease, preventing or slowing the onset of one or more symptoms associated with a disease, reducing and / or alleviating one or more symptoms associated with a disease, reducing the severity and / or duration of a disease and / or any symptoms associated therewith and / or preventing further increases in the severity of a disease and / or any symptoms associated therewith, preventing, reducing, or reversing any physiological damage caused by a disease, and generally any pharmacological effect that is beneficial to the patient being treated. For example, the cis-acting element of the present application, the expression cassette of the present application, the modified virus of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, the pharmaceutical composition of the present application, and / or the agent for preventing and / or treating a disease of the present application need not achieve complete cure or eradication of any symptom or manifestation of a disease. As recognized in the relevant art, a drug used as a therapeutic agent can reduce the severity of a given disease state, but need not eliminate every manifestation of a disease to be considered a useful therapeutic agent. Similarly, a therapeutic agent prophylactically administered can be a viable preventative agent without being completely effective in preventing the onset of a disorder. The prevention and / or treatment described in the present application can include simply reducing the impact of a disease in a subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood of disease occurrence or worsening.

[0312] The cis-acting element of the present application, the expression cassette of the present application, the modified virus of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, the pharmaceutical composition of the present application, and / or the agent for preventing and / or treating a disease of the present application can prevent and / or treat a tumor.

[0313] For example, the cis-acting element of the present application, the expression cassette of the present application, the modified virus of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, the pharmaceutical composition of the present application, and / or the agent for preventing and / or treating a disease of the present application can have good therapeutic effects on a variety of tumors, especially on ETS family transcription factor abnormality-related tumors. For example, the modified virus of the present application can have good therapeutic effects on a variety of ETS family transcription factor abnormality-related tumors except Ewing sarcoma.

[0314] In the present application, the tumor can include Ewing sarcoma, lung cancer, breast cancer, prostate cancer, melanoma, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer and / or brain tumor. For example, the lung cancer described in the present application can include lung adenocarcinoma. For example, the breast cancer described in the present application can include drug-resistant breast cancer. For example, the drug-resistant breast cancer can include doxorubicin-resistant breast cancer.

[0315] Without wishing to be bound by any theory, the examples below are merely to illustrate the cis-acting element, the modified virus, the expression cassette, the preparation method and the use of the present application, and are not used to limit the scope of the present application.

[0316] Examples

[0317] Example 1 Construction of cis-acting element

[0318] By analyzing the respective DNA binding sites of the 28 members of the ETS family, it is found that the binding sites are all centered on GGAA, and the bases at both ends can vary. It is speculated that GGAA can be the common binding sequence of ETS family transcription factors. Therefore, the binding site (GGAA) is inserted in front of the minimum promoter (for example, the minimum CMV promoter: P hCMVmini ) upstream of the target gene n to test the effectiveness of the cis-acting element (i.e. (GGAA) n -P hCMVmini , denoted as (GGAA) n in the following and the drawings), and to adjust different n values (1-23 copies) to optimize the cis-acting element.

[0319] This example illustrates that, taking ETV4, EHF (which needs a MAPK activator to activate the transcription activation function), EWS-FLI1, EWS-ERG, EWS-ETV1 and EWS-ETV4 as examples, the cis-acting element capable of binding to the ETS family transcription factor described in the present application can activate the expression of the downstream gene operably connected thereto in response to the ETS family transcription factor.

[0320] SEAP as a reporter gene, a corresponding (GGAA) n -SEAP vector is constructed. 293T cells are plated in a 24-well plate, and after overnight, ETV4 or EHF (100 ng / well) is taken, (GGAA) n -SEAP (50 ng / well), pCDNA3.1 (150 ng / well) is mixed, PEI (0.9 μL / well) is added, and transfection is performed in a 24-well plate. After 6 h, the liquid is changed, and after 24 h, the supernatant is aspirated to determine the SEAP value. The results show that the increase in n value significantly activates the cis-acting element, and (GGAA) 21The best, high response and very low leakage (Figure 1A). Since the fusion protein EWS-FLI1 and the like have strong transcriptional activation function, the fusion protein EWS-FLI1 and the like (0.5 ng / well) are taken, (GGAA) n -SEAP (50 ng / well), pCDNA3.1 (249.5 ng / well) are mixed, PEI (0.9 μL / well) is added, and the SEAP value is measured after transfection into 293T cells. The results also show that with the increase of n value, the cis-acting element is significantly activated, and (GGAA) 21 is the best (Figures 1B and 1C).

[0321] Subsequently, by adjusting the transfection amount of ETS family transcription factors (0, 1, 5, 10, 20, 40, 80 ng), the cis-acting element constructed in the present application (GGAA) 21 -SEAP cis-acting element whether only responds to high concentration of ETS family transcription factors (except transcriptional repressor ETV6, ETV3, ETV7, ERF, FEV), the transfection step is the same as above, and the results show that: ETS family members cannot activate the cis-acting element at a low concentration of 1 ng, but can activate (GGAA) 21 -SEAP (Figures 1D, 1E, 1F, 1G and 1H).

[0322] Example 2 Verification of the function of the cis-acting element

[0323] To verify whether the cis-acting element of the present application can respond to the fusion protein or high concentration of ETS family transcription factors in cancer cells. The cis-acting element is loaded with red fluorescent protein mCherry, and (GGAA) 21 -mCherry is packaged into a lentivirus and infects ETS high expression lung cancer cell line A549, fusion protein positive cell line A673 and other negative cell lines 293T, RD, RH30. At the same time, a positive control lentivirus (P hCMV -mCherry) is set up. After 48 hours, the red fluorescence is observed under a fluorescence microscope. The results show that the positive control group confirms that the lentivirus can infect A549, A673, 293T, RD and RH30 cells, and the red fluorescent protein mCherry in the test group is only expressed in A549 and A673 cells, and no red fluorescence is observed in 293T, RD and RH30 (Figure 2A), indicating that the cis-acting element designed in the present application can specifically respond to the fusion protein and high concentration of ETS family transcription factors in cancer cells.

[0324] Subsequently, to verify whether the cis-acting element constructed in the present application can control the proliferation of adenovirus, the key replication gene E1A of adenovirus is inserted downstream of the cis-acting element, and (GGAA) 21- E1A (i.e. "switch"), packaged into lentivirus, infected A549, A673, RD cells, 24h later added non-replicative adenovirus (with AD ΔE1A or AD E1A - ), 4 days later collected cells, extracted genome, qPCR detected viral load. The results showed that the switch-containing lentivirus + AD ΔE1A group in A549, A673 cells AD ΔE1A appeared hundreds of times of proliferation (Figure 2B), indicating that the cis-acting element constructed in the present application can be activated by fusion protein or high concentration of ETS family transcription factor to control the replication of adenovirus.

[0325] To test the effect of virus proliferation essential genes as therapeutic genes, the cis-acting element described in the present application was operably linked to other therapeutic genes (i.e. HSV-TK / GCV suicide gene or Bax suicide gene) to construct (GGAA) 21 - HSV-TK (the nucleotide sequence of which is shown in SEQ ID NO: 34) and (GGAA) 21 - Bax (the nucleotide sequence of which is shown in SEQ ID NO: 35), respectively packaged into lentivirus, infected A549 cells, and compared the killing effects of different ways. The results showed that the operable linkage of the cis-acting element and the adenovirus replication key gene E1A can achieve high-efficiency killing effect of adenovirus on A549 cancer cells, which is significantly better than HSV-TK / GCV or Bax (Figure 2C), indicating that the oncolytic virus containing the virus proliferation essential gene operably linked to the cis-acting element described in the present application has excellent killing effect as a treatment method.

[0326] To verify the in vivo specificity of the cis-acting element constructed in the present application, the cis-acting element was operably linked to luciferase to construct (GGAA) 21 - Fluc (the nucleotide sequence of which is shown in SEQ ID NO: 33), packaged into different adeno-associated virus vectors (AAV8, AAV9, AAV PHP.eB ). Through tail vein injection (1 x 10 11 VP only), and set up positive control group (strongly expressed luciferase, P hCMV - Fluc), negative control group (P hCMVmini driven luciferase, P hCMVmini-Fluc). The results showed that similar luciferase signals (none or minimal) were observed in the test groups as in the negative control group, while strong signals were observed in different organ tissues in the positive control group (AAV8 liver; AAV9 heart, liver, spleen, lung, intestine; AAVPHP.eB-liver, lung, brain) (Figure 2D). These data indicated that the cis-acting elements constructed in the present application exhibited excellent specificity.

[0327] In addition, to verify whether the cis-acting elements constructed in the present application can distinguish cancer cells from normal rapidly proliferating healthy cells, the cis-acting elements were operably linked to the red fluorescent protein mCherry to construct (GGAA) 21 -mCherry (P ETS* -mCherry, the nucleotide sequence of which is shown as SEQ ID NO: 38), while the mCherry operably linked to the currently clinically tested cancer-specific promoter hSurv (survivin) was constructed (GGAA) hSurv -mCherry, the nucleotide sequence of which is shown as SEQ ID NO: 39), and the positive and negative controls (P hCMVmin -driven mCherry, P hCMVmin -mCherry, the nucleotide sequence of which is shown as SEQ ID NO: 40; P hCMV -driven mCherry, P hCMV -mCherry, the nucleotide sequence of which is shown as SEQ ID NO: 41). The plasmids were mixed with Lip3000 transfection reagent and subcutaneously injected into the scar tissues of the established skin wound healing model mice, respectively. After 48 h, the skin near the scar tissues of the mice in each group was taken and sectioned to observe the mCHerry expression. The results showed that the traditional cancer-specific promoter hSurv drove the expression of fluorescent protein in healthy proliferating tissues, while the cis-acting elements constructed in the present application remained silent (Figure 11A), indicating that the cis-acting elements constructed in the present application can successfully distinguish cancer cells from healthy proliferating tissues. Further, the E1A operably linked to the currently clinically tested cancer-specific promoters hTERT (telomerase) and hSurv (survivin) was constructed (GGAA) hTERT -E1A, the nucleotide sequence of which is shown as SEQ ID NO: 42; P hSurv -E1A, the nucleotide sequence of which is shown as SEQ ID NO: 43), were packaged into lentiviruses, respectively, and used to infect normal retinal epithelial cell line ARPE-19 cells. After 24 h, non-replicative adenoviruses (AD ΔE1A or AD E1A -The results show that the cells in the traditional cancer-specific promoter hTERT and hSurv groups were obviously killed, and the cells in the hSurv group were almost completely dead, while the cells in the cis-acting element group constructed by the present application were in good condition (Figure 11B), which again indicates that the cis-acting element constructed by the present application can successfully distinguish cancer cells and healthy proliferative tissues.

[0328] The above results show that the cis-acting element constructed by the present application can achieve the desired cancer / tumor selective detection at a high standard without impairing the overall efficacy of the treatment target gene expression.

[0329] Example 3 Preparation of modified virus

[0330] Based on the classic AdEasy-1 system, the modified oncolytic adenovirus was produced in HEK-293 cells and obtained, mainly including the following steps (as shown in Figure 3):

[0331] (1) The cis-acting element ((GGAA) 21 -E1A) regulating the expression of the E1 gene in Example 2 was constructed and subcloned into the shuttle vector pAdTrack.

[0332] (2) The shuttle vector plasmid constructed successfully in (1) was linearized by Pme I enzyme and dephosphorylated by alkaline phosphatase to obtain the linearized and dephosphorylated plasmid fragment containing the cis-acting element of the E1 gene.

[0333] (3) The linearized plasmid fragment in (2) was transformed into the BJ5183 bacterial strain carrying the adenovirus backbone pAdEasy-1 plasmid to directly perform recombination, and the recombinant clones were selected on the plate, the corresponding plasmid was extracted and identified by Pac I enzyme digestion, and the plasmid with two fragments of large (~ 30 Kb) and small (~ 4 Kb) visible after Pac I enzyme digestion electrophoresis was obtained, that is, the correct adenovirus recombinant.

[0334] (4) The correct adenovirus recombinant identified in (3) was amplified in large quantities, and the amplified plasmid was linearized by Pac I enzyme and then transfected into HEK-293 cells. After 6-8 hours of transfection, the liquid was changed, and the cells were further cultured for 7-10 days. During the process, the cells were observed under a microscope to show a cluster of green fluorescent cells resembling meteors (as shown in Figure 4), indicating that the initial virus had been produced and replicated in the HEK-293 cells. The mixture containing the initial adenovirus was collected after the cells in the dish appeared to be obviously broken and floating.

[0335] (5) The adenovirus mixture collected in (4) was repeatedly frozen and thawed, and the supernatant obtained after centrifugation was the P0 adenovirus, which was stored at -80°C after aliquoting and used for subsequent amplification and purification.

[0336] The modified P0 generation oncolytic adenovirus obtained needs to be amplified, purified and detected before being used for subsequent efficacy research. The experimental steps are as follows:

[0337] (1) The P0 generation adenovirus is added to a new batch of HEK-293 cells in a 15 cm culture dish. After infection, multiple cells are observed to express green fluorescence in a cluster-like manner similar to meteors under a microscope. The mixture containing the adenovirus is collected after a large number of cell plaques appear or the cells are obviously broken and floating. After repeated freeze-thawing and centrifugation, the obtained supernatant is the P1 generation adenovirus. Similarly, the P1 generation adenovirus is used to infect more HEK-293 cells in a 15 cm dish to obtain the P2 generation adenovirus. The infection is continuously performed for four generations until a large amount of P4 generation adenovirus supernatant is obtained for subsequent in vitro purification and concentration.

[0338] (2) 50 mL of virus precipitation solution (20% PEG8000, 2.5 M NaCl) is added to every 100 mL of P4 generation virus supernatant, and the mixture is placed at 4°C overnight to precipitate the virus. The mixture is centrifuged at 12000 rpm for 20 min, and the supernatant is discarded. The precipitate is completely resuspended with 10 mL of CsCl solution (solvent: 20 mM Tris-HCl, pH 8.0) with a density of 1.10 g / mL, and centrifuged at 7000 rpm at 4°C for 5 min. The virus suspension is collected.

[0339] (3) In a ultracentrifuge tube, 2 mL of CsCl solution (solvent: same as above) with a density of 1.40 g / mL is added, followed by 3 mL of CsCl solution with a density of 1.30 g / mL, and finally 5 mL of virus suspension obtained in (2). After centrifugation at 22800 rpm at 4°C for 2.5 h, the virus band with a density of 1.30-1.40 g / mL is collected into a dialysis bag (the dialysis bag is boiled in 10 mM EDTA Na2 for 10 min before use). After dialysis in dialysis buffer (50 g sucrose, 10 mL 1 M Tris-HCl solution, pH 8.0, 2 mL 1 M MgCl2 solution, and water to 1000 mL) at 4°C overnight (with one change of dialysis buffer), the virus is collected, aliquoted, and stored at -80°C.

[0340] (4) The titer of the modified oncolytic adenovirus finally obtained in (3) is determined according to the experimental requirements: VP determination method (virus particles), the absorbance of virus particles at 260 nm is determined, and 1 OD value is equivalent to 1.1 x 10 12 PFU determination method (plaque forming unit), the number of virus plaques formed in the infected monolayer HEK-293 cell culture is determined.

[0341] Example 4 In vitro functional verification of modified virus

[0342] cis-acting elements are successfully packaged into adenovirus to prepare new programmable oncolytic adenovirus (AD T-ETS ) which can specifically kill cancer cells. T-ETS After the preparation of AD ΔE1A and AD T-ETS , they are used to infect positive cancer cell lines A549, A673, MCF-7 / ADR and negative cancer cell lines RD, MCF-7, Hela, and the specific killing ability of AD T-ETS is verified in vitro by CCK8 apoptosis rate detection technology, crystal violet staining technology and qPCR technology. The results show that after the addition of AD T-ETS , there is no obvious death phenomenon in negative cell lines, while positive cancer cell lines A549, A673, MCF-7 / ADR show obvious death phenomenon (Figure 5A), and the death phenomenon becomes more significant with the increase of MOI (Figure 5B), and the crystal violet staining results also show that AD T-ETS only kills positive cancer cell lines (Figure 5C), and AD T-ETS shows explosive proliferation in positive cancer cell lines (at least 400 times) (Figure 5D). In addition, after mixing the A549 stable cell line stably expressing mCherry with negative cells RD at 1:1, adding AD ΔE1A (test group) or AD T-ETS (negative control group), after 4 days, the proportion of red cells is observed under a fluorescence microscope, and the results show that the blank control group and the negative control group still contain a large number of red fluorescent cells, but the red fluorescent cells in the test group disappear, indicating that AD T-ETS can specifically kill A549 cells but not RD cells (Figure 5E). The above results show that AD T-ETS can specifically kill fusion protein positive cell lines and ETS family transcription factor high expression cell lines.

[0343] Example 5 In vivo functional verification of modified virus

[0344] To verify the in vivo oncolytic effect of AD 3 , A549 and RD cells (3 million per mouse) are subcutaneously injected into the back of NSG mice to establish A549 and RD tumor-bearing mouse models, respectively, and divided into test and control groups. After the tumor grows to 50 mm T-ETS , the test group is injected with AD ΔE1A , and the control group is injected with AD 9 (negative control group) intratumorally, with an injection dose of 1×10 2 / mouse, and injected once every 2 days for a total of 4 times. The tumor size (V = width 3Alternatively, the endpoint could be 30 days after drug administration, with tumor analysis performed to identify differences between groups (experimental procedure shown in Figure 6A). Results showed that in A549 tumor-bearing mice, compared to those injected with AD... ΔE1A AD injection T-ETS Subsequently, the tumor on the mouse's back remained at 50 mm. 3 The tumor weight was significantly reduced at the treatment endpoint (Figures 6B and 6C); however, in RD tumor-bearing mice, regardless of AD injection... ΔE1A or AD T-ETS Tumors on the backs of mice increased in size over time, and there was no significant difference in tumor size or weight between the two groups at the treatment endpoint (Figures 6E and 6F). Furthermore, intratumoral injection of AD... T-ETS Subsequently, the virus remained concentrated within the tumor (Figure 6D) and did not spread to other organs. No significant difference was observed in the mouse body weight, indicating that AD... T-ETS It has good safety. The above results indicate that AD... T-ETS It exhibits excellent oncolytic effects in vitro and has good safety profile.

[0345] AD T-ETS Therapeutic effects can be achieved through various administration methods, such as inhalation (tracheal administration). A lung metastasis model was established by tail vein injection of A549 cells stably expressing luciferase (2 million cells / cell). Seven days after cell injection, AD cells were administered every other day. ΔE1A or AD T-ETS Administered via trachea (7 doses total) orally into the lungs (2×10⁻⁶). 9 VP / animal), with 28 days post-cell injection as the endpoint, and differences between groups were analyzed (Figure 6G). Results showed that AD T-ETS No bioluminescent signal was observed in the administered mice, while AD ΔE1A Significant bioluminescent signals were detected in the administered mice (Fig. 6H). Furthermore, H&E staining of lung sections showed a correlation with AD. ΔE1A Compared with the treated mice, AD T-ETS The number and size of metastatic nodules in the lungs of the treated mice were significantly reduced (Fig. 6I). Notably, complete disappearance of tumor cells was observed in the lungs of one of the mice (Fig. 6I). Furthermore, no significant difference in body weight was observed between the two groups, and AD... T-ETS It accumulates only in lung tissue, showing more than a thousand-fold increase in proliferation compared to the control group (Figure 6J).

[0346] Data from tracheal administration indicate that AD T-ETS It exhibits excellent antitumor efficacy. However, a key concern is the potential lung tissue toxicity associated with this route of administration. Therefore, the safety of this delivery method was evaluated. PBS and AD were administered using the same route and dosage. ΔE1A or AD T-ETSinjected into the lungs of C57BL / 6J mice. Body weight gain, viral load and histological analysis were analyzed at day 7 post virus injection. AD ΔE1A or AD T-ETS administration did not affect the body weight gain (Figure 6K), and the presence of the virus was limited to the lungs, without spreading to other tissues. Notably, compared to AD ΔE1A , AD T-ETS exhibited very low proliferation in the lungs (3.95 fold) (Figure 6L). Moreover, histopathological analysis of lung tissue samples obtained at day 7 post tracheal administration indicated that AD T-ETS post-injection lung tissue structure was not disrupted. AD ΔE1A and AD T-ETS Both groups exhibited a small amount of inflammatory cell infiltration, which can be attributed to the nature of the virus itself (Figure 6M). These results indicate that AD T-ETS is safe when administered via the trachea.

[0347] Example 6 Killing effect of modified virus on multiple tumors

[0348] This example demonstrates that the modified virus constructed in this application has a killing effect on multiple tumors. Abnormal amplification of ETS family transcription factors is associated with multiple cancers. For example, abnormal amplification of ETS2 and ELF4 is observed in colorectal cancer, amplification of ETV1 is observed in melanoma, and abnormal amplification of ETS1 exists in drug-resistant breast cancer. Therefore, to verify the killing effect of AD T-ETS on these cancer cells, cancer cells overexpressing ETS family transcription factors (e.g., SW480, overexpressing ETS2; LOVO, overexpressing ELF4; SK-MEL-28, overexpressing ETV1; MCF-7 / ADR, overexpressing ETS1; gastric cancer cells; pancreatic cancer cells; colorectal cancer cells; liver cancer cells) were incubated with AD ΔE1A or AD T-ETS for 4 days, and then the apoptosis rate was evaluated using crystal violet staining or CCK8 assay, and the viral proliferation was detected using qPCR. The results showed that AD T-ETS had a significant cytolysis effect on these cancer cells (Figures 7A, B), accompanied by explosive proliferation of AD T-ETS (Figure 7C). In addition, AD T-ETS inhibited the growth of MCF-7 / ADR cancer cell tumor-bearing mice (Figures 7D, E). Therefore, these results indicate that AD T-ETS has the potential to be used for the treatment of multiple tumors, especially malignant tumors with abnormal amplification of ETS family transcription factors.

[0349] Example 7 Long-term administration of modified virus

[0350] This embodiment shows that the modified virus constructed in the application can control lung cancer cells in a small range for a long time, further showing its effectiveness, safety and tolerance. The experimental process is shown in FIG. 8A. A549 cells stably expressing luciferase (2 million per mouse) were injected into the tail vein to establish a lung metastasis model. Seven days after cell injection, AD ΔE1A or AD T-ETS (2x10 9 VP per mouse) was given through the trachea. The first 7 times were given every two days, and then once a week, and the observation was continued for 140 days after administration. During the period, the lung tumor growth was observed by in vivo imaging on D0, 20, 40, 60, 84 after administration, and after D60 observation, the lung tissues of mice in each group were taken for sectioning and HE staining to detect the lung tumor cell growth.

[0351] The results show that the cancer cell signal of the AD T-ETS group mice remained at a low level throughout the experiment (FIG. 8B, C), in contrast to the AD ΔE1A group mice, the cancer cell signal increased significantly over time (FIG. 8B, C). This sharp growth of cancer cells is the reason why all AD ΔE1A group mice died within D76, in contrast to the AD T-ETS group mice, the cancer cells were controlled in a small range, which is the reason why the mice survived to D140 (FIG. 8D). The HE section of lung tissue of mice in each group on D60 also showed that the AD T-ETS group mice had only a small amount of cancer cells in the lungs, in contrast to the AD ΔE1A group mice, which contained a large number of cancer cells (FIG. 8E).

[0352] Example 8: Dose exploration of modified virus administration

[0353] This embodiment shows that the modified virus constructed in the application can achieve certain therapeutic effect at different administration doses. The experimental process is shown in FIG. 9A. A549 cells stably expressing luciferase (2 million per mouse) were injected into the tail vein to establish a lung metastasis model. Seven days after cell injection, AD T-ETS (2x10 9 VP per mouse, group A; 1x10 9 VP per mouse, group B; PBS, group C) was given through the trachea. The first 7 times were given every two days, and then once a week, and the observation was continued for 30 days after administration. During the period, the lung tumor growth was observed by in vivo imaging on D30 after administration, and after observation, the lung tissues of mice in each group were taken for sectioning and HE staining to detect the lung tumor cell growth.

[0354] [Corrected according to Rule 91 on 01.07.2025] The results showed that the lung cancer cell signals of groups A and B were significantly lower than that of group C (Figure 9B), and the lung cancer cell signals of group A were significantly lower than that of group B (Figure 9B). The HE sections of lung tissues of mice in each group showed that the lung cancer cell nodules of groups A and B were significantly lower than that of group C (Figure 9C), and the lung cancer cell nodules of group A were also significantly lower than that of group B (Figure 9C). In addition, after administration of AD T-ETS , the virus was only concentrated in the lungs (Figure 9D) and did not spread to other organs, and there was no significant difference in the body weight of mice (Figure 9E), indicating that AD T-ETS has good safety. The above results show that 2 x 10 9 VP / mouse and 1 x 10 9 VP / mouse have certain therapeutic effects.

[0355] Example 9: Modified virus further expressing a cytokine or hyaluronidase

[0356] This example shows that the modified virus expressing a cytokine or hyaluronidase constructed in the present application has better oncolytic effect. On the basis of (GGAA) 21 -E1A, a cytokine (such as GMCSF) or hyaluronidase (such as PH20) is co-expressed, and (GGAA) 21 -E1A-P2A-GMCSF (the nucleotide sequence of which is shown in SEQ ID NO: 44) and (GGAA) 21 -E1A-P2A-PH20 (the nucleotide sequence of which is shown in SEQ ID NO: 45) are transfected into a positive cancer cell line A549 in vitro, and after 48 h, the cDNA of each group of cells is extracted, qRT-PCR is used to verify the mRNA expression amount of GMCSF and PH20 genes, and the expression efficiency is verified in vitro. The results show that the cis-acting elements described in the present application can be used for co-expression of genes, and the expression amount can be up to thousands of times (Figure 10), and the virus co-expressing a cytokine or hyaluronidase on the basis of expressing a key gene for virus replication can have a better oncolytic effect.

[0357] Example 10: Modified virus further expressing a second regulatory system

[0358] On the basis of the original cis-acting element, a controllable second regulatory system for preventing leakage is inserted to further regulate the expression of the first cis-element.

[0359] 10.1 Second regulatory system containing shRNA

[0360] The anti-leakage mode selection shRNA knockdown system in this embodiment, using small molecule Grazoprevir to inhibit the self-cleavage of NS3 enzyme, we designed to insert NS3 enzyme between GAL4-VP64, forming GAL4-NS3-VP64, in E1A shRNA upstream insertion UAS sequence (for binding GAL4), when no Grazoprevir is added, NS3 enzyme self-cleavage, resulting in GAL4 unable to connect with transcription activator VP64, E1A shRNA expression. When Grazoprevir is added, NS3 enzyme self-cleavage is inhibited, GAL4-NS3-VP64 binds to (UAS)5, driven by VP64, E1A shRNA expression, knock down the expression of E1A, and thus reduce the proliferation efficiency of the virus, to reduce the purpose of leakage (Figure 12A).

[0361] By changing different target genes and corresponding shRNA, the function of the second regulation system is tested. The target gene is SEAP, and pA-SEAP shRNA -(UAS)5-pA-SEAP-P hCMVmin -(GGAA) 21 -CMV-GAL4-NS3-VP64-pA (the nucleotide sequence is shown as SEQ ID NO: 50), transfected into 293T cells (100 ng / well), 6 h later, add Grazoprevir, 24 h later, the supernatant is aspirated, SEAP is determined, the results show that with the increase of Grazoprevir concentration, SEAP value decreases significantly, 20 μm Grazoprevir can reduce the leakage of the first regulation system to a very low state (Figure 12B). At the same time, the second regulation system can also close the activated first regulation system (Figure 12C). The target gene is EGFP, and pA-EGFP shRNA -(UAS)5-pA-EGFP-P hCMVmin -(GGAA) 21 -CMV-GAL4-NS3-VP64-pA (the nucleotide sequence is shown as SEQ ID NO: 51), the results also show that the second regulation system can reduce the green fluorescence caused by the leakage of the first regulation system and the green fluorescence caused by the activation of the first regulation system (Figure 12D). The target gene is replaced by E1A, and pA-E1A shRNA -(UAS)5-pA-E1A-P hCMVmin -(GGAA) 21 -CMV-GAL4-NS3-VP64-pA (the nucleotide sequence is shown as SEQ ID NO: 52), transfected into pre-prepared A549 cells (24-well plate, 200 ng / well), 24 h later,E1A - Cells were collected after 2d, Q-PCR was used to determine the AD in each group E1A - The proliferation effect, the results showed that when the Grazo concentration reached 40um, the second regulatory system could completely close the virus proliferation caused by the activation of the first regulatory system (Figure 12E). The above results prove the successful construction of the second regulatory system, which can close the virus proliferation caused by the activation of the first regulatory system, and provide stronger safety for future clinical application.

[0362] 10.2 Second regulatory system containing RNA binding protein

[0363] In this embodiment, the anti-leakage mode can also select the knockdown system of RNA binding protein, such as L7Ae-C / DBOX system. L7Ae is inserted downstream of UAS sequence ((UAS)5-L7Ae), and (C / DBOX)4 is inserted upstream of E1A ((GGAA) 21 -PhCMVmin-(CDBOX)4-E1A) (the nucleotide sequence is shown as SEQ ID NO: 59), when Grazoprevir is not added, NS3 enzyme autocleavage, causing GAL4 unable to connect with transcription activator VP64, L7Ae unable to express. When Grazoprevir is added, NS3 enzyme autocleavage is inhibited, GAL4-NS3-VP64 binds to (UAS)5, under the drive of VP64, L7Ae expresses, binds to C / DBOX, inhibits the expression of E1A, and then reduces the proliferation efficiency of the virus, achieving the purpose of reducing leakage (Figure 13A).

[0364] By replacing different target genes, the function of the second regulation of L7Ae-C / DBOX system is tested. The target gene is SEAP, and pA-L7Ae-(UAS)5-pA-SEAP-(C / DBOX)4-P hCMVmin -(GGAA) 21 -CMV-GAL4-NS3-VP64-pA (the nucleotide sequence is shown as SEQ ID NO: 60), transfected into 293T cells (100 ng / well), 6h after changing the liquid, adding Grazoprevir, 24h after absorbing the supernatant, determining SEAP, the results show that with the increase of Grazoprevir concentration, SEAP value decreases significantly, 5um Grazoprevir can reduce the leakage of the first regulatory system to a very low state (Figure 13B). At the same time, the second regulatory system can also close the activated first regulatory system (Figure 13B). The target gene is mCherry, and pA-L7Ae-(UAS)5-pA-mCherry-(C / DBOX)4-P hCMVmin -(GGAA)21 -CMV-GAL4-NS3-VP64-pA (the nucleotide sequence of which is shown as SEQ ID NO: 61), and the results also showed that the second regulatory system can reduce the red fluorescence caused by the leakage of the first regulatory system and the red fluorescence caused by the activation of the first regulatory system (Figure 13C). The target gene was replaced with E1A, and pA-L7Ae-(UAS)5-pA-E1A-(C / DBOX)4-P hCMVmin -(GGAA) 21 -CMV-GAL4-NS3-VP64-pA (the nucleotide sequence of which is shown as SEQ ID NO: 61), and the results also showed that the second regulatory system can reduce the red fluorescence caused by the leakage of the first regulatory system and the red fluorescence caused by the activation of the first regulatory system (Figure 13C). The target gene was replaced with E1A, and pA-L7Ae-(UAS)5-pA-E1A-(C / DBOX)4-P - , 2d after collection of cells, Q-PCR was used to determine the ADE1A - proliferation effect, and the results showed that when the concentration of Grazo reached 10um, the second regulatory system could completely close the virus proliferation caused by the activation of the first regulatory system (Figure 13D). The above results prove that the L7Ae-C / DBOX system as the second regulatory system can also close the virus proliferation caused by the activation of the first regulatory system, and is better than the shRNA knockdown system.

Claims

1. A modified virus comprising a first regulatory system, the first regulatory system comprising a first cis-acting element and a first target gene operatively linked thereto, wherein the first cis-acting element is capable of binding to an ETS family transcription factor, and the first target gene comprises a gene essential for viral replication.

2. The modified virus according to claim 1, wherein the expression and / or activity of at least one endogenous viral replication-essential gene of the virus is downregulated in the modified virus.

3. The modified virus according to claim 2, wherein at least one gene essential for endogenous viral replication is downregulated by gene editing and / or gene recombination.

4. The modified virus according to claim 3, wherein the gene editing comprises using antisense RNA, siRNA, shRNA and / or a CRISPR / Cas system.

5. The virus according to any one of claims 1-4, wherein the virus is selected from the group consisting of: adenovirus, vaccinia virus, herpes simplex virus, parvovirus, reovirus, Coxsackie virus, Seneca Valley virus, Semliki Forest virus, poliovirus, measles virus, Newcastle disease virus, vesicular stomatitis virus, Venezuelan equine encephalitis virus, chicken anemia virus, Malaba virus, and echovirus.

6. The modified virus according to any one of claims 1-5, wherein: The virus is an adenovirus, and the first target gene is selected from one or more of the following groups of genes essential for viral replication: early protein 1A, early protein 1B19K, early protein 1B55K, capsid protein Iva2, DNA polymerase, terminal protein precursor pTP, capsid protein 52K, capsid protein precursor pIIIa, pentazoc matrix, core protein pVII, core protein precursor pX, core protein precursor pVI, hexazoc, protease, single-stranded DNA-binding protein, hexamer assembly protein 100K, protein 33K, capsid protein 22K, capsid protein precursor, protein U, fibrin, regulatory protein E4 open reading frame 6 / 7, regulatory protein E4 34K, regulatory protein E4 open reading frame 4, regulatory protein E4 open reading frame 3, regulatory protein E4 open reading frame 2, and regulatory protein E4 open reading frame 1.

7. The modified virus according to any one of claims 1-6, wherein the first target gene is the early protein 1A gene.

8. The modified virus according to any one of claims 1-7, wherein the first target gene comprises the nucleotide sequence shown in SEQ ID NO: 21, or encodes the amino acid sequence shown in SEQ ID NO:

22.

9. The modified virus according to any one of claims 1-8, wherein the first regulatory system further comprises a second target gene, the second target gene comprising a target gene capable of enhancing the antitumor efficacy of the modified virus.

10. The modified virus according to claim 9, wherein the second target gene comprises a target gene that enhances antigen presentation and / or stimulates an immune response.

11. The modified virus according to any one of claims 9-10, wherein the second target gene comprises a gene encoding a cytokine.

12. The modified virus according to claim 11, wherein the cytokines include interleukins, tumor necrosis factors, interferons, chemokines, lymphokines, and / or growth factors.

13. The modified virus according to any one of claims 9-12, wherein the second target gene comprises a gene encoding hyaluronidase and / or a gene encoding granulocyte-macrophage colony-stimulating factor.

14. The modified virus according to any one of claims 9-13, wherein the first target gene and the second target gene are located in the same expression cassette, and the expression of both the first target gene and the second target gene is regulated by the first cis-acting element.

15. The modified virus according to claims 9-14, wherein the first target gene and the second target gene are linked by a nucleotide sequence encoding a linker.

16. The modified virus of claim 15, wherein the linker comprises a 2A peptide.

17. The modified virus according to any one of claims 9-16, wherein the second target gene comprises the nucleotide sequence shown in SEQ ID NO: 46 or 48, or encodes the amino acid sequence shown in SEQ ID NO: 47 or 49.

18. The modified virus according to any one of claims 1-17, wherein the first regulatory system further comprises a polyadenylated nucleotide signal sequence located at the 3' end of the first target gene and / or the second target gene.

19. The modified virus according to any one of claims 1-18, wherein the first regulatory system comprises the nucleotide sequence of any one of SEQ ID NO: 32 and SEQ ID NO: 44-45.

20. The modified virus according to any one of claims 1-19, wherein the copy number of the first regulatory system is one or more.

21. The modified virus according to any one of claims 1-20, wherein the first regulatory system is integrated into the genome of the modified virus.

22. The modified virus of claim 22, wherein the integration comprises using gene editing and / or gene recombination.

23. The modified virus according to any one of claims 1-22, wherein the cis-acting element comprises a domain that binds to ETS family transcription factors.

24. The modified virus according to claim 23, wherein the domain that binds to ETS family transcription factors is capable of binding to the DNA-binding domain of ETS family transcription factors.

25. The modified virus according to any one of claims 1-24, wherein the cis-acting element comprises the nucleotide sequence shown in SEQ ID NO: 1 or n GGAA sequences, wherein n is an integer greater than or equal to 1.

26. The modified virus according to any one of claims 1-25, wherein the cis-acting element comprises the nucleotide sequence shown in any one of SEQ ID NO: 2-9.

27. The modified virus according to any one of claims 1-26, wherein the cis-acting element comprises the nucleotide sequence shown in SEQ ID NO:

8.

28. The modified virus according to any one of claims 1-27, wherein the cis-acting element comprises a promoter and / or an enhancer.

29. The modified virus according to any one of claims 1-28, wherein the cis-acting element is a promoter, the promoter comprising a domain that binds to ETS family transcription factors and a minimal promoter.

30. The modified virus according to claims 1-29, wherein the minimal promoter is located at the 3' end of the ETS family transcription factor binding domain.

31. The modified virus according to any one of claims 29-30, wherein the minimum promoter comprises at least one TATA box element.

32. The modified virus according to any one of claims 29-31, wherein the minimal promoter comprises the minimal CMV promoter.

33. The modified virus according to any one of claims 29-32, wherein the minimum promoter comprises the nucleotide sequence shown in SEQ ID NO:

10.

34. The modified virus according to any one of claims 1-33, wherein the cis-acting element comprises the nucleotide sequence shown in any one of SEQ ID NO: 11-18.

35. The modified virus according to any one of claims 1-34, wherein the ETS family transcription factor includes an aberrant ETS family transcription factor.

36. The modified virus according to any one of claims 1-35, further comprising a second regulatory system capable of regulating the expression of the first regulatory system, wherein the second regulatory system comprises: a) A gene encoding a second transcription factor that responds to regulatory molecules; and b) A response module comprising a second cis-acting element and a regulatory sequence operatively linked thereto, wherein the second cis-acting element is capable of binding the second transcription factor, and the regulatory sequence is capable of regulating the expression level and / or activity of the target gene in the first regulatory system.

37. The modified virus according to claim 36, wherein the regulatory sequence is capable of directly or indirectly binding to the target gene or its expression product in the first regulatory system.

38. The modified virus according to any one of claims 36-37, wherein the expression product of the target gene in the first regulatory system comprises mRNA.

39. The modified virus according to any one of claims 36-38, wherein the expression product of the target gene in the first regulatory system comprises a binding site of the regulatory sequence.

40. The modified virus according to any one of claims 36-39, wherein the regulatory sequence comprises an RNA interference targeting sequence that targets the target gene or its expression product in the first regulatory system.

41. The modified virus according to any one of claims 36-40, wherein the regulatory sequence comprises an RNA interference targeting sequence that targets the expression product of the target gene in the first regulatory system, the expression product of the target gene in the first regulatory system comprising a target site of the RNA interference targeting sequence.

42. The modified virus according to claim 41, wherein the target site is located at the 3'UTR of the mRNA of the target gene in the first regulatory system.

43. The modified virus according to any one of claims 36-42, wherein the regulatory sequence comprises a nucleotide sequence encoding an antisense RNA, shRNA, siRNA, and / or miRNA.

44. The modified virus according to any one of claims 36-39, wherein the regulatory sequence comprises a gene encoding an RNA-binding protein capable of binding the expression product of the target gene in the first regulatory system.

45. The modified virus of claim 44, wherein the expression product of the target gene in the first regulatory system comprises a binding site for an RNA-binding protein.

46. ​​The modified virus according to claim 45, wherein the binding site of the RNA-binding protein is located at the 5'UTR of the mRNA of the target gene in the first regulatory system.

47. The modified virus according to any one of claims 44-45, wherein the RNA-binding protein comprises MCP, L7Ae and / or PP7.

48. The modified virus according to any one of claims 36-47, wherein the second cis-acting element comprises a domain capable of binding to the second transcription factor.

49. The modified virus according to any one of claims 36-48, wherein the second cis-acting element comprises an inducible promoter.

50. The modified virus according to any one of claims 36-49, wherein the regulatory molecule comprises a small molecule compound.

51. The modified virus according to any one of claims 36-50, wherein the regulatory molecule comprises Grazoprevir.

52. The modified virus according to any one of claims 36-51, wherein the second transcription factor comprises a domain capable of responding to the regulatory molecule.

53. The modified virus according to any one of claims 36-52, wherein the second transcription factor comprises a DNA-binding domain, a domain responsive to the regulatory molecule, and a transcriptional activation domain.

54. The modified virus according to any one of claims 36-53, wherein the second transcription factor comprises a DNA-binding domain of GAL4, an NS3 enzyme or a fragment thereof, and a VP64 transcription activation domain.

55. The modified virus according to any one of claims 36-54, wherein the second transcription factor comprises the amino acid sequence shown in SEQ ID NO: 54, or is encoded by the nucleotide sequence shown in SEQ ID NO:

53.

56. The modified virus according to any one of claims 36-55, wherein the second cis-acting element comprises a GAL4 binding sequence.

57. The modified virus of claim 56, wherein the GAL4 binding sequence comprises the nucleotide sequence shown in SEQ ID NO:

55.

58. The modified virus according to any one of claims 1-57, comprising the amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO:

62.

59. An expression cassette comprising, in a 5' to 3' orientation, a cis-acting element capable of binding to ETS family transcription factors and a target gene operatively linked to said cis-acting element, wherein said target gene includes a gene essential for viral replication.

60. An isolated nucleic acid molecule comprising, in a 5' to 3' orientation, a nucleotide sequence encoding a cis-acting element capable of binding to an ETS family transcription factor and a nucleotide sequence of a target gene operatively linked to said cis-acting element, wherein said target gene is an essential gene for viral replication or a gene encoding a reporter protein.

61. The isolated nucleic acid molecule according to claim 60, wherein the cis-acting element comprises the nucleotide sequence shown in SEQ ID NO:

1.

62. The isolated nucleic acid molecule according to any one of claims 60-61, wherein the cis-acting element comprises the nucleotide sequence shown in any one of SEQ ID NO: 2-9.

63. The isolated nucleic acid molecule according to any one of claims 60-62, wherein the cis-acting element comprises the nucleotide sequence shown in SEQ ID NO:

8.

64. The isolated nucleic acid molecule according to any one of claims 60-63, wherein the target gene nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO: 21 or the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:

22.

65. An isolated nucleic acid molecule encoding the modified virus according to any one of claims 1-58.

66. A vector comprising the isolated nucleic acid molecule of any one of claims 60-65.

67. The vector according to claim 66, wherein the vector comprises a viral vector.

68. A cell comprising the modified virus of any one of claims 1-58, the expression cassette of claim 59, the isolated nucleic acid molecule of any one of claims 60-65, and / or the vector of any one of claims 66-67.

69. The cell according to claim 68, which is capable of producing the modified virus according to any one of claims 1-58.

70. A pharmaceutical composition comprising the modified virus of any one of claims 1-58, the expression cassette of claim 59, the isolated nucleic acid molecule of any one of claims 60-65, and / or the vector of any one of claims 66-67 and / or the cell of any one of claims 68-69, and optionally a pharmaceutically acceptable carrier.

71. Use of the modified virus of any one of claims 1-58, the expression cassette of claim 59, the isolated nucleic acid molecule of any one of claims 60-65, the vector of any one of claims 66-67, and / or the cell of any one of claims 68-69 in the preparation of a medicament, wherein the medicament is for the treatment of tumors.

72. Use of the modified virus of any one of claims 1-58, the expression cassette of claim 59, the isolated nucleic acid molecule of any one of claims 60-65, the vector of any one of claims 66-67, the cell of any one of claims 68-69, and / or the pharmaceutical composition of claim 70 in the treatment of tumors.

73. A method of treating a tumor, comprising administering to a subject in need an effective amount of any of the modified virus of claims 1-58, the expression cassette of claim 58, the isolated nucleic acid molecule of any of claims 60-65, the vector of any of claims 66-67, the cell of any of claims 68-69, and / or the pharmaceutical composition of claim 70.

74. The use according to any one of claims 71-72 or the method according to claim 73, wherein the tumor comprises a tumor associated with abnormal transcription factors of the ETS family.

75. The use according to any one of claims 71-72 or the method according to claim 73, wherein the tumor is selected from one or more of the group consisting of Ewing sarcoma, lung cancer, breast cancer, prostate cancer, melanoma, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, and brain tumor.

76. The use according to any one of claims 71-72 or the method according to claim 73, wherein the tumor comprises lung adenocarcinoma.

77. The use according to any one of claims 71-72 or the method according to claim 73, wherein the tumor comprises drug-resistant breast cancer.

78. The use according to any one of claims 71-72 or the method according to claim 73, wherein the tumor comprises doxorubicin-resistant breast cancer.

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