Functional fragments, combinations, and applications 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] This invention belongs to the fields of biotechnology and gene therapy and relates to functional fragments, combinations, and applications for reprogramming recombinant oncolytic viruses. More specifically, it relates to a method for differentiating tumor cells into non-tumor-forming cells using an oncolytic virus vector, and a method for treating tumors using this method. [Background technology]

[0002] The main characteristic of tumor cells is a runaway cell cycle, leading to malignant proliferation. Reprogramming techniques aim to treat tumors by differentiating tumor cells into non-tumor-forming cells that have stopped dividing, effectively "returning to the right path" from their harmful behavior. To date, the most successful example of tumor differentiation induction therapy has been the treatment of acute promyelocytic leukemia using all-trans retinoic acid, which resulted in a high remission rate.

[0003] The treatment of tumors with oncolytic viruses is attracting increasing attention. The principle involves creating specialized oncolytic viruses by genetically modifying several naturally occurring, less pathogenic viruses. These viruses selectively infect tumor cells by inactivating or defecting tumor suppressor genes in target cells, where they replicate in large numbers and ultimately destroy the tumor cells. Furthermore, they can stimulate an immune response, attracting more immune cells to continue killing any remaining cancer cells. Oncolytic virus therapy has attracted widespread attention in recent decades, and related research has made tremendous progress. Currently, various oncolytic viruses, including adenoviruses, herpes simplex viruses, coxsackieviruses, poxviruses, polioviruses, measles viruses, and reoviruses, are already in clinical trials. They recognize and infect tumor cells, ultimately causing them to swell and destroy them. However, they cannot replicate in normal living cells without killing them, theoretically resulting in higher antitumor efficacy and lower side effects.

[0004] Oncolytic viruses are now widely accepted as an important branch of immunotherapy and have demonstrated great potential in basic and clinical research for the treatment of malignant tumors. In 2015, the U.S. 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 approval for the treatment of malignant tumors with oncolytic viruses. Currently, various oncolytic viruses are being used in clinical research for tumor treatment, and their therapeutic efficacy and safety have been confirmed.

[0005] Currently, oncolytic viruses are thought to mediate antitumor effects primarily through the following mechanisms: 1) They specifically proliferate within tumor cells and directly lyse them. 2) Lysed tumor cells release viral particles, stimulating systemic antitumor immunity through numerous pathways, such as promoting tumor antigen presentation, increasing the infiltration of immune cells into the tumor microenvironment, controlling the tumor microenvironment, activating immune cells, and activating the body's immune system through immobilized immune regulatory factors. Furthermore, it has been reported that some viruses can indirectly achieve antitumor effects by infecting tumor-associated vascular endothelial cells, thereby inhibiting tumor angiogenesis.

[0006] Oncolytic adenoviruses are currently the most widely used oncolytic viruses, and many strategic modifications have been made to enhance their tumor targeting. These modifications include targeted transcriptional regulation that controls the E1A gene using mutations and / or tumor-specific promoters of functional genes (e.g., E1A or E1B) involved in the control of cell cycle nodes in the adenovirus genome; targeted transduction regulation that alters how oncolytic adenoviruses enter tumor cells using different serotype adenoviruses or RGD motifs; and delivery of oncolytic adenoviruses to distant tumor sites using cell vectors. As vectors, oncolytic adenoviruses deliver immunoregulatory or therapeutic genes, generating synergistic antitumor effects by enhancing antitumor immunity or inducing apoptosis or suicide in tumor cells. Current oncolytic viruses often possess immunoregulatory factors and suicide genes, which can further enhance the effectiveness of tumor treatment, but the problem of insufficient efficacy remains.

[0007] Gliomas, also known as gliomas, broadly refer to all tumors derived from neuroepithelial cells, and narrowly refer to tumors derived from various glial cells. Gliomas are one of the most deadly malignant tumors, the most common primary central nervous system tumors, 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's (WHO) 1999 classification system, they are divided into astrocytoma, oligodendroglioma, ependymoma, mixed glioma, choroid plexus tumor, neuroepithelial tumor of unknown cause, neuronal and neuron-glial mixed tumors, pineal parenchymal tumors, embryonic tumors, and neuroblastomas. Gliomas and normal nerve tissue grow alternately, the boundaries are unclear, the tumor tissue is difficult to completely remove, and recurrence is common. Furthermore, due to the presence of the blood-brain barrier, conventional antitumor drugs are not very effective. Treatment of glioblastoma remains an unmet clinical need in the medical community. In recent years, some studies have found that certain neurogenic transcription factors or combinations of transcription factors can convert glioma cells into neuron-like cells in vitro or in vivo, thereby limiting their proliferative capacity. Currently used delivery vectors, such as adeno-associated virus (AAV) vectors, cannot self-replicate and are unable to adequately introduce foreign genes into tumor cells, making them difficult to use in the clinical treatment of tumors.

[0008] Therefore, by finding and utilizing an appropriate replication-type expression vector, loading it with the appropriate reprogramming factor, infecting tumor cells and replicating within the cells, releasing the virus to infect more tumor cells, some tumor cells lyse and die, some tumor cells die due to anti-tumor immunity, and some non-dead tumor cells differentiate into non-tumor-forming cells that have stopped dividing, achieving an effective synergistic effect, and currently representing a new therapeutic option for treating tumors. [Overview of the project] [Problems that the invention aims to solve]

[0009] In view of the drawbacks of the prior art, an object of the present invention is to provide functional fragments, combinations, and their applications for reprogramming recombinant oncolytic viruses. The present invention provides a set of transcription factors, combinations of transcription factors, methods by the expression of oncolytic viruses, and applications of the set of transcription factors carried by oncolytic viruses in the preparation of tumor disease drugs, which synergistically promote the differentiation and transformation of tumor cells and reprogram non-tumorigenic cells.

Means for Solving the Problems

[0010] To achieve the object of this invention, the present invention adopts the following technical means.

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

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

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

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

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

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

[0017] In another preferred example, the recombinant nucleic acid includes a set of functional fragments that synergistically promote the reprogramming / differentiation conversion of tumor cells into non-tumorigenic cells. The functional fragments contain at least two functional fragments that promote the expression of transcription factors, and the functional fragments are selected from functional fragments that promote the expression of transcription factors such as NeuroD1, Brn2, Ascl1, and Ngn2.

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

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

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

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

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

[0023] Preferably, the functional fragment that synergistically promotes the reprogramming / differentiation of glial cells contained in the oncolytic virus or the expression of a transcription factor is derived from a mammal, more preferably a human or non-human primate mammal.

[0024] In other preferred examples, a functional fragment that can synergistically promote the reprogramming / differentiation of tumor cells contained in the recombinant oncolytic virus 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 denoted by SEQ ID NO.1 or SEQ ID NO.2, and the polynucleotide sequence encoding the functional NeuroD1 protein being denoted by SEQ ID NO.3 or SEQ ID NO.4.

[0025] In other preferred examples, the functional fragment that can synergistically promote the differentiation of tumor cells contained in the recombinant oncolytic virus is a polynucleotide encoding a functional protein, the functional protein is a functional Brn2 protein, the amino acid sequence of the functional Brn2 protein is shown by SEQ ID NO. 5 or SEQ ID NO. 6, and the polynucleotide sequence encoding the functional Brn2 protein is shown by SEQ ID NO. 7 or SEQ ID NO. 8.

[0026] In other preferred examples, the functional fragment that can synergistically promote the differentiation of tumor cells contained in the recombinant oncolytic virus is a polynucleotide encoding a functional protein, the functional protein is a functional Ascl1 protein, the amino acid sequence of the functional Ascl1 protein is shown by SEQ ID NO.9, SEQ ID NO.10, or SEQ ID NO.18, and the polynucleotide sequence encoding the functional Ascl1 protein is shown by SEQ ID NO.11, SEQ ID NO.12, or SEQ ID NO.19.

[0027] In other preferred examples, the functional fragment that can synergistically promote the differentiation of tumor cells contained in the recombinant oncolytic virus 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 other preferred examples, if the functional fragment contained in the recombinant oncolytic virus that can synergistically promote the differentiation of tumor cells is a polynucleotide encoding a functional protein, then the sequence identity between the amino acid sequence of the functional protein and 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 is 85% or more, such as 85%, 87%, 89%, 90%, 91%, 93%, 95%, 97%, or 99%.

[0029] More preferably, the sequence identity between the amino acid sequence of the functional protein and 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 is 95% or higher, such as 95%, 96%, 97%, 98%, or 99%.

[0030] Most preferably, the sequence identity between the amino acid sequence of the functional protein and 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 is 99% or higher.

[0031] In other preferred examples, if the functional fragment contained in the recombinant oncolytic virus that can synergistically promote the differentiation of tumor cells is a polynucleotide encoding a functional protein, then the sequence identity between the polynucleotide sequence encoding the functional protein and 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 is 75% or more, such as 75%, 77%, 79%, 80%, 85%, 87%, 89%, 90%, 91%, 93%, 95%, 97%, or 99%.

[0032] More preferably, the sequence identity between the polynucleotide sequence encoding the functional protein and 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 is 85% or higher, such as 85%, 87%, 89%, 90%, 91%, 93%, 95%, 97%, or 99%.

[0033] Most preferably, the sequence identity between the polynucleotide sequence encoding the functional protein and 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 is 95% or higher, such as 95%, 96%, 97%, 98%, or 99%.

[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 expressed separately using different expression vectors.

[0035] Preferably, the recombinant oncolytic virus includes selectively replicating recombinant oncolytic viruses.

[0036] Preferably, the selectively replicating recombinant oncolytic virus is derived from an adenovirus, poxvirus, herpes simplex virus, measles virus, Semryki forest virus, varicella stomatitis virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus, or maraba virus, all of which have oncolytic activity.

[0037] Preferably, the tumor cells are 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 other tumors of human or non-human mammalian origin. It is any of the following: ureteral 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, trophoblast cancer, endometrial cancer, vaginal cancer, vulvar cancer, insulinoma, hematological cancer, peritoneal cancer, or pleural cancer.

[0038] Preferably, the reprogramming / differentiation refers to reprogramming or differentiating tumor cells into non-tumor-forming cells.

[0039] A second aspect of the present invention provides a method for promoting the reprogramming / differentiation of tumor cells into non-tumor-forming cells by an oncolytic virus, comprising the step of contacting tumor cells with a recombinant oncolytic virus described in the first aspect of the present invention to cause the tumor cells to be reprogrammed / differentiated into non-tumor-forming cells.

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

[0041] In other preferred examples, the method is an in vitro method.

[0042] In other preferred examples, the method is an in vivo method.

[0043] In other preferred examples, the method is therapeutic. Furthermore, the method can be used in combination with existing tumor therapies such as immunotherapy, CAR-T therapy, and electro-transmission therapy (TTF), without conflicting with existing tumor therapies.

[0044] Preferably, the tumor cells are 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 other tumors of human or non-human mammalian origin. It is any of the following: ureteral 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, trophoblast cancer, endometrial cancer, vaginal cancer, vulvar cancer, insulinoma, hematological cancer, peritoneal cancer, or pleural cancer.

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

[0046] In the present invention, the delivery system is the oncolytic virus, and the selectively replicating recombinant oncolytic virus is derived from adenovirus, poxvirus, herpes simplex virus, measles virus, Semryki forest virus, vesicular stomatitis virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus, or marabavirus, all of which have oncolytic activity.

[0047] In other preferred examples, the expression vector carrying the recombinant oncolytic virus transcription factor polynucleotide may also carry other functional fragments, which may be reporter genes or other transcription factor functional fragments having reprogramming function, and may be selected from, but are not limited to, NeuroD1, Brn2, Ascl1, or Ngn2.

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

[0049] In other preferred examples, the recombinant oncolytic virus comprises a set of functional fragments capable of synergistically promoting the reprogramming / differentiation 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 expressed separately using different expression vectors.

[0050] In a third embodiment of the present invention, (A) Recombinant oncolytic virus as described in the first embodiment, (B) The present invention provides compositions for treating cancer, comprising pharmaceutically acceptable excipients.

[0051] In some embodiments, the composition is (C) Further containing an antitumor agent, The aforementioned antitumor agent includes temozolomide and bevacizumab, or both.

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

[0053] In other preferred examples, the pharmaceutical composition is an injectable preparation.

[0054] A fourth aspect of the present invention provides the use of the recombinant oncolytic virus described in the first aspect in the preparation of a drug for treating tumors.

[0055] In other preferred examples, in the use described above, the recombinant oncolytic virus is formulated as a therapeutic agent to be administered intratumor or near a tumor, and the method of administering the therapeutic agent includes injection, hydrogel administration, convection-enhanced delivery (CED), Ommaya reservoir, intraperitoneal administration, subarachnoid administration, or intravenous administration.

[0056] Preferably, the tumors 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, bile duct cancer, bladder cancer, ureteral cancer, and nerve cancer. This includes 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 tumors, 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, hematological cancer, peritoneal cancer, or pleural cancer.

[0057] The numerical range described in this invention includes not only the numerical values ​​listed above, but also any numerical values ​​between the above numerical ranges that are not listed. For the sake of brevity and clarity, this invention does not comprehensively list all specific numerical values ​​included in the range. [Brief explanation of the drawing]

[0058] [Figure 1A] Figure 1A shows the killing curves of Ad5-AN oncolytic adenoviruses with different infection multiplicities against U87 cells in Example 2. [Figure 1B] Figure 1B shows the killing curves of Ad5-AN oncolytic adenoviruses with different infection multiplicities against U118 cells in Example 2. [Figure 2A] Figure 2A shows that in Example 3, Ad5-AN can differentiate glioma cells U251 into non-tumor-forming neurons. [Figure 2B] Figure 2B shows that Ad5-AN in Example 3 can differentiate glioma cells U251 into non-tumor-forming neurons. [Figure 3A] Figure 3A shows the inhibition of tumor cell proliferation in an ectopic inoculation model of glioma mice by an oncolytic adenovirus type 5 vector expressing a reprogramming factor in Example 4. [Figure 3B]Figure 3B shows the experimental results when the number of experimental days was extended compared to Figure 3A. [Figure 3C] Figure 3C shows the inhibition of tumor cell proliferation in an ectopic inoculation model of glioma mice by an oncolytic adenovirus type 5 vector expressing a reprogramming factor in Example 4. [Figure 3D] Figure 3D shows the inhibition of tumor cell proliferation in an ectopic inoculation model of glioma mice by an oncolytic adenovirus type 5 vector expressing a reprogramming factor in Example 4. [Figure 4A] Figure 4A shows the experimental results in Example 5 when used in combination with an oncolytic virus expressing a reprogramming factor. [Figure 4B] Figure 4B shows the experimental results when the number of experimental days was extended compared to Figure 4A. [Figure 5] Figure 5 shows the parallel experiment of seven groups of mice receiving temozolomide (TMZ) in Example 5. The tumors in all seven mice, numbered A through G respectively, recurred 56 days after administration (7 / 7). [Figure 6] Figure 6 shows the parallel experiment of seven groups of mice in Example 5 receiving Ad5-AN and temozolomide in combination. The tumors in the seven mice numbered A through G did not recur 93 days after administration (0 / 7). [Modes for carrying out the invention]

[0059] Through extensive and detailed research, the inventors have discovered that tumor cells can be differentiated in vitro or in vivo into non-tumor-forming cells that have ceased to divide by using an oncolytic virus expression vector and incorporating a batch of transcription factors or a combination of transcription factors with differentiation reprogramming function. Based on this discovery, the inventors further investigated the application of this method to the development of oncology drugs, and observed that in animal models of gliomas, the oncolytic virus expression vector achieved an effective synergistic effect between oncolytic therapy and reprogramming action, enhancing the antitumor effect, significantly reducing tumor size in animals, and significantly extending survival time. Therefore, the use of this batch of oncolytic virus expression vectors with differentiation reprogramming / reprogramming function, or a combination of transcription factors, is expected to be applied to the development of oncology drugs, particularly glioma drugs.

[0060] term The term "administration" means physically introducing the product of the present invention into a target area using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intracerebral, intratumoral, intramuscular, subcutaneous, intraperitoneal, intrathecal, or other parenteral administration routes such as injection or infusion.

[0061] The term “approximately” may mean a value or composition that lies within a tolerance range for a particular value or composition as determined by those skilled in the art, and depends in part on how the value or composition is measured or determined. Typically, “approximately” means ±10% or ±20%. For example, approximately 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 the process of controlling or altering the biological activity of a cell, changing the cell from one biological state to another, and typically includes processes that alter the fate of a cell, such as differentiation (from progenitor cells to terminal cells), dedifferentiation (from terminal cells to pluripotent stem cells), transdifferentiation (from one terminal cell to another), dedifferentiation (from terminal cells to progenitor cells), and deterministic transdifferentiation (from one progenitor cell to another terminal cell that naturally differentiates).

[0062] In the present invention, the terms "differentiation conversion," "reprogramming," "differentiation conversion reprogramming," "differentiation conversion / reprogramming," or "reprogramming / differentiation conversion" specifically refer to the process from one terminal cell to another, particularly the process from tumor cells to non-tumor-forming cells.

[0063] Transcription factor The present invention provides a set of transcription factors having reprogramming function, and these transcription factors and their combinations have excellent differentiation ability and can be used to promote the efficient differentiation 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 necessary for neuronal differentiation, selected from the group consisting of NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and Otx2.

[0065] Preferably, the transcription factor of the present invention includes at least two of the aforementioned transcription factors.

[0066] NeuroD1 functional fragments are polynucleotides or protein fragments expressing the neurogenic differentiation 1 transcription factor of mammalian origin. NeuroD1 is a bHLH (basic helix-loop-helix) transcription factor. For example, the human-derived NeuroD1 molecule has GenBank ID# 4760, and its protein sequence is indicated by SEQ ID NO.1. The NCBI Reference Sequence is NM_002500.5, and its CDS sequence is indicated by SEQ ID NO.3.

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

[0068] The Ascl1 functional fragment is a polynucleotide or a protein fragment expressing the same that encodes the Achaete-scute homolog 1 transcription factor, derived from mammals. Ascl1 is a bHLH (basic helix-loop-helix) transcription factor. For example, the human Ascl1 molecule has an ID# of 429 in GenBank, and its protein sequence is indicated by SEQ ID NO. 9. The NCBI Reference Sequence is NM_004316.4, and its CDS sequence is indicated by SEQ ID NO. 11.

[0069] The Ngn2 functional fragment, also known as Neurog2, is a polynucleotide or its expression protein fragment that encodes the neurogenin-2 transcription factor, derived from mammals. Ngn2 is a bHLH (basic helix-loop-helix) type transcription factor. For example, the human Ngn2 molecule has GenBank ID# 63973, and its protein sequence is shown as SEQ ID NO. 13. The NCBI Reference Sequence is NM_024019.4, and its CDS sequence is shown as SEQ ID NO. 15.

[0070] Any method for promoting the increased expression of transcription factors for glial cell transdifferentiation includes, but is not limited to, promoting the increased expression of any of the NeuroD1, Brn2, Ascl1, or Ngn2 transcription factors in glial cells by directly contacting or introducing a functional fragment that promotes the expression of an inducer or transcription factor into the tumor cells, thereby promoting the transdifferentiation of the tumor cells into non-tumoric cells. The method for promoting the increased expression of the functional fragment of the transcription factor may further be obtained by CRISPR / dCas9 targeting the expression of the DNA activating gene of the relevant transcription factor, or by CRISPR / Cas13 targeting the relevant transcription factor RNA to enhance the expression of the transcription factor functional protein.

[0071] Those skilled in the art can screen for methods of promoting the above-mentioned transcription factors based on existing databases. Based on the function of transcription factors on tumor cell reversal disclosed in this invention, those skilled in the art should understand that they can reasonably predict that any substance that promotes the above-mentioned transcription factors will function on tumor cell reversal.

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

[0073] Oncolytic viruses The oncolytic viruses described in the present invention are recombinant oncolytic viruses derived from adenoviruses, poxviruses, herpes simplex viruses, measles viruses, Semryki forest viruses, varicella stomatitis viruses, polioviruses, retroviruses, reoviruses, Seneca Valley viruses, echoviruses, coxsackieviruses, Newcastle disease viruses, and Maraba viruses, all of which have oncolytic activity.

[0074] The 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 more. It is any of the following: cancer, ureteral 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, trophoblast cancer, endometrial cancer, vaginal cancer, vulvar cancer, insulinoma, hematological cancer, peritoneal cancer, or pleural cancer.

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

[0077] Gliomas, also known as gliomas, broadly refer to all tumors derived from neuroepithelial cells, and narrowly refer to tumors derived from various glial cells. Gliomas are one of the most deadly malignant tumors, the most common primary central nervous system tumors, 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's (WHO) 1999 classification system, they are divided into astrocytoma, oligodendroglioma, ependymoma, mixed glioma, choroid plexus tumor, neuroepithelial tumor, neuronal and neuron-glial mixed tumors, pineal parenchymal tumor, embryonic tumor, and neuroblastoma.

[0078] Pharmaceutical composition and administration method The present invention further provides a pharmaceutical composition for treating cancer, the pharmaceutical composition comprising a recombinant oncolytic virus containing a combination of functional fragments that promote the expression of tumor cell differentiation and conversion transcription factors, and a pharmaceutically acceptable vector.

[0079] A pharmaceutically acceptable vector means a vector for administration of a therapeutic agent containing various excipients and diluents.

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

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

[0082] Pharmacochemically acceptable vectors may include one or a combination of at least two of the following: a coating layer, a capsule, a microcapsule, or a 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 mentioned above, or the anti-cancer-promoting molecule expressed by the oncolytic virus). In some examples, the vector can protect the main component in the composition to mitigate or avoid inactivation or degradation of the main component under certain negative conditions (e.g., oxidation, denaturation by strong acids or strong bases). For example, enzymes in gastric juice or relatively low pH values ​​may lead to 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, vectors can be used to control the release of a major component (e.g., an oncolytic virus). Release control may include, but is not limited to, sustained release, prolonged release, or targeted release. For example, vectors may include hydrogel capsules, microcapsules, or nanocapsules made from one or at least two of the following: collagen, gelatin, chitosan, alginate, polyvinyl alcohol, polyethylene oxide, starch, or cross-linked starch.

[0084] In some embodiments, pharmaceutically acceptable vectors may include dispersion media (e.g., solvents), coatings, buffers, stabilizing agents, isotonic agents, or absorption retarders. Exemplary pharmaceutically acceptable vectors may include phosphate-buffered saline, water, emulsions (e.g., oil-water emulsions), various types of wetting agents, sterile solutions, gels, or bioabsorbable matrix materials, or other suitable materials, or any combination thereof.

[0085] In some examples, the excipients include, but are not limited to, water, physiological saline, polyethylene glycol, hyaluronic acid, ethanol, and pharmaceutically acceptable salts, such as salts of inorganic acids (e.g., hydrochloric acid, hydrobromide, phosphate, or sulfate) or salts of organic acids (e.g., acetate, propionate, or benzoate).

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

[0087] The method of administering the composition described in the present invention is not particularly limited, but typical examples include, but are not limited to, injection into or near a tumor, hydrogel administration, convection-enhanced delivery (CED), Ommaya reservoir, intraperitoneal administration, subarachnoid administration, or intravenous administration.

[0088] The pharmaceutical composition can be administered to subjects with cancer, such as humans or animals. In some embodiments, the pharmaceutical composition can be administered to subjects by one or more methods of administration. The one or more methods of administration include, but are not limited to, oral administration, injection administration, or topical administration. Forms of the composition suitable for oral administration include, but are not limited to, tablets, liposome formulations, sustained-release capsules, microparticles, microspheres, or any other suitable form. Forms of the composition suitable for injection administration include, but are not limited to, sterile aqueous solutions or oily formulations. Forms of the composition suitable for topical administration include, but are not limited to, sterile aqueous solutions or oily formulations, suspensions, or emulsions. Taking nasal administration as an example, the form of the composition may include aerosols, mists, powders, solutions, suspensions, or gels.

[0089] In some embodiments, the pharmaceutical composition can be stored at appropriate temperatures, including room temperature (approximately 20°C), 40°C, -20°C, and -80°C. The composition can further be formulated into various forms, such as powder, to facilitate storage and transport. The powder may be sterile powder, and a solvent may 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 contain components that have antibacterial activity but do not significantly adversely affect the survival of oncolytic viruses, and as a result, the pharmaceutical composition can be stable under specific storage conditions such as refrigeration and freezing, and can be protected from contamination by microorganisms such as bacteria and fungi.

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

[0091] The compositions of the present invention can be used before or after administering other pharmaceutical compositions for treating cancer. Optionally, the compositions disclosed herein can be combined with other therapeutic methods to treat the cancer in which the subject has been affected. For example, other therapeutic methods include, but are not limited to, administration of other pharmaceutical compositions capable of treating cancer to the subject, surgical removal of the tumor in the subject, radiotherapy, 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 hormones, targeted agents (e.g., bevacizumab), or immunotherapeutic agents (e.g., monoclonal antibodies and / or tumor vaccines).

[0092] Effects of the invention Through extensive and detailed research, the inventors have discovered that tumor cells can be differentiated in vitro or in vivo into non-tumor-forming cells that have ceased to divide by using an oncolytic virus expression vector and incorporating a batch of transcription factors or a combination of transcription factors with differentiation reprogramming function. Based on this discovery, the inventors further investigated the application of this method to the development of oncology drugs, and observed that in animal models of gliomas, the oncolytic virus expression vector achieved an effective synergistic effect between oncolytic therapy and reprogramming action, enhancing the antitumor effect, significantly reducing tumor size in animals, and significantly extending survival time. Therefore, the use of this batch of oncolytic virus expression vectors with differentiation reprogramming / reprogramming function, or a combination of transcription factors, is expected to be applied to the development of oncology drugs, particularly glioma drugs.

[0093] Compared to conventional technologies, the present invention offers the following competitive advantages: By innovatively utilizing oncolytic viruses in combination with reprogramming activity, the invention obtains transcription factors or combinations with reprogramming function expressed on oncolytic viruses and recombinant oncolytic viruses, and explores the ability of transcription factors and combinations thereof to differentiate tumor cells into non-tumor-forming cells. Such recombinant oncolytic viruses have efficient tumor-killing and tumor-suppressing effects, better treating tumors and preventing tumor recurrence.

[0094] The technical means of the present invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the above embodiments are merely for the purpose of aiding the understanding of the present invention and do not limit the present invention to any specific examples.

[0095] In the following examples, experimental methods for which specific conditions are not indicated generally follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning. A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturer.

[0096] General method Materials and methods amino acid sequence SEQ ID NO.1 (hNeuroD1 amino acid sequence) is MTKSYSESGLMGEPQPQGPPSWTDECLSSQDEEHEADKKEDDLETMNAEEDSLRNGGEEEDEDEDLEEEEEEEEEDDDQKPKRRGPKKKKMTKARLERFKLRRMKANARERNRMHGLNAALDNLRKVVPCYSKTQKLSKIETLRLAKNYIWALSEILRSGKSPDLVSFVQTLCKGLSQPTTNLVAGCLQLNPRTFLPEQNQDMPPHLPTASASFPVHPYSYQSPGLPSPPYGTMDSSHVFHVKPPPHAYSAALEPFFESPLTDCTSPSFDGPLSPPLSINGNFSFKHEPSAEFEKNYAFTMHYPAATLAGAQSHGSIFSGTAAPRCEIPIDNIMSFDSHSHHERVMSAQLNAIFHD.

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

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

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

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

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

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

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

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

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

[0106] SEQ ID NO.11 (hAscl1 nucleotide sequence) is 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) is MFVKSETLELKEEEEVLMLLGSASPASATLTPMSSSADEEEDEELRRPGSARGQRGAEAGQGVQGSPASGAGGCRPGRLLGLMHECKRRPSRSRAVSRGAKTAETVQRIKKTRRLKANNRERNRMHNLNAALDALREVLPTFPEDAKLTKIETLRFAHNYIWALTETLRLADHCAGAGGLQGALFTEAVLLSPGAALGASGDSPSPPSSWSCTNSPASSSNSTSPYSCTLSPASPGSDVDYWQPPPPEKHRYAPHLPLARDCI.

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

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

[0116] Immunochromosis Immunochromism of cultured cells was performed according to "Direct conversion of fibroblasts to functional neurons by defined factors" (Vierbuchen, T. et al. Nature 463, 1035-1041 (2010)), and immunostaining experiments on tissue sections were performed according to published methods. Primary antibodies used for immunohistochemistry include 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), chick 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 experimental method On day 1, the cell density was increased to 5 × 10⁻⁶. 4The culture medium was adjusted to cells / mL, and 96-well plates were plated with 100 μL / well. On day 2, the medium was removed, and the corresponding viruses were incubated in 5 × 10⁻¹⁶ units so that the MOI value was a multiple of 10. 3 The cells were serially diluted at a dose of cells / well and added to each well to infect the cells, setting up three replicates for each MOI. On day 7, the supernatant was gently removed, 50 μL of medium was added to each well, followed by 20 μL of 5 mg / mLMTT solution. After 3 hours, 100 μL of lysis solution was added, and the formed formazan crystals were dissolved overnight at 37°C. The OD value 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 passed through twice. Tumor masses were excised from tumor-bearing mice under sterile conditions, cut into uniformly sized rice-grain-sized pieces, and inoculated subcutaneously into the armpits of nude mice using an insertion needle, with the tumors reaching 100 mm. 3 Once the tumors had grown to a certain extent, nude mice with appropriate tumor masses were selected and randomly grouped before administration began. All samples were lysed in PBS, and the intratumoral injection volume was 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 × Width) 2 ) / 2

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

[0120] Subsequently, the animals were slaughtered, tumor masses were collected and weighed, and biochemical and molecular tests were performed.

[0121] The mice used in the orthotopic glioma transplantation model were 7-week-old NOD-scid mice. Human glioma cells were induced to digest with 0.25% trypsin for 3 days, or not induced, and the supernatant was removed by centrifugation to concentrate the cells to a density of approximately 2.5 × 10⁶.5 This results in cells / μL, with 2μL applied to each mouse's striatum for a total of 5 × 10 5 Individual cells were transplanted. Histochemical testing was performed three weeks after transplantation, or the virus was injected one week later, followed by immunohistochemical testing.

[0122] (Example 1) Vector construction and packaging of recombinant oncolytic virus (i.e., oncolytic virus containing recombinant nucleic acid) strains. In this embodiment, 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 as 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, and constructed into the vector. P2A is a self-cleaving polypeptide, enabling efficient co-expression of hAscl1 and Ngn2, and Ad5-AN was obtained. The empty vector Ad5-Vector served as the experimental control vector.

[0123] Adenovirus packaging and purification were performed by cotransforming adenovirus packaging backbone plasmids and shuttle plasmids into HEK-293 cells. After the cells became plaque-like, the cell supernatant and cell lysates were collected, and the virus was purified by concentration or cesium chloride density gradient centrifugation. Adenovirus titer was measured by enzyme immunoassay, and the viral titer was calculated by counting the number of positive cells that stained brown after infection.

[0124] (Example 2) Selective killing effect of recombinant oncolytic viruses on cancer cells In this example, taking human glioblastoma cells as an example, glioma cell lines U251 and U87, as well as normal human astrocytes HA, were inoculated into T25 tissue culture flasks, and the complete culture medium was DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. On day 1, the cell density was 5 × 10⁶. 4 The culture medium was adjusted to cells / mL, and 96-well plates were plated with 100 μL / well. On day 2, the medium was removed, and the corresponding viruses (Ad5-vector, Ad5-AN) were added in 5 × 10⁻¹⁶ layers so that the MOI value was a multiple of 10. 3 The cells were serially diluted at a dose of cells / well and added to each well to infect the cells, setting up three replicates for each MOI. On day 7, the supernatant was gently removed, 50 μL of medium was added to each well, followed by 20 μL of 5 mg / mLMTT solution. After 3 hours, 100 μL of lysis solution was added, and the formed formazan crystals were dissolved overnight at 37°C. The OD value was measured at 570 nm.

[0125] The results are shown in Figures 1A and 1B, which show the killing curves of Ad5-AN oncolytic adenoviruses with different infection multiplicities against U87 and U118 cells. The IC50 values ​​of Ad5-AN oncolytic adenovirus against U87 and U118 cells were 0.9 and 0.6, respectively, demonstrating excellent tumor-killing ability. On the other hand, the IC50 value against normal human astrocyte HA was 136, indicating that Ad5-AN oncolytic adenoviruses exhibit excellent specificity and safety, providing a broad spatial range for subsequent dosages. The IC50 values ​​of Ad5-vector oncolytic adenovirus against U87 and U118 cells were 0.8 and 0.7, respectively, and the IC50 value against normal human astrocyte HA was 116, demonstrating similarly specific tumor-killing ability.

[0126] (Example 3) Reprogramming effect of recombinant oncolytic viruses on cancer cells This example investigates how transcription factors or combinations of transcription factors with high transformation efficiency incorporated into recombinant oncolytic viruses can promote the differentiation of glioma cells into non-tumor-forming cells, using the combination of Ascl1 and Ngn2 factors with Ad5-AN oncolytic adenovirus as an example.

[0127] Human glioma cells were seeded and cultured for 24 hours. Adenovirus was then added, and a low titer infection mode (MOI of 0.05) was employed to observe the effect of adenovirus on the differentiation of glioma cells into non-tumor-forming cells. To better show infected cells, the tumor cells were co-infected with the lentivirus FUGW-IRES-EGFP, which has green fluorescent protein. 24 hours after infection, the culture 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 infection of cultured human glioma cells U251 with the virus, some Tuj1-positive cells (Tuj1 is a neuronal marker molecule) appeared by cell immunofluorescence detection, exhibiting neuronal morphology. As shown in Figures 2A and 2B, the results in Figures 2A and 2B indicate that Ad5-AN can differentiate U251 glioma cells into non-tumor-forming neurons, with a transformation efficiency of 75.2%. In Ad5-vector-infected glioma cells, cell immunofluorescence detection showed no appearance of Tuj1-positive cells, indicating that the differentiation of glioma cells into non-tumor-forming neurons is specifically mediated by a combination of the factors Ascl1 and Ngn2.

[0128] (Example 4) Inhibition of tumor cell proliferation in an ectopic inoculation model of glioma mice using an oncolytic adenovirus type 5 vector expressing a reprogramming factor. To verify the combined effect of oncolytic viruses and redifferentiation therapy, we will use an oncolytic virus vector (using oncolytic adenovirus type 5 as an example) to express a reprogramming factor, and then promote its proliferation within tumor cells via the specificity of oncolytic adenovirus type 5, thereby achieving a synergistic effect between the oncolytic action in suppressing gliomas and the in vivo differentiation method.

[0129] This example employs a human glioma U87 BALB / CA-nu mouse ectopic inoculation model. Cultured U87 human glioma cells are inoculated into the armpits of nude mice during the logarithmic growth phase, and the tumor size is 100 mm. 3 Once the tumor has grown to a certain extent, nude mice with a suitable tumor mass are selected and randomly divided into groups. After group division, administration is started, with the PBS group serving as the control and the Ad5-AN-low group receiving a dose of 3 × 10⁻¹⁶. 8 The PFU was administered in the Ad5-vector-high and Ad5-AN-high groups at a dose of 1 × 10⁻⁶. 9 PFU was administered every other day for five consecutive days. The volume of the tumor mass was measured and calculated every three days. Subsequently, the animals were euthanized, the tumor mass was collected and weighed, and biochemical and molecular tests were performed.

[0130] The results are shown in Figures 3A, 3B, and 3C. Figures 3A, 3B, and 3C demonstrate the inhibition of tumor cell proliferation in an ectopic inoculation model of glioma mice by an oncolytic adenovirus vector expressing a reprogramming factor. As a result, compared to the control PBS group, the tumor volume of the Ad5-vector-high group decreased by 33.37%, the tumor volume of the Ad5-AN-low group decreased by 32.25%, and the tumor volume of the Ad5-AN-high group decreased by 67.49% (Figure 3A). We found that the addition of the reprogramming factor significantly enhanced the inhibitory ability of the oncolytic adenovirus against gliomas and significantly reduced tumor cell proliferation. Realtime PCR analysis also showed a significant increase in the expression of the initial neuron marker molecule DCX in Ad5-AN-high group cells (Figure 3B), and HE coloration also indicated that glioma proliferation was inhibited (Figure 3C). The mouse tumors were 2000 mm. 3When the tumors proliferated to a certain extent, the animals were sacrificed. The average duration of life was 21.3 days for the control PBS group, 24.5 days for the Ad5 empty vector group, 23.6 days for the Ad5-AN-low group, and 35.4 days for the Ad5-AN-high group (Figure 3A-2). These results demonstrate that the synergistic effect of oncolytic activity and in vivo reprogramming therapy achieves a more significant ability to inhibit glioma growth.

[0131] Furthermore, in the case of 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, respectively) to alanine-proline (AP) (enhancer, SEQ ID NO 18 in the protein sequence, SEQ ID NO 19 in the nucleotide sequence) can further enhance the inhibitory effect of Ad5-AN on gliomas.

[0132] (Example 5) Combined experiments using oncolytic viruses that express reprogramming factors To investigate the combined effect of oncolytic viruses expressing reprogramming factors with existing therapies, this example uses human glioblastoma cells as an example, expressing reprogramming factors using an oncolytic virus (oncolytic adenovirus type 5 as an example) vector, proliferating in tumor cells via the specificity of oncolytic adenovirus type 5, and then using temozolomide in combination.

[0133] This example employs a human glioma U87 BALB / CA-nu mouse ectopic inoculation model. Cultured U87 human glioma cells are inoculated into the armpits of nude mice during the logarithmic growth phase, and the tumor size is 100 mm. 3 Once the tumor has grown to a certain extent, nude mice with a suitable tumor mass are selected and randomly divided into groups. After group division, administration is started, with the PBS group serving as the control and the Ad5-AN group receiving a dose of 1 × 10⁻⁶. 9The patients received PFUs (proliferative fungicides), specifically temozolomide (TMZ) in a gastric dose (15 mg / kg, once daily for 5 doses followed by 2 interruptions), and Ad5-AN in combination with temozolomide (Ad5-AN + TMZ). Ad5-AN was administered every other day for 5 consecutive doses. The volume of the tumor mass was measured and calculated every 3 days. After that, the animals were sacrificed, the tumor mass was collected and weighed, and biochemical and molecular tests were performed.

[0134] The results are shown in Figure 4, with Figure 4A showing the combined treatment experiment with an oncolytic virus expressing a reprogramming factor. The results showed that compared to the control group (PBS), the tumor volume in the Ad5-AN group was significantly reduced, and the tumor volume in the temozolomide group was also significantly reduced. However, the tumor volume in the Ad5-AN and temozolomide combination group was significantly reduced more than that of the Ad5-AN and temozolomide monotherapy groups, and the tumors essentially disappeared (4 / 6). Furthermore, after the extension experiment (Figure 4B), the temozolomide group experienced gradual tumor recurrence and regrowth around 35 days after administration. In particular, 56 days after administration, all tumors in the mice in the seven parallel experiments recurred (Figure 5, 7 / 7), and around 60 days, tumor growth reached 2000 mm². 3 It was found that the tumor volume approached this level, which is also consistent with the high recurrence rate of gliomas after temozolomide chemotherapy in clinical practice. The tumor volume in the Ad5-AN and temozolomide combination group was significantly more effective than in the Ad5-AN group or the temozolomide group alone, with the tumors essentially disappearing (7 / 7). More importantly, when observed at 93 days, none of the tumors in the mice in the parallel experiment across all 7 groups recurred (Figure 6, 0 / 7). These results demonstrate that oncolytic viruses expressing reprogramming factors can achieve superior antitumor effects when used in combination with existing therapies.

[0135] (Example 6) Orthotopic tumor model of oncolytic viruses expressing reprogramming factors To further confirm the therapeutic ability of oncolytic viruses expressing reprogramming factors against tumors, this example uses a human glioblastoma cell orthotopic brain model as an example, expressing reprogramming factors using an oncolytic virus (oncolytic adenovirus type 5 as an example) vector, and proliferating it in tumor cells via the specificity of oncolytic adenovirus type 5. The synergistic effect of oncolytic activity in inhibiting gliomas and in vivo differentiation conversion was then investigated.

[0136] This example first involves intracerebral transplantation (5 × 10) of glioma cells (U87-luc). 5 The procedure was performed. Seven days after transplantation, the patients were divided into groups and administered the drug. The control group was the PBS group, and the Ad5-AN group received a dose of 1 × 10⁶. 9 The study included a PFU group receiving temozolomide intragastricly (15 mg / kg, once daily for 5 doses followed by 2 interruptions), and a group receiving Ad5-AN in combination with temozolomide. Ad5-AN was administered every 4 days for 3 consecutive doses. Thirty days after viral injection, some brain tissue samples from the Ad5-AN group were collected for immunohistochemical analysis. It was found that virus-infected cells expressed the neuronal marker molecule Tuj1 and exhibited neuronal morphology. Simultaneously, 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). Furthermore, 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 showed that, similarly in the case of orthotopic tumors, oncolytic viruses expressing reprogramming factors can achieve superior antitumor effects and long-lasting anti-recurrence effects when used in combination with existing therapies.

[0137] (Example 7) Inhibition of tumor cell proliferation in a glioma PDX model using an oncolytic viral vector expressing a reprogramming factor. To further investigate the combined effects of oncolytic virus and redifferentiation therapy, this example was tested in 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 reinoculated into the armpits of nude mice during the logarithmic growth phase, until the tumor reached 100 mm. 3 Once the tumor has grown to a certain extent, nude mice with a suitable tumor mass are selected and randomly divided into groups. After group division, administration is started, with the PBS group serving as the control and the Ad5-AN group receiving a dose of 1 × 10⁻⁶. 9 The study included a PFU group receiving temozolomide intragastricly (15 mg / kg, once daily for 5 doses followed by 2 interruptions), and a group receiving Ad5-AN in combination with temozolomide. Ad5-AN was administered every other day for 5 consecutive doses. Tumor mass volume was measured and calculated every 3 days. After euthanasia, the tumor mass was collected, weighed, and subjected to biochemical and molecular tests. The results showed that, similar to the subcutaneous model of glioma cell lines, the tumor volume in the Ad5-AN group was significantly reduced compared to the control PBS group, and the tumor in the temozolomide group was also significantly reduced. However, the tumors gradually recurred and regrowed around 45 days after administration (6 / 6), and by day 72, the tumor growth was 2000 mm. 3 The results showed that the tumor volume in the Ad5-AN and temozolomide combination group was significantly more effective than in the Ad5-AN group or the temozolomide group alone, with the tumors essentially disappearing (72 days, 5 / 6), and more importantly, the long-term antitumor effect was observed to be maintained (102 days, 4 / 6 no recurrence). These results demonstrate that oncolytic viruses expressing reprogramming factors can achieve superior antitumor and long-lasting anti-recurrence effects in patient tissue-derived in vivo models when used in combination with existing therapies.

[0138] (Example 8) Inhibition of glioma proliferation by recombinant herpes simplex virus expressing reprogramming factors. In this example, herpes simplex virus type 1 was used as an oncolytic viral vector, and the human Ascl1 (SEQ ID No. 11) fragment and the 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 viruses, each was subjected to ectopic inoculation model tests in human glioma U87 BALB / CA-nu mice. The tumor size was approximately 100 mm. 3 Once the tumors proliferated, nude mice with suitable tumor masses were selected and randomly divided into groups, and administration was started after group division. The doses for the control PBS group, HSV (empty vector without reprogramming factor), and HSV-AN group were 2 × 10⁻⁶. 6 The treatment groups were PFU, temozolomide intragastric group (15 mg / kg, once daily for 5 doses followed by 2 interruptions), and HSV-AN combined with temozolomide. HSV was administered every other day for 5 consecutive doses. Tumor mass volume was measured and calculated every 3 days, after which the animals were sacrificed, the tumor mass was collected and weighed, and biochemical and molecular tests were performed. The results showed that, compared to the control PBS group, the tumor volume of the HSV empty vector group decreased by 25.6% after 10 days of administration, and the tumor volume of the HSV-AN group decreased by 58.9%, indicating that the addition of reprogramming factors significantly increased the inhibitory ability of oncolytic herpesvirus against gliomas. In the temozolomide group, tumor volume decreased by 73.6%, and the recurrence rate at 2 months post-administration was (6 / 6). In the group receiving HSV-AN in combination with temozolomide, tumor volume decreased by 92.5%, and the recurrence rate at 2 months post-administration was (1 / 6). These results all indicate that the synergistic effect of the oncolytic effect of oncolytic herpesvirus drugs and the in vivo differentiation of tumor cells significantly enhances the ability to inhibit glioma proliferation, demonstrating that superior antitumor and long-lasting anti-recurrence effects can be achieved when used in combination with the existing treatment TMZ.

[0139] Although 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 can make various equivalent modifications or substitutions that fall within the scope of the claims of the present invention without departing from the spirit of the invention.

[0140] (Note) (Note 1) It contains recombinant nucleic acids that include functional fragments that promote the reprogramming / differentiation of tumor cells into non-tumoric cells. Recombinant oncolytic virus, characterized in that the functional fragment contains at least one functional fragment that promotes the expression of a transcription factor, and the functional fragment is selected from functional fragments that can promote the expression of at least one transcription factor among NeuroD1, Brn2, Ascl1, or Ngn2.

[0141] (Note 2) The recombinant nucleic acid comprises a set of functional fragments that synergistically promote the reprogramming / differentiation of tumor cells into non-tumoric cells. The recombinant oncolytic virus according to Appendix 1, characterized in that the functional fragment comprises at least two functional fragments that promote the expression of transcription factors, and the functional fragment is selected from functional fragments that can promote the expression of at least two transcription factors among NeuroD1, Brn2, Ascl1, or Ngn2.

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

[0143] (Note 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. Preferably, the recombinant oncolytic virus according to Appendix 1 is characterized in that 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] (Note 5) The aforementioned 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, and SEQ ID NO.16. Preferably, the recombinant oncolytic virus according to Appendix 4 is characterized in that 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] (Note 6) The functional fragment is a polynucleotide encoding a functional protein, having a sequence identity of 75% or more with SEQ ID NO.19, or is a polynucleotide encoding a transcription factor, or The aforementioned 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, having a sequence identity of 85% or more with SEQ ID NO.19, or a polynucleotide encoding a transcription factor. The aforementioned 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, having a sequence identity of 95% or more with SEQ ID NO.19, or a polynucleotide encoding a transcription factor. The recombinant oncolytic virus according to 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] (Note 7) The recombinant oncolytic virus according to Appendix 1, characterized in that the expression system of the functional fragment that promotes the expression of the transcription factor is constructed under the same expression vector or expressed separately using different expression vectors.

[0147] (Note 8) The recombinant oncolytic virus according to any one of Appendix 1 to 7, characterized in that the recombinant oncolytic virus includes a selectively replicating recombinant oncolytic virus.

[0148] (Note 9) The recombinant oncolytic virus described in Appendix 8 is characterized in that the selectively replicating recombinant oncolytic virus is derived from an adenovirus, poxvirus, herpes simplex virus, measles virus, Semryki forest virus, varicella stomatitis virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus, or marabavirus, which have oncolytic activity.

[0149] (Note 10) A method for promoting the reprogramming / differentiation of tumor cells into non-tumor-forming cells using an oncolytic virus, A method characterized by comprising the step of reprogramming tumor cells into non-tumor-forming cells by contacting them with a recombinant oncolytic virus described in any one of appendices 1 to 9.

[0150] (Note 11) A composition for treating cancer, (A) Recombinant oncolytic virus as described in any one of Appendix 1 to 9, (B) A composition characterized by comprising a pharmaceutically acceptable excipient.

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

[0152] (Note 13) Use of recombinant oncolytic viruses as described in any one of Appendix 1 to 9 in the preparation of drugs for the treatment of tumors.

[0153] (Note 14) The use described in Appendix 13, characterized in that, in the use described above, the recombinant oncolytic virus is formulated as a therapeutic agent to be administered within or near a tumor, and the method of administering the therapeutic agent includes one of the following: injection, intraperitoneal administration, subarachnoid administration, or intravenous administration.

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

[0155] (Note 16) The use described in Appendix 13 is characterized in that the tumors include 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, ureteral 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, hematological cancer, peritoneal cancer, or pleural cancer.

[0156] (Note 17) The use described in Appendix 13 is characterized in that the tumor includes one or more types of glioma, astrocytoma, astroblastoma, medulloblastoma, schwannoma, and brain metastatic cancer.

Claims

1. A recombinant oncolytic virus constructed by using an adenovirus or herpes simplex virus having oncolytic properties as a vector and inserting a recombinant nucleic acid into the genome of the vector, The recombinant nucleic acid comprises a functional fragment that promotes the expression of two transcription factors, Ascl1 and Ngn2. The aforementioned functional fragment is a polynucleotide encoding a functional protein, The sequences of the polynucleotides are such that they have a sequence identity of 90% or more with SEQ ID NO. 11 and SEQ ID NO. 15, respectively, or that the sequences of the polynucleotides are such that they have a sequence identity of 90% or more with SEQ ID NO. 19 and SEQ ID NO. 15, respectively, or The sequences of the functional proteins have a sequence identity of 90% or more with SEQ ID NO. 9 and SEQ ID NO. 13, respectively, or the sequences of the functional proteins have a sequence identity of 90% or more with SEQ ID NO. 18 and SEQ ID NO. 13, respectively. A recombinant oncolytic virus characterized by the following:

2. The recombinant oncolytic virus according to claim 1, characterized in that the expression system for the functional fragments that promote the expression of the two transcription factors Ascl1 and Ngn2 is constructed under the same expression vector or expressed separately using different expression vectors.

3. A composition for treating cancer, (A) Recombinant oncolytic virus according to claim 1, (B) A composition comprising a pharmaceutically acceptable excipient.

4. The aforementioned composition, (C) The composition according to claim 3, further comprising an antitumor agent, wherein the antitumor agent comprises one or both of temozolomide and bevacizumab.

5. Use of the recombinant oncolytic virus according to claim 1 in the preparation of a drug for treating a tumor.

6. The use according to claim 5, characterized in that, in the use described above, the recombinant oncolytic virus is formulated as a therapeutic agent to be administered within or near a tumor, and the method of administering the therapeutic agent includes one of the following: injection, intraperitoneal administration, subarachnoid administration, or intravenous administration.

7. The use according to claim 5, wherein the recombinant oncolytic virus is formulated as a therapeutic agent to be administered within or near a tumor, and the method of administering the therapeutic agent includes one or more types of hydrogel administration, convection-enhanced drug delivery, and Ommaya reservoirs.

8. The use according to claim 5, characterized in that the tumor includes glioblastoma, neuroblastoma, meningioma, spinal cord tumor, or retinoblastoma.

9. The use according to claim 5, characterized in that the tumor includes one or more types of glioma, astrocytoma, astroblastoma, medulloblastoma, schwannoma, and brain metastatic cancer.

Images