Functional fragments, combinations and applications thereof for reprogramming recombinant oncolytic viruses

Abstract

The present invention provides a functional fragment, combination and use of recombinant oncolytic virus reprogramming.The present invention provides a set of transcription factors, combination of transcription factors, and a method of expressing oncolytic virus that synergistically promotes the transdifferentiation of tumor cells and reprograms them into non-tumor cells, which can achieve effective synergistic effect between oncolytic therapy and reprogramming effect, and can be effectively applied to restrict tumor progression, and further provides the application of the transcription factors carried by the recombinant oncolytic virus in the preparation of tumor disease drugs.

Description

[Technical Field]

[0001] The present invention belongs to the fields of biotechnology and gene therapy, and relates to functional fragments and combinations for reprogramming recombinant oncolytic viruses and their applications, particularly to a method for transdifferentiating tumor cells into non-tumorigenic cells using oncolytic viral vectors, and a method for treating tumors using this method. [Background technology]

[0002] A key characteristic of tumor cells is runaway cell cycle progression, which leads to malignant growth. Reprogramming can be used to induce tumor cells to differentiate into non-tumorigenic cells that no longer divide, thereby enabling tumor cells to "wash away their evil deeds and return to the right path," thereby achieving the goal of tumor treatment. The most successful example of tumor differentiation therapy to date is the use of all-trans retinoic acid to treat acute promyelocytic leukemia, which achieved a high remission rate.

[0003] Oncolytic virus therapy for tumors has attracted increasing attention. Its principle is to create specialized oncolytic viruses by genetically modifying naturally occurring viruses with low pathogenicity. These viruses selectively infect tumor cells by inactivating or deleting tumor suppressor genes in target cells, allowing them to replicate extensively within tumor cells and ultimately destroy them. This then stimulates the immune response, attracting more immune cells to continue killing residual cancer cells. Over the past few decades, oncolytic virus therapy has attracted widespread attention, and significant progress has been made in related research. Currently, a variety of oncolytic viruses, including adenovirus, herpes simplex virus, coxsackievirus, poxvirus, poliovirus, measles virus, and reovirus, have entered clinical trials. These viruses recognize and infect tumor cells, ultimately causing them to swell and destroy them, but are unable to replicate within normal living cells without causing lethal effects. This theoretically provides superior antitumor efficacy and fewer side effects.

[0004] Oncolytic viruses are now generally accepted as an important branch of immunotherapy and have proven to have great potential in basic and clinical research for the treatment of malignant tumors. In 2015, the US Food and Drug Administration and the European Medicines Agency successively approved the oncolytic HSV-1 virus T-VEC for the treatment of melanoma, demonstrating the maturity of oncolytic virus technology and the approval of oncolytic viruses for the treatment of malignant tumors. Currently, various oncolytic viruses are being used in clinical studies for tumor treatment, and their therapeutic efficacy and safety have been confirmed.

[0005] Currently, oncolytic viruses are thought to mediate their antitumor effects primarily through the following mechanisms: 1) They specifically replicate in tumor cells and directly lyse them; 2) Lysed tumor cells release viral particles, which stimulate systemic antitumor immunity through multiple pathways, such as promoting tumor antigen presentation, increasing immune cell infiltration into the tumor microenvironment, controlling the tumor microenvironment, activating immune cells, and activating the immune system through their embedded immune regulatory factors. Furthermore, some viruses have been reported to achieve indirect antitumor effects by inhibiting tumor angiogenesis through infection of tumor-associated vascular endothelial cells.

[0006] Oncolytic adenoviruses are currently the most commonly used oncolytic viruses. Many strategies have been developed to enhance tumor targeting of oncolytic adenoviruses, including targeted transcriptional regulation of the E1A gene by mutating functional genes involved in cell cycle node control in the adenoviral genome (e.g., E1A or E1B) and / or using tumor-specific promoters; targeted transduction regulation by using different serotypes of adenoviruses or RGD motifs to alter the way oncolytic adenoviruses enter tumor cells; and delivery of oncolytic adenoviruses to distant tumor sites using cellular vectors. Oncolytic adenoviruses can be used as vectors to deliver immunoregulatory or therapeutic genes, enhancing antitumor immunity or inducing tumor cell apoptosis or suicide, thereby generating synergistic antitumor effects. Current oncolytic viruses often contain immunoregulatory or suicide genes, which can further enhance the efficacy of tumor treatment, but their efficacy remains limited.

[0007] Gliomas, also known as gliomas, broadly refer to all tumors derived from neuroepithelial cells, and more narrowly refer to various tumors derived from glial cells. Gliomas are one of the most lethal malignant tumors and the most common primary central nervous system tumor, accounting for 30% of brain and central nervous system tumors and 80% of malignant brain tumors, posing a serious threat to human health. According to the World Health Organization (WHO) 1999 classification system, gliomas are classified as astrocytomas, oligodendrogliomas, ependymomas, mixed gliomas, choroid plexus tumors, neuroepithelial tumors of unknown etiology, neuronal and mixed neuronal-glial tumors, pineal parenchymal tumors, embryonal tumors, and neuroblastomas. Gliomas grow alternately with normal neural tissue, resulting in unclear boundaries, making complete tumor resection difficult and prone to recurrence. Furthermore, the presence of the blood-brain barrier limits the therapeutic efficacy of conventional antitumor agents. Treatment of glioblastoma remains an unmet clinical need in the medical community. In recent years, some studies have found that some neurogenic transcription factors or combinations of transcription factors can transform glioma cells into neuron-like cells in vitro or in vivo and limit the proliferation ability of glioma cells.Currently commonly used delivery vectors, such as adeno-associated virus (AAV) vectors, are unable to self-replicate and are unable to sufficiently introduce foreign genes into tumor cells, making them difficult to use in clinical tumor treatment.

[0008] Therefore, by searching for and utilizing an appropriate replicative expression vector, which is loaded with appropriate reprogramming factors, it infects tumor cells and replicates within the cells, the virus is released and infects more tumor cells, some tumor cells lyse and die, some tumor cells die due to anti-tumor immunity, and some surviving tumor cells differentiate into non-tumorigenic cells that no longer divide, achieving an effective synergistic effect and currently being a new therapeutic option for treating tumors. Summary of the Invention [Problem to be solved by the invention]

[0009] In view of the shortcomings of the prior art, the present invention aims to provide a functional fragment, combination, and application of recombinant oncolytic virus reprogramming. The present invention provides a set of transcription factors, a combination of transcription factors, and a method for expressing oncolytic viruses that synergistically promote the transdifferentiation of tumor cells and reprogram them into non-tumorigenic cells, as well as the application of the set of transcription factors carried by oncolytic viruses in the preparation of tumor disease drugs. [Means for solving the problem]

[0010] In order to achieve the object of the present invention, the present invention employs the following technical means.

[0011] In a first aspect of the present invention, a recombinant oncolytic virus is provided that contains a recombinant nucleic acid comprising a functional fragment that promotes reprogramming / transdifferentiation of tumor cells.

[0012] The functional fragments contain at least one functional fragment that promotes expression of a transcription factor, and the functional fragments are selected from functional fragments that can promote expression of at least one transcription factor selected from NeuroD1, Brn2, Ascl1, or Ngn2.

[0013] In another preferred embodiment, the functional fragment that promotes expression of a transcription factor contained in the recombinant oncolytic virus includes at least a functional fragment that promotes expression of an Ascl1 transcription factor.

[0014] In another preferred embodiment, the functional fragment that promotes expression of a transcription factor contained in the recombinant oncolytic virus includes at least a functional fragment that promotes expression of NeuroD1 transcription factor.

[0015] In another preferred embodiment, the functional fragment that promotes expression of a transcription factor contained in the recombinant oncolytic virus includes at least a functional fragment that promotes expression of Brn2 transcription factor.

[0016] In another preferred embodiment, the functional fragment that promotes expression of a transcription factor contained in the recombinant oncolytic virus includes at least a functional fragment that promotes expression of Ngn2 transcription factor.

[0017] In another preferred embodiment, the recombinant nucleic acid comprises a set of functional fragments that synergistically promote reprogramming / transdifferentiation of tumor cells into non-tumorigenic cells, the functional fragments comprising at least two functional fragments that promote expression of transcription factors, the functional fragments being selected from functional fragments that promote expression of transcription factors such as NeuroD1, Brn2, Ascl1, and Ngn2.

[0018] In another preferred embodiment, the functional fragment that promotes expression of a transcription factor contained in the recombinant oncolytic virus includes at least a functional fragment that promotes expression of NeuroD1 transcription factor and a functional fragment that promotes expression of another transcription factor, and the functional fragment that promotes expression of the other transcription factor is selected from any of functional fragments that promote expression of transcription factors such as Ascl1, Ngn2, or Brn2.

[0019] More preferably, the functional fragment that promotes the expression of another transcription factor is selected from any of functional fragments that promote the expression of a transcription factor such as Ascl1 or Ngn2.

[0020] In another preferred embodiment, the functional fragment that promotes expression of a transcription factor contained in the recombinant oncolytic virus includes at least a functional fragment that promotes expression of the Ngn2 transcription factor and a functional fragment that promotes expression of another transcription factor, and the functional fragment that promotes expression of the other transcription factor is selected from any of the functional fragments that promote expression of transcription factors such as NeuroD1, Brn2, and Ascl1.

[0021] More preferably, the functional fragment that promotes expression of another transcription factor is selected from any of functional fragments that promote expression of transcription factors such as Ascl1 or NeuroD1. Even more preferably, the functional fragment that promotes expression of another transcription factor is a functional fragment that promotes expression of the Ascl1 transcription factor, i.e., the recombinant oncolytic virus contains a recombinant nucleic acid comprising a functional fragment that promotes reprogramming / transdifferentiation of tumor cells, and the functional fragment simultaneously contains functional fragments that promote expression of the Ascl1 transcription factor and the Ngn2 transcription factor.

[0022] In another preferred embodiment, the functional fragment contained in the recombinant oncolytic virus that can synergistically promote reprogramming / transdifferentiation of tumor cells or the functional fragment that promotes expression of a transcription factor is a polynucleotide encoding a functional protein, and the functional protein is a functional protein of a transcription factor such as NeuroD1, Brn2, Ascl1, or Ngn2.

[0023] Preferably, the functional fragments contained in the oncolytic viruses that can synergistically promote reprogramming / transdifferentiation of glial cells or that promote expression of transcription factors are derived from mammals, more preferably mammals such as humans or non-human primates.

[0024] In another preferred embodiment, the functional fragment contained in the recombinant oncolytic virus and capable of synergistically promoting tumor cell reprogramming / transdifferentiation is a polynucleotide encoding a functional protein, the functional protein being a functional NeuroD1 protein, the amino acid sequence of the functional NeuroD1 protein being represented by SEQ ID NO. 1 or SEQ ID NO. 2, and the polynucleotide sequence encoding the functional NeuroD1 protein being represented by SEQ ID NO. 3 or SEQ ID NO. 4.

[0025] In another preferred embodiment, the functional fragment contained in the recombinant oncolytic virus and capable of synergistically promoting tumor cell transdifferentiation is a polynucleotide encoding a functional protein, the functional protein being a functional Brn2 protein, the amino acid sequence of the functional Brn2 protein being represented by SEQ ID NO.5 or SEQ ID NO.6, and the polynucleotide sequence encoding the functional Brn2 protein being represented by SEQ ID NO.7 or SEQ ID NO.8.

[0026] In another preferred embodiment, the functional fragment contained in the recombinant oncolytic virus and capable of synergistically promoting tumor cell transdifferentiation is a polynucleotide encoding a functional protein, wherein the functional protein is a functional Ascl1 protein, the amino acid sequence of the functional Ascl1 protein is represented by SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.18, and the polynucleotide sequence encoding the functional Ascl1 protein is represented by SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.19.

[0027] In another preferred embodiment, the functional fragment contained in the recombinant oncolytic virus and capable of synergistically promoting tumor cell transdifferentiation is a polynucleotide encoding a functional protein, the functional protein being a functional Ngn2 protein, the amino acid sequence of the functional Ngn2 protein being represented by SEQ ID NO. 13 or SEQ ID NO. 14, and the polynucleotide sequence encoding the functional Ngn2 protein being represented by SEQ ID NO. 15 or SEQ ID NO. 16.

[0028] In another preferred embodiment, when the functional fragment capable of synergistically promoting tumor cell transdifferentiation contained in the recombinant oncolytic virus is a polynucleotide encoding a functional protein, the amino acid sequence of the functional protein has sequence identity of 85% or more with SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.14 or SEQ ID NO.18, such as 85%, 87%, 89%, 90%, 91%, 93%, 95%, 97% or 99%.

[0029] More preferably, the amino acid sequence of the functional protein has a sequence identity of 95% or more, such as 95%, 96%, 97%, 98% or 99%, with SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.14 or SEQ ID NO.18.

[0030] Most preferably, the amino acid sequence of the functional protein has a sequence identity of 99% or more with SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.14 or SEQ ID NO.18.

[0031] In another preferred embodiment, when the functional fragment capable of synergistically promoting tumor cell transdifferentiation contained in the recombinant oncolytic virus is a polynucleotide encoding a functional protein, the sequence of the polynucleotide encoding the functional protein has a sequence identity of 75% or more, such as 75%, 77%, 79%, 80%, 85%, 87%, 89%, 90%, 91%, 93%, 95%, 97% or 99%, with SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.16 or SEQ ID NO.19.

[0032] More preferably, the polynucleotide sequence encoding the functional protein has a sequence identity of 85% or more with SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.16 or SEQ ID NO.19, such as 85%, 87%, 89%, 90%, 91%, 93%, 95%, 97% or 99%.

[0033] Most preferably, the polynucleotide sequence encoding the functional protein has a sequence identity of 95% or more, such as 95%, 96%, 97%, 98% or 99%, with SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.16 or SEQ ID NO.19.

[0034] Preferably, the expression systems for the functional fragments that promote the expression of the transcription factor are constructed under the same expression vector, or are expressed separately using different expression vectors.

[0035] Preferably, the recombinant oncolytic virus comprises a selectively replicating recombinant oncolytic virus.

[0036] Preferably, the selectively replicating recombinant oncolytic virus is derived from an oncolytic adenovirus, poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, vesicular stomatitis virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus or Maraba virus.

[0037] Preferably, the tumor cells are selected from the group consisting of glioblastoma, glioma, astrocytoma, astroblastoma, medulloblastoma, schwannoma, brain metastasis cancer, neuroblastoma, chordoma, meningioma, teratoma, spinal tumor, breast cancer, head and neck tumor, kidney cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, bile duct cancer, bladder cancer, The cancer is any of ureteral cancer, glioma, osteochondroma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, cervical cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, ovarian cancer, pancreatic endocrine tumor, glucagonoma, pancreatic cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymic cancer, trophoblastic cancer, endometrial cancer, vaginal cancer, vulvar cancer, insulinoma, blood cancer, peritoneal cancer, or pleural cancer.

[0038] Preferably, said reprogramming / transdifferentiation refers to reprogramming or transdifferentiating tumor cells into non-tumorigenic cells.

[0039] In a second aspect of the present invention, there is provided a method for promoting oncolytic virus-mediated reprogramming / transdifferentiation of tumor cells into non-tumorigenic cells, comprising the step of contacting tumor cells with a recombinant oncolytic virus according to the first aspect of the present invention, thereby reprogramming / transdifferentiating said tumor cells into non-tumorigenic cells.

[0040] In other preferred embodiments, the method is non-therapeutic and non-diagnostic.

[0041] In another preferred embodiment, the method is an in vitro method.

[0042] In another preferred embodiment, the method is an in vivo method.

[0043] In another preferred embodiment, the method is therapeutic, and can be used in conjunction with existing therapies such as immunotherapy, CAR-T, and electric field therapy (TTF) without conflicting with existing tumor therapies.

[0044] Preferably, the tumor cells are selected from the group consisting of glioblastoma, glioma, astrocytoma, astroblastoma, medulloblastoma, schwannoma, brain metastasis cancer, neuroblastoma, chordoma, meningioma, teratoma, spinal tumor, breast cancer, head and neck tumor, kidney cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, bile duct cancer, bladder cancer, The cancer is any of ureteral cancer, glioma, osteochondroma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, cervical cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, ovarian cancer, pancreatic endocrine tumor, glucagonoma, pancreatic cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymic cancer, trophoblastic cancer, endometrial cancer, vaginal cancer, vulvar cancer, insulinoma, blood cancer, peritoneal cancer, or pleural cancer.

[0045] In another preferred embodiment, any method for promoting increased expression of transcription factors for glial cell transdifferentiation includes, but is not limited to, promoting increased expression of any of NeuroD1, Brn2, Ascl1, and Ngn2 transcription factors in glial cells by directly contacting or introducing an inducer or a functional fragment that promotes expression of the transcription factor into the tumor cells, thereby promoting transdifferentiation of the tumor cells into non-tumorigenic cells.

[0046] In the present invention, the delivery system is the oncolytic virus, and the selectively replicating recombinant oncolytic virus is derived from an oncolytic virus such as adenovirus, poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, vesicular stomatitis virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus, or Maraba virus.

[0047] In another preferred embodiment, the expression vector carrying the transcription factor polynucleotide of the recombinant oncolytic virus may simultaneously carry other functional fragments, wherein the other functional fragments may be reporter genes or functional fragments of other transcription factors having reprogramming function, including, but not limited to, those selected from NeuroD1, Brn2, Ascl1, or Ngn2.

[0048] Preferably, at least two polynucleotide fragments of transcription factors may be carried in the same vector, and the polynucleotide fragments of the two transcription factors may be expressed separately under one tumor cell-specific promoter or two tumor cell-specific promoters. When two or more transcription factors are present in the transcription product of a single promoter, the promoter and the open reading frames of the multiple transcription factors are connected in tandem by a polycistronic element, and the transcription factors are separated by an IRES or polypeptide 2A (P2A) element to achieve expression of multiple transcription factors (Pharmaceutics 2019, 11(11), 580; the IRES sequence used in the present invention is copied from Addgene #69550, and the P2A sequence is copied from Addgene #130692).

[0049] In another preferred embodiment, the recombinant oncolytic virus comprises a set of functional fragments that can synergistically promote the reprogramming / transdifferentiation of tumor cells, and the expression systems of the functional fragments that promote the expression of transcription factors are constructed under the same expression vector or are expressed separately using different expression vectors.

[0050] In a third aspect of the present invention, (A) a recombinant oncolytic virus according to the first aspect; (B) a pharmaceutically acceptable excipient.

[0051] In some embodiments, the composition comprises: (C) further comprising an antitumor agent; The antitumor agent includes one or both of temozolomide and bevacizumab.

[0052] In another preferred embodiment, the pharmaceutical composition is a liquid formulation or a lyophilized formulation.

[0053] In another preferred embodiment, the pharmaceutical composition is an injection.

[0054] In a fourth aspect of the present invention there is provided the use of a recombinant oncolytic virus according to the first aspect in the preparation of a medicament for treating a tumour.

[0055] In another preferred embodiment, in the use, the recombinant oncolytic virus is formulated as a therapeutic agent to be administered into or near a tumor, and the method of administration of the therapeutic agent includes any of injection, hydrogel administration, convection-enhanced delivery (CED), Ommaya reservoir, intraperitoneal administration, intrathecal administration, or intravenous administration.

[0056] Preferably, the tumor includes glioblastoma, glioma, astrocytoma, astroblastoma, medulloblastoma, schwannoma, brain metastasis cancer, neuroblastoma, chordoma, meningioma, teratoma, spinal tumor, breast cancer, head and neck tumor, kidney cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, bile duct cancer, bladder cancer, ureter cancer, neurocarcinoma, These include glioma, osteochondroma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, cervical cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, ovarian cancer, pancreatic endocrine tumor, glucagonoma, pancreatic cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymic cancer, trophoblastic cancer, endometrial cancer, vaginal cancer, vulvar cancer, insulinoma, blood cancer, peritoneal cancer, or pleural cancer.

[0057] The numerical ranges described in the present invention include not only the numerical values ​​recited above, but also any numerical values ​​between the numerical ranges that are not recited above, and for the sake of conciseness and clarity, the present invention does not exhaustively recite specific numerical values ​​included in the ranges. [Brief explanation of the drawings]

[0058] [Figure 1A] FIG. 1A shows the killing curves of Ad5-AN oncolytic adenovirus at different multiplicities of infection against U87 cells in Example 2. [Figure 1B] FIG. 1B shows the killing curves of Ad5-AN oncolytic adenovirus at different multiplicities of infection against U118 cells in Example 2. [Figure 2A] FIG. 2A shows that Ad5-AN in Example 3 can transdifferentiate glioma cells U251 into non-tumorigenic neuronal cells. [Figure 2B] FIG. 2B shows that Ad5-AN in Example 3 can transdifferentiate glioma cells U251 into non-tumorigenic neuronal cells. [Figure 3A] FIG. 3A shows the inhibition of tumor cell growth in an ectopic inoculation model of glioma mice by the oncolytic type 5 adenoviral vector expressing the reprogramming factor in Example 4. [Figure 3B]FIG. 3B shows the results of an experiment in which the number of days of the experiment in FIG. 3A was extended. [Figure 3C] FIG. 3C shows the inhibition of tumor cell growth in an ectopic inoculation model of glioma mice by the oncolytic type 5 adenoviral vector expressing the reprogramming factor in Example 4. [Figure 3D] FIG. 3D shows the inhibition of tumor cell growth in an ectopic inoculation model of glioma mice by the oncolytic type 5 adenoviral vector expressing the reprogramming factors in Example 4. [Figure 4A] FIG. 4A shows the experimental results of Example 5 in which reprogramming factors were used in combination with an oncolytic virus expressing the reprogramming factors. [Figure 4B] FIG. 4B shows the results of an experiment in which the number of days of the experiment in FIG. 4A was extended. [Figure 5] FIG. 5 shows a parallel experiment of seven groups of temozolomide (TMZ) in Example 5, in which tumors in seven mice numbered A to G all recurred 56 days after administration (7 / 7). [Figure 6] Figure 6 shows the seven parallel groups of the Ad5-AN and temozolomide combination group in Example 5, in which tumors in seven mice numbered A to G did not recur 93 days after administration (0 / 7). DETAILED DESCRIPTION OF THE INVENTION

[0059] After extensive and detailed research, the present inventors have found that by using an oncolytic viral expression vector to deliver a transcription factor or a combination of transcription factors with transdifferentiation / reprogramming function, tumor cells can be differentiated into non-tumorigenic cells that no longer divide, either in vitro or in vivo. Based on this discovery, the present inventors further investigated the application of this method to the development of tumor drugs, and observed that, particularly in animal models of glioma, the oncolytic viral expression vector achieved a synergistic effect between oncolytic therapy and reprogramming activity, enhancing the anti-tumor effect and significantly reducing tumor size and prolonging survival time in the animals. Therefore, this batch of oncolytic viral expression vectors and a combination of transcription factors with transdifferentiation / reprogramming function are expected to be useful in the development of tumor therapeutics, particularly for glioma therapeutics.

[0060] term The term "administering" refers to the physical introduction of a product of the present invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intracerebral, intratumoral, intramuscular, subcutaneous, intraperitoneal, intrathecal, or other parenteral routes of administration such as injection or infusion.

[0061] The term "about" can refer to a value or composition that is within a tolerance of a particular value or composition as determined by one of ordinary skill in the art, and depends in part on how the value or composition is measured or determined. Typically, "about" means ±10% or ±20%. For example, about 1:1 means (1±0.2):(1±0.2) or (1±0.1):(1±0.1). As used herein, the term "reprogramming" generally refers to a process that controls or alters the biological activity of a cell, changing the cell from one biological state to another, and typically includes processes that change the fate of a cell, such as differentiation (from a progenitor cell to a terminal cell), dedifferentiation (from a terminal cell to a pluripotent stem cell), transdifferentiation (from one terminal cell to another terminal cell), retrodifferentiation (from a terminal cell to a progenitor cell), or transcommitment (from one progenitor cell to another terminal cell to which a progenitor cell naturally differentiates).

[0062] In the present invention, the term "transdifferentiation" or "reprogramming" or "transdifferentiation reprogramming" or "transdifferentiation / reprogramming" or "reprogramming / transdifferentiation" specifically refers to the process of converting one terminal cell into another terminal cell, in particular the process of converting a tumor cell into a non-tumorigenic cell.

[0063] transcription factors The present invention provides a set of transcription factors with reprogramming function, and these transcription factors and their combinations have excellent transdifferentiation ability and can be used to promote the efficiency of transdifferentiation of glial cells into neurons.

[0064] As used herein, the term "transcription factor of the present invention" refers to one or a set of transcription factors required for neuronal differentiation selected from the group consisting of NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and Otx2.

[0065] Preferably, the transcription factors of the present invention include at least two of the above transcription factors.

[0066] The NeuroD1 functional fragment is a mammalian polynucleotide encoding the Neurogenic differentiation 1 transcription factor or an expressed protein fragment thereof. NeuroD1 is a bHLH (basic helix-loop-helix) transcription factor. For example, the human NeuroD1 molecule has a GenBank ID number of 4760, and its protein sequence is represented by SEQ ID NO. 1. NCBI Reference Sequence: NM_002500.5, and its CDS sequence is represented by SEQ ID NO. 3.

[0067] A Brn2 functional fragment, also known as POU3F2, Oct7, or N-Oct3, is a polynucleotide encoding a mammalian Pou class 3 homeobox 2 transcription factor or its expressed protein fragment. Brn2 is a neuron-specific POU-III transcription factor family member. For example, the human Brn2 molecule has GenBank ID# 5454, and its protein sequence is represented by SEQ ID NO. 5. NCBI Reference Sequence: NM_005604.4, and its CDS sequence is represented by SEQ ID NO. 7.

[0068] The Ascl1 functional fragment is a polynucleotide encoding a mammalian Achaete-scute homolog 1 transcription factor or an expressed protein fragment thereof. Ascl1 is a bHLH (basic helix-loop-helix) transcription factor. For example, the human Ascl1 molecule has a GenBank ID number of 429, and its protein sequence is represented by SEQ ID NO. 9. NCBI Reference Sequence: NM_004316.4, and its CDS sequence is represented by SEQ ID NO. 11.

[0069] The Ngn2 functional fragment, also known as Neurog2, is a mammalian polynucleotide encoding the Neurogenin-2 transcription factor or its expressed protein fragment. Ngn2 is a bHLH (basic helix-loop-helix) transcription factor. For example, the human Ngn2 molecule has a GenBank ID number of 63973, and its protein sequence is represented by SEQ ID NO. 13. NCBI Reference Sequence: NM_024019.4, and its CDS sequence is represented by SEQ ID NO. 15.

[0070] Any method for promoting increased expression of transcription factors for glial cell transdifferentiation includes, but is not limited to, promoting increased expression of any of NeuroD1, Brn2, Ascl1, and Ngn2 transcription factors in glial cells by directly contacting or introducing an inducer or a functional fragment that promotes the expression of the transcription factor into the tumor cells, thereby promoting the transdifferentiation of the tumor cells into non-tumorigenic cells. The method for promoting increased expression of the functional fragment of the transcription factor can also be achieved by using CRISPR / dCas9 to target the expression of DNA-activated genes of the related transcription factor, or by using CRISPR / Cas13 to target the related transcription factor RNA and increase the expression of the transcription factor functional protein.

[0071] Those skilled in the art can screen for methods of promoting the above transcription factors based on existing databases. Based on the functions of transcription factors on tumor cell transdifferentiation disclosed in the present invention, those skilled in the art can reasonably predict that any substance that promotes the above transcription factors will function on tumor cell transdifferentiation.

[0072] Preferably, the reprogramming function transcription factors of the present invention can be used in combination with modified expression elements to further increase expression of the transcription factors of the present invention.

[0073] Oncolytic viruses The oncolytic viruses described in the present invention are replicative recombinant oncolytic viruses derived from adenovirus, poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, vesicular stomatitis virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus and Maraba virus, which have oncolytic activity.

[0074] Oncolytic viruses that can be used in the present invention are not particularly limited, but are preferably derived from adenovirus type 5, poxvirus, and herpes simplex virus type 1 or 2.

[0075] tumor cells As used herein, tumor cells include glioblastoma, glioma, astrocytoma, astroblastoma, medulloblastoma, schwannoma, brain metastasis cancer, neuroblastoma, chordoma, meningioma, teratoma, spinal cord tumor, breast cancer, head and neck tumor, kidney cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, cholangiocarcinoma, bladder cancer, and the like, derived from a human or non-human mammal. cancer, ureteral cancer, glioma, osteochondroma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, cervical cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, ovarian cancer, pancreatic endocrine tumor, glucagonoma, pancreatic cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymic cancer, trophoblastic cancer, endometrial cancer, vaginal cancer, vulvar cancer, insulinoma, blood cancer, peritoneal cancer, or pleural cancer.

[0076] Tumor cells that can be used in the present invention are not particularly limited, but preferably include various gliomas derived from the central nervous system of mammals, such as glioblastoma, astrocytoma, oligodendroglioma, ependymoma, or neuroblastoma.

[0077] Gliomas, also known as gliomas, broadly refer to all tumors derived from neuroepithelial cells, and more narrowly refer to various tumors derived from glial cells. Gliomas are one of the most lethal malignant tumors and the most common primary central nervous system tumor, accounting for 30% of brain and central nervous system tumors and 80% of malignant brain tumors, posing a serious threat to human health. According to the 1999 World Health Organization (WHO) classification system, gliomas are divided into astrocytomas, oligodendrogliomas, ependymomas, mixed gliomas, choroid plexus tumors, neuroepithelial tumors, neuronal and mixed neuronal-glial tumors, pineal parenchymal tumors, embryonal tumors, and neuroblastoma tumors.

[0078] Pharmaceutical compositions and methods of administration The present invention further provides a pharmaceutical composition for treating cancer, the pharmaceutical composition comprising a recombinant oncolytic virus comprising a combination of functional fragments that promote expression of a transdifferentiation transcription factor in tumor cells, and a pharmaceutically acceptable vector.

[0079] A pharmaceutically acceptable vector refers to a vector for administration of a therapeutic agent, including various excipients and diluents.

[0080] The pharmaceutical composition of the present invention generally contains 10 6 or 10 9 PFU / g of adenovirus type 5 particles, preferably 10 6 or 10 8 PFU / g of adenovirus type 5 particles, more preferably 10 7 or 10 8 Contains PFU / g of adenovirus type 5 particles.

[0081] The pharmaceutical composition of the present invention generally contains 10 5 or 10 8 PFU / g of herpes simplex virus type 1 particles, preferably 10 6 or 10 8 PFU / g of herpes simplex virus type 1 particles, more preferably 10 6 or 10 7 Contains PFU / g of herpes simplex virus type 1 particles.

[0082] A pharmaceutically acceptable vector may include any one or a combination of at least two of a coating layer, capsule, microcapsule, or nanocapsule. It should be noted that the vector must be non-toxic and not significantly affect the activity of the main component in the composition (e.g., the oncolytic virus or the molecule with anti-cancer promoting activity expressed by the oncolytic virus). In some embodiments, the vector can protect the main component in the composition to reduce or avoid inactivation or degradation of the main component under some negative conditions (e.g., oxidation, denaturation by strong acid or strong base, etc.). For example, enzymes in gastric juice or a relatively low pH value may result in degradation or inactivation of the main component. By protecting the main component in the composition, the vector can help maintain or improve the efficacy of the pharmaceutical composition.

[0083] In some embodiments, the vector can be used for controlled release of the main component (e.g., an oncolytic virus). Controlled release can include, but is not limited to, sustained release, sustained release, or targeted release. For example, the vector can include a hydrogel capsule, microcapsule, or nanocapsule made from any one or a combination of at least two of collagen, gelatin, chitosan, alginate, polyvinyl alcohol, polyethylene oxide, starch, or cross-linked starch.

[0084] In some examples, pharmaceutically acceptable vectors can include dispersion media (e.g., solvents), coatings, buffers, stabilizing formulations, isotonicity or absorption delaying agents, etc. Exemplary pharmaceutically acceptable vectors can include phosphate buffered saline, water, emulsions (e.g., oil-water emulsions), various types of wetting agents, sterile solutions, gels or bioresorbable matrix materials, or other suitable materials, or any combination thereof.

[0085] In some embodiments, the excipient includes, but is not limited to, water, saline, polyethylene glycol, hyaluronic acid, ethanol, a pharmaceutically acceptable salt, such as a salt of an inorganic acid (e.g., hydrochloride, hydrobromide, phosphate, or sulfate) or a salt of an organic acid (e.g., acetate, propionate, or benzoate).

[0086] Generally, the pharmaceutical composition of the present invention can be obtained by mixing the expression vector with a pharmaceutically acceptable vector.

[0087] The method of administration of the compositions described in the present invention is not particularly limited, and representative examples include, but are not limited to, intratumoral or peritumoral injection, hydrogel administration, convection-enhanced delivery (CED), Ommaya reservoir, intraperitoneal administration, intrathecal administration, or intravenous administration.

[0088] The pharmaceutical composition can be administered to a subject, such as a human or animal, with cancer. In some embodiments, the pharmaceutical composition can be administered to a subject by one or more administration methods. The one or more administration methods include, but are not limited to, oral administration, injection administration, or topical administration. Composition forms suitable for oral administration include, but are not limited to, tablets, liposome formulations, sustained-release capsules, microparticles, microspheres, or any other suitable form. Composition forms suitable for injectable administration include, but are not limited to, sterile aqueous solutions or oily formulations. Composition forms suitable for topical administration include, but are not limited to, sterile aqueous solutions or oily formulations, suspensions, or emulsions. For nasal administration, for example, the composition forms can include aerosols, mists, powders, solutions, suspensions, gels, etc.

[0089] In some embodiments, the pharmaceutical composition can be stored at an appropriate temperature, including room temperature (about 20°C), 40°C, -20°C, and -80°C. The composition can also be formulated into various forms, such as powder, for ease of storage and transportation. The powder may be a sterile powder, and a solvent can be added to the sterile powder and mixed uniformly before use to prepare a solution for oral, injectable, or topical administration. In some embodiments, the pharmaceutical composition may further include a component that has antibacterial activity but does not significantly adversely affect the viability of the oncolytic virus, such that the pharmaceutical composition is stable under certain storage conditions, such as refrigeration and freezing, and can prevent contamination by microorganisms such as bacteria and fungi.

[0090] Therapeutic applications The recombinant oncolytic virus according to the present invention comprises any molecular entity that promotes the expression or enhanced activity of a functional fragment of said transcription factor, or comprises a delivery system for a molecular entity with an enhanced activity or functional fragment of said transcription factor, and can be used for the preparation of an antitumor drug.

[0091] The compositions of the present invention can be used before or after the administration of other pharmaceutical compositions for treating cancer. If desired, the compositions disclosed in the present invention can be combined with other therapeutic methods to treat cancer in a subject. For example, other therapeutic methods include, but are not limited to, administering to a subject other pharmaceutical compositions capable of treating cancer, surgically removing a tumor in a subject, radiation therapy, or electric field therapy. Specifically, pharmaceutical compositions for treating cancer include, but are not limited to, cytotoxic anticancer agents and / or non-cytotoxic anticancer agents. Non-cytotoxic anticancer agents may include hormonal agents, targeted agents (e.g., bevacizumab), or immunotherapeutic agents (e.g., monoclonal antibodies and / or tumor vaccines).

[0092] Effect of the invention After extensive and detailed research, the present inventors have found that by using an oncolytic viral expression vector to deliver a transcription factor or a combination of transcription factors with transdifferentiation / reprogramming function, tumor cells can be differentiated into non-tumorigenic cells that no longer divide, either in vitro or in vivo. Based on this discovery, the present inventors further investigated the application of this method to the development of tumor drugs, and observed that, particularly in animal models of glioma, the oncolytic viral expression vector achieved a synergistic effect between oncolytic therapy and reprogramming activity, enhancing the anti-tumor effect and significantly reducing tumor size and prolonging survival time in the animals. Therefore, this batch of oncolytic viral expression vectors and a combination of transcription factors with transdifferentiation / reprogramming function are expected to be useful in the development of tumor therapeutics, particularly for glioma therapeutics.

[0093] Compared with the prior art, the present invention mainly has the following competitive advantages: By innovatively utilizing oncolytic viruses in combination with reprogramming functions, transcription factors or combinations with reprogramming functions that are carried and expressed by oncolytic viruses and recombinant oncolytic viruses are obtained, and the ability of transcription factors and combinations thereof to transdifferentiate tumor cells into non-tumorigenic cells is explored; such recombinant oncolytic viruses have efficient tumor-killing and tumor-suppressing effects, which can better treat tumors and prevent tumor recurrence.

[0094] The technical solutions of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the above examples are only intended to help understand the present invention and are not intended to specifically limit the present invention.

[0095] Experimental methods for which no specific conditions are given in the following examples generally follow conventional conditions, e.g., those described in Sambrook et al., Molecular Cloning. A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or those recommended by the manufacturer.

[0096] General-purpose method Materials and Methods Amino acid sequence SEQ ID NO. 1 (hNeuroD1 amino acid sequence) MTKSYSESGLMGEPQPQGPPSWTDECLSSQDEEHEADKKEDDLETMNAEEDSLRNGGEEEDEDEDLEEEEEEEEEDDDQKPKRRGPKKKKMTKARLERFKLRRMKANARERNRMHGLNAALDNLRKVVPCYSKTQKLSKIETLRLAKNYIWALSEILRSGKSPDLVSFVQTLCKGLSQPTTNLVAGCLQLNPRTFLPEQNQDMPPHLPTASASFPVHPYSYQSPGLPSPPYGTMDSSHVFHVKPPPHAYSAALEPFFESPLTDCTSPSFDGPLSPPLSINGNFSFKHEPSAEFEKNYAFTMHYPAATLAGAQSHGSIFSGTAAPRCEIPIDNIMSFDSHSHHERVMSAQLNAIFHD.

[0097] SEQ ID NO. 2 (mNeuroD1 amino acid sequence) MTKSYSESGLMGEPQPQGPPSWTDECLSSQDEEHEADKKEDELEAMNAEEDSLRNGGEEEEEDEDLEEEEEEEEEEEDQKPKRRGPKKKKMTKARLERFKLRRMKANARERNRMHGLNAALDNLRKVVPCYSKTQKLSKIETLRLAKNYIWALSEILRSGKSPDLVSFVQTLCKGLSQPTTNLVAGCLQLNPRTFLPEQNPDMPPHLPTASASFPVHPYSYQSPGLPSPPYGTMDSSHVFHVKPPPHAYSAALEPFFESPLTDCTSPSFDGPLSPPLSINGNFSFKHEPSAEFEKNYAFTMHYPAATLAGPQSHGSIFSSGAAAPRCEIPIDNIMSFDSHSHHERVMSAQLNAIFHD.

[0098] SEQ ID NO. 3 (hNeuroD1 nucleotide sequence)

[0099] SEQ ID NO. 4 (mNeuroD1 nucleotide sequence)

[0100] SEQ ID NO. 5 (hBrn2 amino acid sequence) MATAASNHYSLLTSSASIVHAEPPGGMQQGAGGYREAQSLVQGDYGALQSNGHPLSHAHQWITALSHGGGGGGGGGGGGGGGGGGGDGSPWSTSPLGQPDIKPSVVVQQ GGRGDELHGPGALQQQHQQQQQQQQQQQQQQQQQQQQQRPPHLVHHAANHHPGPGAWRSAAAAAHLPPSMGASNGGLLYSQPSFTVNGMLGAGGQPAGLHHHGLRDAHDEPH HADHHPHPHSHPHQQPPPPPPPQGPPGHPGAHHDPHSDEDTPTSDDLEQFAKQFKQRRIKLGFTQADVGLALGTLYGNVFSQTTICRFEALQLSFKNMCKLKPLLNKWLEEADSSSGSPTSIDKIAAQGRKRKKRTSIEVSVKGALESHFLKCPKPSAQEITSLADSLQLEKEVVRVWFCNRRQKEKRMTPPGGTLPGAEDVYGGSRDTPPHHGVQTPVQ.

[0101] SEQ ID NO. 6 (mBrn2 amino acid sequence) MATAASNHYSLLTSSASIVHAEPPGGMQQGAGGYREAQSLVQGDYGALQSNGHPLSHAHQWITALSHGGGGGGGGGGGGGGGGGGGDGSPWSTSPLGQPDIKPSVVVQQG GRGDELHGPGALQQQHQQQQQQQQQQQQQQQQQQQQQQRPPHLVHHAANHHPGPGAWRSAAAAAHLPPSMGASNGGLLYSQPSFTVNGMLGAGGQPAGLHHHGLRDAHDEP HHADHHPHPHSHPHQQPPPPPPPQGPPGHPGAHHDPHSDEDTPTSDDLEQFAKQFKQRRIKLGFTQADVGLALGTLYGNVFSQTTICRFEALQLSFKNMCKLKPLLNKWLEEADSSSGSPTSIDKIAAQGRKRKKRTSIEVSVKGALESHFLKCPKPSAQEITSLADSLQLEKEVVRVWFCNRRQKEKRMTPPGGTLPGAEDVYGGSRDTPPHHGVQTPVQ.

[0102] SEQ ID NO. 7 (hBrn2 nucleotide sequence)

[0103] SEQ ID NO. 8 (mBrn2 nucleotide sequence)

[0104] SEQ ID NO. 9 (hAscl1 amino acid sequence) MESSAKMESGGAGQQPQPQPQQPFLPPAACFFATAAAAAAAAAAAAAQSAQQQQQQQQQQAPQLRPAADGQPSGGGHKSAPKQVKRQRSSSPELMRCKRRLNFSGFGYSLPQQQPAAV ARRNERERNRVKLVNLGFATLREHVPNGAANKKMSKVETLRSAVEYIRALQQLLDEHDAVSAAFQAGVLSPTISPNYSNDLNSMAGSPVSSYSSDEGSYDPLSPEEQELLDFTNWF.

[0105] SEQ ID NO. 10 (mAscl1 amino acid sequence) MESSGKMESGAGQQPQPPQPFLPPAACFFATAAAAAAAAAAAAQSAQQQQPQAPPQQAPQLSPVADSQPSGGGHKSAAKQVKRQRSSSPELMRCKRRLNFSGFGYSLPQQQPAAVARRNERERNRVKLVNLGFATLREHVPNGAANKKMSKVETLRSAVEYIRALQQLLDEHDAVSAAFQAGVLSPTISPNYSNDLNSMAGSPVSSYSSDEGSYDPLSPEEQELLDFTNWF.

[0106] SEQ ID NO. 11 (hAscl1 nucleotide sequence) atggaaagctctgccaagatggagagcggcggcgccggccagcagccccagccgcagccccagcagcccttcctgccgcccgcagcctgtttctttgccacggccgcagccgcggcggccgcagccgccgcagcggcagcgcagagcgcgcagcagcagcagcagcagcagcagcagcagcagcaggcgccgcagctgagaccggcggccgacggccagccctcagggggcggtcacaagtcagcgcccaagcaagtcaagcgacagcgctcgtcttcgcccgaactgatgcgctgcaaacgccggctcaacttcagcggctttggctacagcctgccgcagcagcagccggccgccgtggcgcgccgcaacgagcgcgagcgcaaccgcgtcaagttggtcaacctgggctttgccacccttcgggagcacgtccccaacggcgcggccaacaagaagatgagtaaggtggagacactgcgctcggcggtcgagtacatccgcgcgctgcagcagctgctggacgagcatgacgcggtgagcgccgccttccaggcaggcgtcctgtcgcccaccatctcccccaactactccaacgacttgaactccatggccggctcgccggtctcatcctactcgtcggacgagggctcttacgacccgctcagccccgaggagcaggagcttctcgacttcaccaactggttctga is as follows.

[0107] SEQ ID NO. 12 (mAscl1 nucleotide sequence) is atggagagctctggcaagatggagagtggagccggccagcagccgcagcccccgcagcccttcctgcctcccgcagcctgcttctttgcgaccgcggcggcggcggcagcggcggcggccgcggcagctcagagcgcgcagcagcaacagccgcaggcgccgccgcagcaggcgccgcagctgagcccggtggccgacagccagccctcagggggcggtcacaagtcagcggccaagcaggtcaagcgccagcgctcgtcctctccggaactgatgcgctgcaaacgccggctcaacttcagcggcttcggctacagcctgccacagcagcagccggccgccgtggcgcgccgcaacgagcgcgagcgcaaccgggtcaagttggtcaacctgggttttgccaccctccgggagcatgtccccaacggcgcggccaacaagaagatgagcaaggtggagacgctgcgctcggcggtcgagtacatccgcgcgctgcagcagctgctggacgagcacgacgcggtgagcgctgcctttcaggcgggcgtcctgtcgcccaccatctcccccaactactccaacgacttgaactctatggcgggttctccggtctcgtcctactcctccgacgagggatcctacgaccctcttagcccagaggaacaagagctgctggactttaccaactggttctga is as follows.

[0108] SEQ ID NO. 13 (hNgn2 amino acid sequence) is MFVKSETLELKEEEDVLVLLGSASPALAALTPLSSSADEEEEEEPGASGGARRQRGAEAGQGARGGVAAGAEGCRPARLLGLVHDCKRRPSRARAVSRGAKTAETVQRIKKTRRLKANNRERNRMHNLNAALDALREVLPTFPEDAKLTKIETLRFAHNYIWALTETLRLADHCGGGGGGLPGALFSEAVLLSPGGASAALSSSGDSPSPASTWSCTNSPAPSSSVSSNSTSPYSCTLSPASPAGSDMDYWQPPPPDKHRYAPHLPIARDCI.

[0109] SEQ ID NO. 14 (mNgn2 amino acid sequence) MFVKSETLELKEEEEVLMLLGSASPASATLTPMSSSADEEEDEELRRPGSARGQRGAEAGQGVQGSPASGAGGCRPGRLLGLMHECKRRPSRSRAVSRGAKTAETVQRIKKTRRLKANNRERNRMHNLNAALDALREVLPTFPEDAKLTKIETLRFAHNYIWALTETLRLADHCAGAGGLQGALFTEAVLLSPGAALGASGDSPSPPSSWSCTNSPASSSNSTSPYSCTLSPASPGSDVDYWQPPPPEKHRYAPHLPLARDCI.

[0110] SEQ ID NO. 15 (hNgn2 nucleotide sequence) atgttcgtcaaatccgagaccttggagttgaaggaggaagaggacgtgttagtgctgctcggatcggcctcccccgccttggcggccctgaccccgctgtcatccagcgccgacgaagaagaggaggaggagccgggcgcgtcaggcggggcgcgtcggcagcgcggggctgaggccgggcagggggcgcggggcggcgtggctgcgggtgcggagggctgccggcccgcacggctgctgggtctggtacacgattgcaaacggcgcccttcccgggcgcgggccgtctcccgaggcgccaagacggccgagacggtgcagcgcatcaagaagacccgtagactgaaggccaacaaccgcgagcgaaaccgcatgcacaacctcaacgcggcactggacgcgctgcgcgaggtgctccccacgttccccgaggacgccaagctcaccaagatcgagaccctgcgcttcgcccacaactacatctgggcactcaccgagaccctgcgcctggcggatcactgcgggggcggcggcgggggcctgccgggggcgctcttctccgaggcagtgttgctgagcccgggaggagccagcgccgccctgagcagcagcggagacagcccctcgcccgcctccacgtggagttgcaccaacagccccgcgccgtcctcctccgtgtcctccaattccacctccccctacagctgcactttatcgcccgccagcccggccgggtcagacatggactattggcagcccccacctcccgacaagcaccgctatgcacctcacctccccatagccagggattgtatctag is as follows.

[0111] SEQ ID NO. 16 (mNgn2 nucleotide sequence) is atgttcgtcaaatctgagactctggagttgaaggaggaagaggaggtactgatgctgctgggctcggcttccccggcctcggcgaccctgaccccgatgtcctccagcgcggacgaggaggaggacgaggagctgcgccggccgggctccgcgcgtgggcagcgtggagcggaagccgggcagggggtgcagggcagtccggcgtcgggtgccgggggttgccggccagggcggctgctgggcctgatgcacgagtgcaagcgtcgcccgtcgcgctcacgggccgtctcccgaggtgccaagacggcggagacggtgcagcgcatcaagaagacccgcaggctcaaggccaacaaccgcgagcgcaaccgcatgcacaacctaaacgccgcgctggacgcgctgcgcgaggtgctgcccaccttccccgaggatgccaagctcacgaagatcgagacgctgcgcttcgcccacaattacatctgggcgctcaccgagactctgcgcctggcggaccactgcgccggcgccggtggcctccagggggcgctcttcacggaggcggtgctcctgagcccgggagctgcgctcggcgccagcggggacagcccttctccaccttcctcctggagctgcaccaacagcccggcgtcatcctccaactccacgtccccatacagctgcactttatcgcccgctagccccgggtcagacgtggactactggcagcccccacctccggagaagcatcgttatgcgcctcacctgcccctcgccagggactgtatctagである。

[0112] SEQ ID NO.17は、

[0113] SEQ ID NO. 18 (h-SA-Ascl1 amino acid sequence) MESSAKMESGGAGQQPQPQPQQPFLPPAACFFATAAAAAAAAAAAAAQSAQQQQQQQQQQAPQLRPAADGQPSGGGHKSAPKQVKRQRSSAPELMRCKRRLNFSGFGYSLPQQQPAAV ARRNERERNRVKLVNLGFATLREHVPNGAANKKMSKVETLRSAVEYIRALQQLLDEHDAVSAAFQAGVLAPTIAPNYSNDLNSMAGAPVSSYSSDEGSYDPLAPEEQELLDFTNWF.

[0114] SEQ ID NO. 19 (h-SA-Ascl1 nucleotide sequence) atggaaagctctgccaagatggagagcggcggcgccggccagcagccccagccgcagccccagcagcccttcctgccgcccgcagcctgtttctttgccacggccgcagccgcggcggccgcagccgccgcagcggcagcgcagagcgcgcagcagcagcagcagcagcagcagcagcagcagcaggcgccgcagctgagaccggcggccgacggccagccctcagggggcggtcacaagtcagcgcccaagcaagtcaagcgacagcgctcgtctgcacccgaactgatgcgctgcaaacgccggctcaacttcagcggctttggctacagcctgccgcagcagcagccggccgccgtggcgcgccgcaacgagcgcgagcgcaaccgcgtcaagttggtcaacctgggctttgccacccttcgggagcacgtccccaacggcgcggccaacaagaagatgagtaaggtggagacactgcgctcggcggtcgagtacatccgcgcgctgcagcagctgctggacgagcatgacgcggtgagcgccgccttccaggcaggcgtcctggcacccaccatcgcacccaactactccaacgacttgaactccatggccggcgcaccggtctcatcctactcgtcggacgagggctcttacgacccgctcgcacccgaggagcaggagcttctcgacttcaccaactggttctga is as follows.

[0115] Cell culture Human glioma cell lines U251 and U87 (purchased from the Shanghai Institutes for Biological Sciences Cell Bank, Chinese Academy of Sciences) and normal human astrocyte HA cells (purchased from Sciencell) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in a 5% CO2 incubator. After virus infection, the entire medium was replaced with induction medium (DMEM, 2% B-27, 1% PS) 12 h after infection, and then replaced again with neural culture medium (DMEM / F-12, 2% B-27, 1% PS, 20 ng / mL BDNF, 20 ng / mL GDNF) 48 h later. Half of the medium was then replaced every 3 days.

[0116] Immunocolorization Immunostaining of cultured cells was performed according to the published method, "Direct conversion of fibroblasts to functional neurons by defined factors" (Vierbuchen, T. et al. Nature 463, 1035-1041 (2010)). Immunostaining of tissue sections was performed according to the published method. Primary antibodies used for immunostaining included mouse anti-NeuN (Millipore, 1:100), rabbit anti-Dsred (Clontech, 1:500), mouse anti-Tuj1 (Covance, 1:500), mouse anti-Map2 (Sigma, 1:500), rabbit anti-GFP (Invitrogen, 1:1,000), chicken anti-GFP (Invitrogen, 1:1,000), rabbit anti-Ki67 (1:200; RM-9106; Thermo Fisher Scientific), and mouse anti-BrdU (1:200; B2531; Sigma). FITC-, Cy3-, and Cy5-conjugated secondary antibodies were purchased from Jackson Immunoresearch.

[0117] MTT experiment method On day 1, the cell density was increased to 5 x 10 4The medium was removed on day 2, and the corresponding virus was added to a 96-well plate at a multiplicity of infection (MOI) of 5 × 10 cells / mL, with the MOI value being a multiple of 10. 3 The cells were infected with serial dilutions of 100 μL / well, with each MOI set to three replicates. On day 7, the supernatant was gently removed, and 50 μL of medium was added to each well, followed by 20 μL of 5 mg / mL MTT solution. After 3 hours, 100 μL of lysis solution was added, and the formazan crystals were dissolved overnight at 37°C. The OD was measured at 570 nm.

[0118] Glioma model In the subcutaneous glioma model, human U87 human brain glioma cells were cultured and inoculated into the armpits of nude mice during the logarithmic growth phase and passaged twice. Tumor masses from tumor-bearing mice were removed under sterile conditions, cut into uniformly sized rice grain-sized masses, and inoculated into the armpits of nude mice using an insert needle until the tumors reached 100 mm. 3 Once tumors reached a certain growth level, nude mice with appropriate tumor masses were selected and randomly divided into groups, and then administration began. All samples were dissolved in PBS and injected intratumorally in a volume of 50 μL / tumor. The length and width of the tumor mass were measured every 3 days, and the tumor volume was calculated using the following formula: Volume = (length x width 2 ) / 2

[0119] The tumor inhibition rate is calculated using the following formula: Tumor inhibition rate % = (V model group - V administration group) / V model group × 100%

[0120] The animals were then sacrificed and the tumor masses were harvested, weighed, and subjected to biochemical and molecular testing.

[0121] The mice used for the orthotopic glioma transplantation model were 7-week-old NOD-scid mice. Human glioma cells were either induced or not to be digested with 0.25% trypsin for 3 days, and the supernatant was removed by centrifugation to concentrate the cells to a density of approximately 2.5 × 10 cells.5 Each mouse brain striatum received 2 μL of the solution, for a total of 5 × 10 cells / μL. 5 Histochemistry was performed 3 weeks after transplantation or 1 week after virus injection followed by immunohistochemistry.

[0122] Example 1 Vector construction and packaging of recombinant oncolytic virus (i.e., oncolytic virus containing recombinant nucleic acid) strains Among the recombinant oncolytic viruses in this example, adenovirus type 5 (Ad5) was used as an example for verification. In the recombinant oncolytic adenovirus genome, the human GFAP promoter sequence (nucleotide sequence shown in SEQ ID NO. 17) was inserted into the human Ascl1 (SEQ ID NO. 11) fragment and the CDS (SEQ ID NO. 15) fragment derived from the human Ngn2 gene, instead of the endogenous promoter of the wild-type E1A gene, to construct the vector. P2A is a self-cleaving polypeptide, which achieved efficient co-expression of hAscl1 and Ngn2. Ad5-AN was obtained, and the empty vector Ad5-Vector was used as the experimental control vector.

[0123] Adenovirus packaging and purification were performed by cotransfection of the adenovirus packaging backbone plasmid and shuttle plasmid into HEK-293 cells. After the cells reached a plaque state, the cell supernatant and lysate were collected and the virus was purified by concentration or cesium chloride density gradient centrifugation. The adenovirus titer was measured by enzyme immunoassay and calculated by counting the number of brown-stained positive cells after infection.

[0124] Example 2 Selective killing of cancer cells by recombinant oncolytic viruses Among the tumor cells in this example, human glioblastoma cells were used as an example. Glioma cell lines U251 and U87, and normal human astrocyte HA were seeded into a T25 tissue culture flask, and the complete culture medium was DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. On day 1, the cell density was adjusted to 5 × 10 4 The medium was removed on day 2, and the corresponding virus (Ad5-vector, Ad5-AN) was added at 5 × 10 to give an MOI of 10. 3 The cells were infected with serial dilutions of 100 μL / well, with each MOI set to three replicates. On day 7, the supernatant was gently removed, and 50 μL of medium was added to each well, followed by 20 μL of 5 mg / mL MTT solution. After 3 hours, 100 μL of lysis solution was added, and the formazan crystals were dissolved overnight at 37°C. The OD was measured at 570 nm.

[0125] The results are shown in Figures 1A and 1B, which show the killing curves of Ad5-AN oncolytic adenovirus at different multiplicities of infection (MOI) in U87 and U118 cells. The IC50 values ​​for Ad5-AN oncolytic adenovirus were 0.9 and 0.6, respectively, demonstrating excellent tumor-killing ability. The IC50 values ​​for normal human astrocyte HA were 136, demonstrating excellent specificity and safety, and providing a wide spatial range for subsequent dosing. The IC50 values ​​for Ad5-vector oncolytic adenovirus were 0.8 and 0.7, respectively, in U87 and U118 cells, and 116, respectively, demonstrating similar specific tumor-killing ability.

[0126] Example 3 Reprogramming effect of recombinant oncolytic viruses on cancer cells In this example, we used the combination of Ascl1 and Ngn2 factors with Ad5-AN oncolytic adenovirus as an example to examine how a transcription factor or combination of transcription factors with high transformation efficiency carried by a recombinant oncolytic virus can promote the transdifferentiation of glioma cells into non-tumorigenic cells.

[0127] After seeding and culturing for 24 h, adenovirus was added. To observe the effect of adenovirus on the transdifferentiation of glioma cells to nontumorigenic cells, a low titer infection mode (MOI: 0.05) was used. To better visualize infected cells, the tumor cells were co-infected with the lentivirus FUGW-IRES-EGFP carrying green fluorescent protein. 24 h after infection, the medium (DMEM / F12, B27, Glutamax, and penicillin / streptomycin) was changed. Brain-derived neurotrophic factor (BDNF; PeproTech, 20 ng / mL) was added to the medium every 3 days. Ten days after virus infection of cultured human U251 glioma cells, immunofluorescence detection revealed the appearance of some Tuj1-positive cells (Tuj1 is a neuronal marker molecule) and neuronal morphology. As shown in Figures 2A and 2B, these results indicate that Ad5-AN can transdifferentiate U251 glioma cells into non-tumorigenic neurons with a transduction efficiency of 75.2%. Immunofluorescence detection of Ad5-vector-infected glioma cells did not reveal any Tuj1-positive cells, indicating that the promotion of glioma cell transdifferentiation into non-tumorigenic neurons is specifically mediated by the combination of Ascl1 and Ngn2.

[0128] Example 4 Oncolytic type 5 adenoviral vectors expressing reprogramming factors inhibit tumor cell growth in an ectopic inoculation model of glioma in mice To verify the combined effect of oncolytic viruses and redifferentiation therapy, reprogramming factors are expressed using oncolytic virus (oncolytic type 5 adenovirus as an example) vectors and propagated within tumor cells via the specificity of oncolytic type 5 adenovirus, thereby achieving a synergistic effect between the oncolytic effect and in vivo redifferentiation therapy in suppressing glioma.

[0129] In this example, a human brain glioma U87 BALB / CA-nu mouse ectopic inoculation model was adopted. Cultured U87 human brain glioma cells were inoculated into the armpits of nude mice in the logarithmic growth phase, and tumors were grown to 100 mm 3 When tumors grew to about 100%, nude mice with suitable tumor masses were selected and randomly divided into groups. After grouping, administration was initiated. The control group was the PBS group, and the Ad5-AN-low group was administered at a dose of 3 × 10 8 PFU, and the dose of Ad5-vector-high and Ad5-AN-high groups was 1 × 10 9 The dose was 1000 PFU and administered every other day for 5 consecutive doses. The tumor mass volume was measured and calculated every 3 days, and then the animals were sacrificed, and the tumor mass was collected and weighed for biochemical and molecular testing.

[0130] The results are shown in Figures 3A, 3B, and 3C. Figures 3A, 3B, and 3C show the inhibition of tumor cell growth in ectopic inoculation of glioma mice by oncolytic type 5 adenoviral vectors expressing reprogramming factors. Compared with the control PBS group, tumor volume was reduced by 33.37% in the Ad5-vector-high group, 32.25% in the Ad5-AN-low group, and 67.49% in the Ad5-AN-high group (Figure 3A). The addition of reprogramming factors significantly enhanced the inhibitory effect of oncolytic adenovirus on gliomas and significantly reduced tumor cell growth. Real-time PCR analysis also revealed significantly increased expression of the early neuronal marker molecule DCX in Ad5-AN-high cells (Figure 3B), and HE staining also demonstrated inhibition of glioma growth (Figure 3C). Mouse tumors reached 2000 mm 3Once the tumor growth reached a critical stage, the animals were sacrificed. The mean days were 21.3 for the control PBS group, 24.5 for the Ad5 empty vector group, 23.6 for the Ad5-AN-low group, and 35.4 for the Ad5-AN-high group (Figure 3A-2). These results demonstrate that the synergistic effect of oncolytic activity and in vivo reprogramming therapy significantly inhibits glioma growth.

[0131] Furthermore, for the human Ascl1 protein, mutating five conserved serine-proline (SP) phosphorylation sites in its protein sequence (located at positions 93, 190, 194, 207, and 223 of the protein sequence, respectively) to alanine-proline (AP) (enhancer, protein sequence SEQ ID NO 18, nucleotide sequence SEQ ID NO 19) can further enhance the inhibitory ability of Ad5-AN against glioma.

[0132] Example 5 Combination experiments with oncolytic viruses expressing reprogramming factors To investigate the combined effects of oncolytic viruses expressing reprogramming factors with existing treatments, this example uses human glioblastoma cells as an example, expressing reprogramming factors using oncolytic virus (oncolytic type 5 adenovirus as an example) vectors, allowing them to proliferate into tumor cells via the specificity of oncolytic type 5 adenovirus, and then administering them in combination with temozolomide.

[0133] In this example, a human brain glioma U87 BALB / CA-nu mouse ectopic inoculation model was adopted. Cultured U87 human brain glioma cells were inoculated into the armpits of nude mice in the logarithmic growth phase, and tumors were grown to 100 mm 3 When tumors grew to a certain extent, nude mice with suitable tumor masses were selected and randomly divided into groups. After grouping, administration was started. The control group was the PBS group, and the Ad5-AN group was administered at a dose of 1 × 10 9The animals were treated with intragastric temozolomide (TMZ) (15 mg / kg, once daily, 5 doses with 2 doses off), and the Ad5-AN group was administered with temozolomide (Ad5-AN + TMZ). Ad5-AN was administered every 2 days for 5 consecutive doses. The tumor volume was measured and calculated every 3 days, after which the animals were sacrificed, and the tumor masses were collected, weighed, and subjected to biochemical and molecular testing.

[0134] The results are shown in Figure 4. Figure 4A shows the combination experiment using an oncolytic virus expressing a reprogramming factor. Compared with the control PBS group, the Ad5-AN group and the temozolomide group showed significantly reduced tumor volume. However, the tumor volume in the Ad5-AN and temozolomide combination group was significantly reduced compared with the single-agent effects of the Ad5-AN and temozolomide groups, with the tumors essentially disappearing (4 / 6). Furthermore, after an extension experiment (Figure 4B), tumors in the temozolomide group gradually recurred and grew again around 35 days after administration. In particular, tumors in all seven mice in the parallel experiment recurred after 56 days (Figure 5, 7 / 7). Around 60 days later, tumor growth reached 2000 mm 3 This was consistent with the clinically observed high recurrence rate of gliomas after temozolomide chemotherapy. The tumor volume in the Ad5-AN and temozolomide combination group was significantly more effective than either the Ad5-AN or temozolomide groups alone, with tumors essentially disappearing (7 / 7). More importantly, after 93 days, none of the tumors in the seven parallel experimental groups had recurred (Figure 6, 0 / 7). These results suggest that oncolytic viruses expressing reprogramming factors can achieve superior antitumor efficacy in combination with existing therapies.

[0135] Example 6 Orthotopic tumor model of oncolytic viruses expressing reprogramming factors To further confirm the therapeutic potential of oncolytic viruses expressing reprogramming factors against tumors, this example uses a human glioblastoma cell brain orthotopic model as an example. Reprogramming factors are expressed using an oncolytic virus (oncolytic type 5 adenovirus as an example) vector, and are propagated into tumor cells via the specificity of oncolytic type 5 adenovirus. The synergistic effect of oncolytic activity and in vivo transdifferentiation in inhibiting gliomas was verified.

[0136] In this example, first, glioma cells (U87-luc) were transplanted into the brain (5 × 10 5 ) was performed. Seven days after transplantation, the mice were divided into groups and administered the PBS group as a control, and the Ad5-AN group was administered at a dose of 1 × 10 9 The mice were treated with PFU, intragastric temozolomide (15 mg / kg, once daily, five doses with two breaks), and the Ad5-AN and temozolomide combination group. Ad5-AN was administered every four days for three consecutive doses. Thirty days after virus injection, brain tissue samples from the Ad5-AN group were analyzed for immunohistochemistry. The virus-infected cells expressed the neural marker Tuj1 and exhibited neuronal morphology. At the same time, the survival time of mice injected with Ad5-AN was significantly longer than that of the PBS group (average 32.6 days vs. 22.3 days). The survival time of the Ad5-AN and temozolomide combination group was significantly longer than that of the temozolomide monotherapy group (average 97.5 days vs. 56.8 days). The results also showed that in the case of orthotopic tumors, oncolytic viruses expressing reprogramming factors could be used in combination with existing therapies to achieve better antitumor and long-lasting anti-recurrence effects.

[0137] Example 7 Oncolytic viral vectors expressing reprogramming factors inhibit the growth of glioma PDX tumor cells To further verify the combined effect of oncolytic virus and regenerative therapy, this example tested a glioma PDX (Patient-derived tumor xenograft) model constructed from patient-derived glioma tissue. PDX tissue was inoculated into the armpits of nude mice and passaged, then re-inoculated into the armpits of nude mice during the logarithmic growth phase. Tumors grew to 100 mm. 3 When tumors grew to a certain extent, nude mice with suitable tumor masses were selected and randomly divided into groups. After grouping, administration was started. The control group was the PBS group, and the Ad5-AN group was administered at a dose of 1 × 10 9 The animals were treated with intragastric temozolomide (15 mg / kg, once daily, 5 doses with 2 doses stopped), and the Ad5-AN and temozolomide combination group, in which Ad5-AN was administered every 2 days for 5 consecutive doses. Tumor volumes were measured and calculated every 3 days. The animals were then sacrificed, and tumors were collected, weighed, and subjected to biochemical and molecular testing. Results showed that, similar to the subcutaneous glioma cell line model, tumor volumes in the Ad5-AN group and the temozolomide group were significantly reduced compared to the control PBS group. However, approximately 45 days after administration, tumors gradually recurred and regrown (6 / 6), and by day 72, tumor growth had reached 2000 mm. 3 The tumor volume in the Ad5-AN and temozolomide combination group was significantly more effective than the Ad5-AN and temozolomide single-agent groups, with tumors essentially disappearing (72 days, 5 / 6), and more importantly, long-term antitumor efficacy was observed (102 days, 4 / 6 without recurrence). The results demonstrated that oncolytic viruses expressing reprogramming factors can be used in combination with existing therapies to achieve superior antitumor and long-term antirecurrence efficacy in in vivo models derived from patient tissue.

[0138] Example 8 Inhibition of glioma growth by recombinant herpes simplex virus expressing reprogramming factors In this example, herpes simplex virus type 1 was used as an oncolytic viral vector, and a human Ascl1 (SEQ ID No. 11) fragment and a CDS (SEQ ID No. 15) fragment derived from the human Ngn2 gene were inserted to achieve efficient co-expression of Ascl1 and Ngn2. After packaging and purifying the recombinant herpes simplex virus, an ectopic inoculation test was conducted in BALB / CA-nu mice with human brain glioma U87. Tumors of approximately 100 mm were observed. 3 Once the tumors had grown to a certain size, nude mice bearing suitable tumor masses were selected and randomly divided into groups. The administration was initiated after grouping. The doses for the control PBS group, HSV (empty vector containing no reprogramming factors), and HSV-AN groups were 2 × 10 6 The animals were treated with PFU, intragastric temozolomide (15 mg / kg, once daily, five doses with two breaks), and a combination of HSV-AN and temozolomide. HSV was administered every two days for five consecutive doses. Tumor volumes were measured and calculated every three days. The animals were then sacrificed, and tumors were collected, weighed, and subjected to biochemical and molecular testing. Compared with the control PBS group, tumor volumes in the HSV empty vector group and HSV-AN group were reduced by 25.6% and 58.9%, respectively, 10 days after administration, demonstrating that the addition of reprogramming factors significantly enhanced the inhibitory effect of oncolytic herpesviruses on gliomas. In the temozolomide group, tumor volume decreased by 73.6%, with a recurrence rate of 6 / 6 two months after administration. In the HSV-AN and temozolomide combination group, tumor volume decreased by 92.5%, with a recurrence rate of 1 / 6 two months after administration. These results demonstrate that the synergistic effect of the oncolytic herpesvirus drug on tumor lysis and in vivo tumor cell transdifferentiation can achieve a more significant ability to inhibit glioma growth, and that when combined with the existing treatment TMZ, it can achieve superior antitumor and long-lasting antirecurrence effects.

[0139] Although the preferred embodiments of the present invention have been specifically described above, the present invention is not limited to the above embodiments, and those skilled in the art may make various equivalent modifications or substitutions within the scope of the claims of the present invention without departing from the spirit of the present invention.

[0140] (Addendum) (Appendix 1) containing a recombinant nucleic acid comprising a functional fragment that promotes reprogramming / transdifferentiation of tumor cells into non-tumorigenic cells, A recombinant oncolytic virus, characterized in that the functional fragment contains at least one functional fragment that promotes expression of a transcription factor, and the functional fragment is selected from functional fragments that can promote expression of at least one transcription factor selected from NeuroD1, Brn2, Ascl1 or Ngn2.

[0141] (Appendix 2) the recombinant nucleic acid comprises a set of functional fragments that synergistically promote the reprogramming / transdifferentiation of tumor cells into non-tumorigenic cells; The recombinant oncolytic virus described in Appendix 1, characterized in that the functional fragments contain at least two functional fragments that promote expression of transcription factors, and the functional fragments are selected from functional fragments that can promote expression of at least two transcription factors selected from NeuroD1, Brn2, Ascl1, or Ngn2.

[0142] (Appendix 3) the recombinant nucleic acid comprises a set of functional fragments that synergistically promote the reprogramming / transdifferentiation of tumor cells into non-tumorigenic cells; The recombinant oncolytic virus described in Appendix 1, characterized in that the functional fragment contains a functional fragment that promotes expression of one or two transcription factors, Ascl1 or Ngn2.

[0143] (Appendix 4) The functional fragment is a polynucleotide encoding a functional protein, and is selected from polynucleotides encoding transcription factors that have 75% or more sequence identity with SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 15, or SEQ ID NO. 16; Preferably, the functional protein is selected from transcription factor functional proteins having 85% or more sequence identity with SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 13 or SEQ ID NO. 14; Preferably, the functional fragment is a polynucleotide encoding a functional protein, and is selected from polynucleotides encoding transcription factors that have 85% or more sequence identity with SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 15, or SEQ ID NO. 16; Preferably, the functional protein is selected from transcription factor functional proteins having a sequence identity of 95% or more with SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.13 or SEQ ID NO.14.

[0144] (Appendix 5) The functional fragment is a polynucleotide encoding a functional protein, and is selected from polynucleotides encoding transcription factors that have 95% or more sequence identity with SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 15, or SEQ ID NO. 16; Preferably, the functional protein is selected from transcription factor functional proteins having a sequence identity of 99% or more with SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.13, and SEQ ID NO.14.

[0145] (Appendix 6) The functional fragment is a polynucleotide encoding a functional protein, such as a transcription factor-encoding polynucleotide having 75% or more sequence identity with SEQ ID NO. 19; or The functional protein is a transcription factor functional protein having a sequence identity of 85% or more with SEQ ID NO. 18; Preferably, the functional fragment is a polynucleotide encoding a functional protein, such as a transcription factor-encoding polynucleotide having 85% or more sequence identity with SEQ ID NO. 19; or The functional protein is a transcription factor functional protein having a sequence identity of 95% or more with SEQ ID NO. 18; More preferably, the functional fragment is a polynucleotide encoding a functional protein, such as a transcription factor-encoding polynucleotide having 95% or more sequence identity with SEQ ID NO. 19; or The recombinant oncolytic virus described in Appendix 1, characterized in that the functional protein is a transcription factor functional protein having a sequence identity of 99% or more with SEQ ID NO. 18.

[0146] (Appendix 7) The recombinant oncolytic virus described in Appendix 1, characterized in that the expression systems of the functional fragments that promote the expression of the transcription factor are constructed under the same expression vector or are expressed separately using different expression vectors.

[0147] (Appendix 8) 8. The recombinant oncolytic virus according to any one of claims 1 to 7, wherein the recombinant oncolytic virus comprises a selectively replicating recombinant oncolytic virus.

[0148] (Appendix 9) The recombinant oncolytic virus according to Appendix 8, characterized in that the selectively replicating recombinant oncolytic virus is derived from an adenovirus, poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, vesicular stomatitis virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus, or Maraba virus, which have oncolytic activity.

[0149] (Appendix 10) 1. A method for promoting reprogramming / transdifferentiation of tumor cells into non-tumorigenic cells by oncolytic viruses, comprising: 10. A method comprising the step of reprogramming tumor cells by contacting the tumor cells with a recombinant oncolytic virus described in any one of appendices 1 to 9, thereby reprogramming / transdifferentiating the tumor cells into non-tumorigenic cells.

[0150] (Appendix 11) 1. A composition for treating cancer, comprising: (A) a recombinant oncolytic virus according to any one of appendices 1 to 9; and (B) a pharmaceutically acceptable excipient.

[0151] (Appendix 12) The composition comprises: (C) The composition described in Appendix 11, further comprising an antitumor agent, wherein the antitumor agent comprises one or both of temozolomide and bevacizumab.

[0152] (Appendix 13) 10. Use of a recombinant oncolytic virus according to any one of appendices 1 to 9 in the preparation of a medicament for treating a tumor.

[0153] (Appendix 14) The use of claim 13, wherein the recombinant oncolytic virus is formulated as a therapeutic agent to be administered into or near a tumor, and the method of administering the therapeutic agent includes any one of injection, intraperitoneal administration, intrathecal administration, and intravenous administration.

[0154] (Appendix 15) The use described in Appendix 13, wherein the recombinant oncolytic virus is formulated as a therapeutic agent administered within or near a tumor, and the method of administering the therapeutic agent includes one or more of hydrogel administration, convection-enhanced drug delivery, and Ommaya reservoir.

[0155] (Appendix 16) 14. The use of claim 13, wherein the tumor is selected from the group consisting of glioblastoma, neuroblastoma, chordoma, meningioma, teratoma, spinal cord tumor, breast cancer, head and neck tumor, kidney cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, bile duct cancer, bladder cancer, ureter cancer, glioma, osteochondroma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, cervical cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, cutaneous squamous cell carcinoma, mesothelioma, ovarian cancer, pancreatic endocrine tumor, glucagonoma, pancreatic cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymic cancer, trophoblastic cancer, endometrial cancer, vaginal cancer, vulvar cancer, insulinoma, blood cancer, peritoneal cancer, and pleural cancer.

[0156] (Appendix 17) 14. The use of claim 13, wherein the tumor comprises one or more of glioma, astrocytoma, astroblastoma, medulloblastoma, schwannoma, and brain metastasis cancer.

Claims

1. containing a recombinant nucleic acid comprising a functional fragment that promotes reprogramming / transdifferentiation of tumor cells into non-tumorigenic cells, The recombinant oncolytic virus, characterized in that the functional fragment contains at least one functional fragment that promotes expression of a transcription factor, and the functional fragment is selected from functional fragments that can promote expression of at least one transcription factor selected from NeuroD1, Brn2, Ascl1, or Ngn2.

2. The recombinant nucleic acid comprises a set of functional fragments that synergistically promote the reprogramming / transdifferentiation of tumor cells into non-tumorigenic cells, The recombinant oncolytic virus of claim 1, characterized in that the functional fragment contains at least two functional fragments that promote expression of a transcription factor, and the functional fragments are selected from functional fragments that can promote expression of at least two of the transcription factors NeuroD1, Brn2, Ascl1 or Ngn2.

3. The recombinant nucleic acid comprises a set of functional fragments that synergistically promote the reprogramming / transdifferentiation of tumor cells into non-tumorigenic cells, The recombinant oncolytic virus of claim 1, characterized in that the functional fragment contains a functional fragment that promotes expression of one or two transcription factors, Ascl1 or Ngn2.

4. The functional fragment is a polynucleotide encoding a functional protein, selected from polynucleotides encoding transcription factors having a sequence identity of 75% or more with SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 15 or SEQ ID NO. 16; Preferably, the functional protein is selected from transcription factor functional proteins having a sequence identity of 85% or more with SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 13 or SEQ ID NO. 14; Preferably, the functional fragment is a polynucleotide encoding a functional protein, selected from polynucleotides encoding transcription factors having a sequence identity of 85% or more with SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 15 or SEQ ID NO. 16; 2. The recombinant oncolytic virus according to claim 1, characterized in that the functional protein is selected from the transcription factor functional proteins having a sequence identity of 95% or more with SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 13 or SEQ ID NO.

14.

5. The functional fragment is a polynucleotide encoding a functional protein, selected from polynucleotides encoding transcription factors having a sequence identity of 95% or more with SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO. 16; The recombinant oncolytic virus according to claim 4, characterized in that the functional protein is selected from the transcription factor functional proteins having a sequence identity of 99% or more with SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 13, SEQ ID NO.

14.

6. The functional fragment is a polynucleotide encoding a functional protein, the polynucleotide encoding a transcription factor having a sequence identity of 75% or more with SEQ ID NO. 19, or The functional protein is a transcription factor functional protein having a sequence identity of 85% or more with SEQ ID NO. 18; Preferably, the functional fragment is a polynucleotide encoding a functional protein, the polynucleotide encoding a transcription factor having a sequence identity of 85% or more with SEQ ID NO. 19, or The functional protein is a transcription factor functional protein having a sequence identity of 95% or more with SEQ ID NO. 18; More preferably, the functional fragment is a polynucleotide encoding a functional protein, the polynucleotide encoding a transcription factor having a sequence identity of 95% or more with SEQ ID NO. 19, or 2. The recombinant oncolytic virus of claim 1, wherein the functional protein is a transcription factor functional protein having a sequence identity of 99% or more with SEQ ID NO.

18.

7. The recombinant oncolytic virus according to claim 1, characterized in that the expression systems of the functional fragments promoting the expression of the transcription factor are constructed under the same expression vector or are expressed separately using different expression vectors.

8. The recombinant oncolytic virus according to any one of claims 1 to 7, characterized in that the recombinant oncolytic virus comprises a selectively replicating recombinant oncolytic virus.

9. The recombinant oncolytic virus of claim 8, characterized in that the selectively replicating recombinant oncolytic virus is derived from an adenovirus, poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, vesicular stomatitis virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus or Maraba virus, which have oncolytic activity.

10. 1. A method for promoting reprogramming / transdifferentiation of tumor cells into non-tumorigenic cells by oncolytic viruses, comprising: A method comprising a step of reprogramming / transdifferentiating tumor cells into non-tumorigenic cells by contacting the tumor cells with a recombinant oncolytic virus described in any one of claims 1 to 9, thereby reprogramming the tumor cells.

11. A composition for treating cancer, comprising: (A) a recombinant oncolytic virus according to any one of claims 1 to 9; (B) a pharma- ceutically acceptable excipient.

12. The composition comprises:

12. The composition of claim 11, further comprising (C) an antitumor agent, the antitumor agent comprising one or both of temozolomide and bevacizumab.

13. 10. Use of a recombinant oncolytic virus according to any one of claims 1 to 9 in the preparation of a medicament for treating a tumor.

14. 14. The use of claim 13, wherein the recombinant oncolytic virus is formulated as a therapeutic agent administered intratumorally or in the vicinity of a tumor, and the method of administration of the therapeutic agent includes one of the following: injection, intraperitoneal, intrathecal, or intravenous administration.

15. The use of claim 13, wherein the recombinant oncolytic virus is formulated as a therapeutic agent administered within or near a tumor, and the method of administering the therapeutic agent includes one or more of hydrogel administration, convection-enhanced drug delivery, and Ommaya reservoir.

16. 14. The use according to claim 13, wherein the tumor comprises glioblastoma, neuroblastoma, chordoma, meningioma, teratoma, spinal tumor, breast cancer, head and neck tumor, kidney cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, bile duct cancer, bladder cancer, ureter cancer, glioma, osteochondroma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, cervical cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, ovarian cancer, pancreatic endocrine tumor, glucagonoma, pancreatic cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymic cancer, trophoblastic cancer, endometrial cancer, vaginal cancer, vulvar cancer, insulinoma, blood cancer, peritoneal cancer or pleural cancer.

17. 14. The use according to claim 13, wherein the tumor comprises one or more of glioma, astrocytoma, astroblastoma, medulloblastoma, schwannoma and brain metastasis cancer.

Images