Reprogrammed functional fragments of recombinant oncolytic viruses, combinations and uses thereof

Abstract

The invention provides a reprogrammed functional fragment of a recombinant oncolytic virus, a composition and application thereof. The invention also provides a group of transcription factors and transcription factor combinations for synergistically promoting tumor cells to transdifferentiate and reprogram into non-tumor cells, effective synergy of an oncolytic therapy and a reprogramming effect is realized through an oncolytic virus expression method, and the transcription factors and transcription factor combinations can be effectively applied to limiting tumor deterioration and improving tumor differentiation. The invention also discloses application of the transcription factor carried by the recombinant oncolytic virus in preparation of medicines for treating tumor diseases.

Description

Functional fragments, combinations and applications of reprogrammed oncolytic viruses Technical Field

[0001] The present invention belongs to the field of biotechnology and gene therapy, and relates to functional fragments, combinations and applications of reprogrammed recombinant oncolytic viruses. Specifically, it relates to a method of using oncolytic virus vectors to transdifferentiate tumor cells into non-tumorigenic cells, and a method of using this method to treat tumors. Background Art

[0002] A hallmark of tumor cells is an uncontrolled cell cycle, leading to malignant proliferation. Reprogramming can be used to induce tumor cells to differentiate into non-tumorigenic cells that no longer divide, thereby "rehabilitating" them and achieving the goal of treating tumors. To date, the most successful example of tumor-induced differentiation therapy is the use of all-trans retinoic acid (ATRA) to treat acute promyelocytic leukemia, achieving a high remission rate.

[0003] Oncolytic virus therapy for tumors is gaining increasing attention. Its principle is to genetically modify some naturally occurring viruses with weak pathogenicity to create specialized oncolytic viruses. These viruses exploit the inactivation or defects of tumor suppressor genes in target cells to selectively infect tumor cells, replicate in large numbers within them, and ultimately destroy them. At the same time, they can also stimulate an immune response, attracting more immune cells to continue killing residual cancer cells. In recent decades, oncolytic virus therapy has attracted widespread attention, and related research has made tremendous progress. Currently, a variety of oncolytic viruses, including adenovirus, herpes simplex virus, coxsackievirus, poxvirus, poliovirus, measles virus, and reovirus, have entered clinical trials. They recognize and infect tumor cells, ultimately causing them to swell and destroy them. However, they are unable to replicate within normal body cells and therefore have no killing effect. In theory, they have a higher anti-tumor effect and fewer side effects.

[0004] Oncolytic viruses are now widely accepted as a key branch of immunotherapy, demonstrating their significant potential in both basic and clinical research for the treatment of malignant tumors. In 2015, the U.S. Food and Drug Administration and the European Medicines Agency approved the HSV-1-like oncolytic virus T-VEC for the treatment of melanoma, marking the maturity of oncolytic virus technology and the recognition of oncolytic viruses for the treatment of malignant tumors. Currently, a variety of oncolytic viruses are being used in clinical studies for tumor treatment, and their efficacy and safety have been verified.

[0005] It is currently believed that oncolytic viruses primarily mediate their anti-tumor effects through the following mechanisms: 1) They specifically multiply within tumor cells and directly lyse them; 2) Lysed tumor cells release viral particles, which stimulate systemic anti-tumor immunity through various pathways, such as promoting tumor antigen presentation, increasing immune cell infiltration in the tumor microenvironment, modulating the tumor microenvironment, activating immune cells, and activating the immune system through the immunomodulatory factors they carry. Furthermore, reports suggest that some viruses can indirectly exert their anti-tumor effects by infecting tumor-associated vascular endothelial cells and inhibiting tumor angiogenesis.

[0006] Oncolytic adenovirus is currently the most commonly used oncolytic virus. There are a variety of strategies to enhance the tumor targeting of oncolytic adenovirus, including mutating functional genes involved in cell cycle node regulation in the adenovirus genome (such as E1A or E1B) and / or using tumor-specific promoters to regulate the targeted transcriptional regulation of the E1A gene, using different serotypes of adenovirus or RGD motifs to change the targeted transduction regulation of the way oncolytic adenovirus enters tumor cells, and using cell vectors to deliver oncolytic adenovirus to distant tumor sites. Oncolytic adenovirus is used as a vector to deliver immunomodulatory genes or therapeutic genes, and produces synergistic anti-tumor effects by enhancing anti-tumor immunity or causing tumor cell apoptosis or suicide. Current oncolytic viruses usually carry immunomodulatory factors or suicide genes, which can further enhance the therapeutic effect of tumors, but there is still a problem of insufficient effectiveness.

[0007] Glioma, also known as glioma, broadly refers to all tumors of neuroepithelial origin, and more narrowly refers to tumors arising from various types of glial cells. Glioma is 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 scheme, gliomas are categorized as astrocytomas, oligodendrogliomas, ependymomas, mixed gliomas, choroid plexus tumors, neuroepithelial tumors of uncertain origin, neuronal and mixed neuronal-glial tumors, pineal parenchymal tumors, embryonal tumors, and neuroblastomas. Glioma growth intersects with normal neural tissue, resulting in unclear boundaries, making tumor tissue difficult to remove and prone to recurrence. Furthermore, the blood-brain barrier (BBB) ​​hinders the efficacy of conventional anti-tumor drugs. Currently, the treatment of gliomas 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 transform glioma cells into neuron-like cells in vitro or in vivo, and limit their proliferation. Currently, commonly used delivery vectors, such as adeno-associated virus (AAV) vectors, cannot replicate themselves and are unable to transfer exogenous genes to a sufficient number of tumor cells, making them difficult to use in clinical tumor treatment.

[0008] Therefore, finding and utilizing appropriate replicative expression vectors that carry appropriate reprogramming factors to infect tumor cells and replicate inside the cells, releasing the virus to infect more tumor cells, causing some tumor cells to be dissolved and killed, some tumor cells to die due to anti-tumor immunity, and some tumor cells that have not died to be differentiated into non-tumorigenic cells that no longer divide, thus achieving effective synergistic effects, is a new treatment option for tumors.

[0009] Summary of the Invention

[0010] In response to the shortcomings of the prior art, the present invention aims to provide a functional fragment, combination, and application of a recombinant oncolytic virus for reprogramming. The present invention provides a set of transcription factors and transcription factor combinations that synergistically promote tumor cell transdifferentiation and reprogramming into non-tumorigenic cells, a method for expressing them through oncolytic viruses, and the use of this set of oncolytic virus-carried transcription factors in the preparation of tumor disease drugs.

[0011] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0012] In a first aspect of the present invention, a recombinant oncolytic virus is provided, wherein the recombinant oncolytic virus contains a recombinant nucleic acid, wherein the recombinant nucleic acid includes a functional fragment that promotes tumor cell reprogramming / transdifferentiation;

[0013] The functional fragment contains at least one functional fragment that promotes the expression of a transcription factor, and the functional fragment is selected from the functional fragment that can promote the expression of at least one transcription factor among NeuroD1, Brn2, Ascl1 or Ngn2.

[0014] In another preferred example, the functional fragments that promote the expression of transcription factors contained in the recombinant oncolytic virus at least include a functional fragment that promotes the expression of Ascl1 transcription factor.

[0015] In another preferred example, the functional fragments that promote the expression of transcription factors contained in the recombinant oncolytic virus at least include a functional fragment that promotes the expression of NeuroD1 transcription factor.

[0016] In another preferred example, the functional fragments that promote the expression of transcription factors contained in the recombinant oncolytic virus at least include a functional fragment that promotes the expression of Brn2 transcription factor.

[0017] In another preferred example, the functional fragments that promote the expression of transcription factors contained in the recombinant oncolytic virus include at least functional fragments that promote the expression of Ngn2 transcription factors.

[0018] In another preferred embodiment, the recombinant nucleic acid includes a group of functional fragments that synergistically promote the reprogramming / transdifferentiation 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.

[0019] In another preferred example, the functional fragments that promote the expression of transcription factors contained in the recombinant oncolytic virus include at least a functional fragment that promotes the expression of NeuroD1 transcription factor and another functional fragment that promotes the expression of transcription factors, and the other functional fragment that promotes the expression of transcription factors is selected from any functional fragment that promotes the expression of transcription factors such as Ascl1, Ngn2 or Brn2.

[0020] More preferably, the another functional fragment that promotes the expression of a transcription factor is selected from any functional fragment that promotes the expression of a transcription factor such as Ascl1 or Ngn2.

[0021] In another preferred example, the functional fragments that promote the expression of transcription factors contained in the recombinant oncolytic virus include at least a functional fragment that promotes the expression of Ngn2 transcription factors and another functional fragment that promotes the expression of transcription factors, and the other functional fragment that promotes the expression of transcription factors is selected from any functional fragment that promotes the expression of transcription factors such as NeuroD1, Brn2, and Ascl1.

[0022] More preferably, the functional fragment that promotes the expression of another transcription factor is selected from any functional fragment that promotes the expression of transcription factors such as Ascl1 or NeuroD1. Further preferably, the functional fragment that promotes the expression of another transcription factor is a functional fragment that promotes the expression of Ascl1 transcription factor, that is, the recombinant oncolytic virus contains a recombinant nucleic acid, the recombinant nucleic acid includes a functional fragment that promotes tumor cell reprogramming / transdifferentiation, and the functional fragment contains functional fragments that promote the expression of Ascl1 transcription factor and Ngn2 transcription factor at the same time.

[0023] In another preferred example, the functional fragment contained in the recombinant oncolytic virus that can synergistically promote tumor cell reprogramming / transdifferentiation or the functional fragment that promotes transcription factor expression is a polynucleotide encoding a functional protein, and the functional protein is a functional protein of transcription factors such as NeuroD1, Brn2, Ascl1, Ngn2, etc.

[0024] Preferably, the functional fragments contained in the oncolytic virus that can synergistically promote glial cell reprogramming / transdifferentiation or the functional fragments that promote transcription factor expression are derived from mammals, and more preferably, the functional fragments are derived from humans or non-human primates.

[0025] In another preferred example, the recombinant oncolytic virus contains a functional fragment that can synergistically promote tumor cell reprogramming / transdifferentiation, and the functional fragment is a polynucleotide encoding a functional protein, and the functional protein is a functional NeuroD1 protein, and the amino acid sequence of the functional NeuroD1 protein is shown in SEQ ID NO.1 or SEQ ID NO.2; the polynucleotide sequence encoding the functional NeuroD1 protein is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0026] In another preferred example, the recombinant oncolytic virus contains a functional fragment that can synergistically promote tumor cell transdifferentiation, and the functional fragment is a polynucleotide encoding a functional protein, and the functional protein is a functional Brn2 protein, and the amino acid sequence of the functional Brn2 protein is shown in SEQ ID NO.5 or SEQ ID NO.6; the polynucleotide sequence encoding the functional Brn2 protein is shown in SEQ ID NO.7 or SEQ ID NO.8.

[0027] In another preferred example, the recombinant oncolytic virus contains a functional fragment that can synergistically promote tumor cell transdifferentiation, and the functional fragment is a polynucleotide encoding a functional protein, and the functional protein is a functional Ascl1 protein, and the amino acid sequence of the functional Ascl1 protein is shown in SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.18; the polynucleotide sequence encoding the functional Ascl1 protein is shown in SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.19.

[0028] In another preferred example, the recombinant oncolytic virus contains a functional fragment that can synergistically promote tumor cell transdifferentiation, and the functional fragment is a polynucleotide encoding a functional protein, and the functional protein is a functional Ngn2 protein, and the amino acid sequence of the functional Ngn2 protein is shown in SEQ ID NO.13 or SEQ ID NO.14; the polynucleotide sequence encoding the functional Ngn2 protein is shown in SEQ ID NO.15 or SEQ ID NO.16.

[0029] In another preferred example, when the functional fragment that can synergistically promote 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 a sequence homology of not less than 85% with respect to 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, for example, it can be 85%, 87%, 89%, 90%, 91%, 93%, 95%, 97% or 99%, etc.

[0030] More preferably, the amino acid sequence of the functional protein has a sequence homology of not less than 95% with that of 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, for example, it can be 95%, 96%, 97%, 98% or 99%.

[0031] Optimally, the amino acid sequence of the functional protein has a sequence homology of not less than 99% with that of 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.

[0032] In another preferred example, when the functional fragment that can synergistically promote tumor cell transdifferentiation contained in the recombinant oncolytic virus is a polynucleotide encoding a functional protein, the polynucleotide sequence encoding the functional protein has a sequence homology of not less than 75% with respect to 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, for example, it can be 75%, 77%, 79%, 80%, 85%, 87%, 89%, 90%, 91%, 93%, 95%, 97% or 99%, etc.

[0033] More preferably, the polynucleotide sequence encoding the functional protein has a sequence homology of not less than 85% 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, for example, it can be 85%, 87%, 89%, 90%, 91%, 93%, 95%, 97% or 99%, etc.

[0034] Optimally, the polynucleotide sequence encoding the functional protein has a sequence homology of not less than 95% 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, for example, it can be 95%, 96%, 97%, 98% or 99%.

[0035] Preferably, the expression system of the functional fragments that promote the expression of transcription factors is constructed under the same expression vector, or expressed separately using different expression vectors.

[0036] Preferably, the recombinant oncolytic virus comprises a selectively replicative recombinant oncolytic virus.

[0037] Preferably, the selectively replicable 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, Ekovirus, Coxsackievirus, Newcastle disease virus or Maraba virus.

[0038] Preferably, the tumor cell is any glioblastoma, glioma, astrocytoma, astroblastoma, medulloblastoma, schwannoma, brain metastasis, 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 from human or non-human mammals. , 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, stomach cancer, thymic cancer, trophoblastic carcinoma, endometrial cancer, vaginal cancer, vulvar cancer, insulinoma, blood cancer, peritoneal cancer, or pleural cancer.

[0039] Preferably, the reprogramming / transdifferentiation refers to reprogramming or transdifferentiation of tumor cells into non-tumorigenic cells.

[0040] In the second aspect of the present invention, a method for promoting the reprogramming / transdifferentiation of tumor cells into non-tumorigenic cells by oncolytic viruses is provided, the method comprising the following steps: contacting the recombinant oncolytic virus described in the first aspect of the present invention with tumor cells, thereby reprogramming / transdifferentiating the tumor cells into non-tumorigenic cells.

[0041] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

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

[0043] In another preferred embodiment, the method is an in vivo method.

[0044] In another preferred embodiment, the method is therapeutic. It should be noted that the method does not conflict with existing tumor therapies and can be used in combination with therapies such as immunotherapy, CAR-T, and TTF.

[0045] Preferably, the tumor cell is any glioblastoma, glioma, astrocytoma, astroblastoma, medulloblastoma, schwannoma, brain metastasis, 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 from human or non-human mammals. , 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, stomach cancer, thymic cancer, trophoblastic carcinoma, endometrial cancer, vaginal cancer, vulvar cancer, insulinoma, blood cancer, peritoneal cancer, or pleural cancer.

[0046] In another preferred embodiment, any method that can promote the increased expression of transcription factors that promote glial cell transdifferentiation includes but is not limited to directly contacting or introducing the inducing factor or a functional fragment that promotes the expression of the transcription factor with the tumor cells, thereby promoting the increased expression of any NeuroD1, Brn2, Ascl1, or Ngn2 transcription factors in glial cells and promoting the transdifferentiation of the tumor cells into non-tumorigenic cells.

[0047] In the present invention, the delivery system is the oncolytic virus, and the selective replicative 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, Ekko virus, Coxsackie virus, Newcastle disease virus or Maraba virus with oncolytic effect.

[0048] In another preferred example, the recombinant oncolytic virus-carrying expression vector of the transcription factor polynucleotide may also carry other functional fragments at the same time. The other functional fragments may be reporter genes or other functional fragments of transcription factors with reprogramming functions, including but not limited to those selected from NeuroD1, Brn2, Ascl1 or Ngn2.

[0049] Preferably, at least two polynucleotide fragments of transcription factors can be carried in the same vector, and the polynucleotide fragments of the two transcription factors can be expressed under a tumor cell-specific promoter, or respectively expressed under two tumor cell-specific promoters. When two or more transcription factors are in the transcript of a single promoter, the promoter and the open reading frames of multiple transcription factors are connected in series through a polycistronic element, wherein the transcription factors are separated by IRES or 2A polypeptide (P2A) elements 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).

[0050] In another preferred embodiment, the recombinant oncolytic virus comprises a group of functional fragments that can synergistically promote tumor cell reprogramming / transdifferentiation, and the expression system of the functional fragments that promote the expression of transcription factors can be constructed under the same expression vector, or expressed separately using different expression vectors.

[0051] In a third aspect of the present invention, a composition for treating cancer is provided, comprising:

[0052] (A) the recombinant oncolytic virus according to the first aspect;

[0053] (B) Pharmaceutically acceptable excipients.

[0054] In some embodiments, the composition further comprises:

[0055] (C) Anti-tumor drugs;

[0056] The anti-tumor drugs include one or both of temozolomide and bevacizumab.

[0057] In another preferred embodiment, the pharmaceutical composition is a liquid preparation or a lyophilized preparation.

[0058] In another preferred embodiment, the pharmaceutical composition is an injection.

[0059] In a fourth aspect of the present invention, there is provided use of the recombinant oncolytic virus as described in the first aspect in the preparation of a medicament for treating tumors.

[0060] In another preferred embodiment, in the use, the recombinant oncolytic virus is formulated into a therapeutic agent for administration through the tumor or near the tumor, and the administration of the therapeutic agent includes any one of injection, hydrogel administration, convection-enhanced delivery (CED), Ommaya capsule administration, intraperitoneal administration, subarachnoid administration or intravenous administration.

[0061] Preferably, the tumor includes: glioblastoma, glioma, astrocytoma, astroblastoma, medulloblastoma, schwannoma, brain metastasis, 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, nerve 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, stomach cancer, thymic cancer, trophoblastic carcinoma, endometrial cancer, vaginal cancer, vulvar cancer, insulinoma, blood cancer, peritoneal cancer, or pleural cancer.

[0062] The numerical range described in the present invention includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] FIG1A and FIG1B show the killing curves of Ad5-AN oncolytic adenovirus at different multiplicity of infection on U87 and U118 cells in Example 2.

[0064] FIG2A and FIG2B show that Ad5-AN in Example 3 can transdifferentiate glioma cells U251 into non-tumorigenic neuronal cells.

[0065] Figures 3A, 3B and 3C show that the type 5 oncolytic adenovirus vector expressing reprogramming factors in Example 4 inhibits the growth of tumor cells in the ectopic inoculation model of glioma mice; Figure 3A-2 is the experimental result after extending the experimental days in Figure 3A.

[0066] FIG4 shows the experimental results of oncolytic virus combined with drug expression of reprogramming factors in Example 5, and FIG4-2 shows the experimental results after the experimental days in FIG4 were extended.

[0067] FIG5 shows seven parallel experiments of the temozolomide group (TMZ) in Example 5, in which the tumors of the seven mice numbered AG all recurred 56 days after administration (7 / 7);

[0068] FIG6 shows seven parallel experiments of the Ad5-AN and temozolomide combination treatment group in Example 5. The seven mice numbered AG had no tumor recurrence 93 days after administration (0 / 7). DETAILED DESCRIPTION

[0069] After extensive and in-depth research, the inventors found that the use of oncolytic virus expression vectors, carrying a group of transcription factors or combinations of transcription factors with transdifferentiation and reprogramming functions, can differentiate tumor cells into non-tumorigenic cells that no longer divide in vitro or in vivo. Based on this discovery, the inventors further explored the application of this method in exploring the development of tumor drugs, especially in glioma animal models, and observed that oncolytic virus expression vectors can achieve effective synergy between oncolytic therapy and reprogramming, enhance the anti-tumor effect, significantly reduce the size of the tumor in animals, and significantly prolong the survival time. Therefore, this batch of transcription factors or combinations of transcription factors with transdifferentiation / reprogramming functions using oncolytic virus expression vectors are expected to be applied to the development of tumor drugs, especially glioma drugs.

[0070] the term

[0071] The term "administering" refers to the physical introduction of the product of the 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, spinal or other parenteral routes of administration, such as by injection or infusion.

[0072] The term "about" may refer to a value or composition within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which will depend 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 of regulating or changing the biological activity of a cell, converting it from one biological state to another, generally including differentiation (from progenitor cells to terminal cells), dedifferentiation (from terminal cells to pluripotent stem cells), transdifferentiation (from one terminal cell to another terminal cell), reverse differentiation (from terminal cells to progenitor cells), transdetermination (from one progenitor cell to another terminal cell naturally differentiated from a progenitor cell), etc., which changes the fate of a cell.

[0073] In the present invention, the "transdifferentiation" or "reprogramming" or "transdifferentiation reprogramming" or "transdifferentiation / reprogramming" or "reprogramming / transdifferentiation" specifically refers to the process from one terminal cell to another terminal cell, specifically, the process of tumor cells converting into non-tumorigenic cells.

[0074] transcription factors

[0075] The present invention provides a group of transcription factors with reprogramming functions. These transcription factors and their combinations have excellent transdifferentiation capabilities and can be used to promote the efficiency of transdifferentiation of glial cells into neurons.

[0076] As used herein, the term "transcription factor of the present invention" refers to one or a group of transcription factors required for neural cell differentiation selected from the group consisting of NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and Otx2.

[0077] Preferably, the transcription factor of the present invention comprises at least two of the aforementioned transcription factors.

[0078] The NeuroD1 functional fragment is a polynucleotide or protein fragment derived from a mammal that encodes the Neurogenic Differentiation 1 transcription factor. NeuroD1 is a bHLH (basic helix-loop-helix) transcription factor. For example, the human NeuroD1 molecule has a GenBank ID# of 4760, a protein sequence shown in SEQ ID NO.1, NCBI Reference Sequence: NM_002500.5, and a CDS sequence shown in SEQ ID NO.3.

[0079] Brn2 functional fragments, also known as POU3F2, Oct7, and N-Oct3, are polynucleotides or protein fragments derived from mammals that encode Pou class 3homeobox 2 transcription factors. Brn2 belongs to the POU-III type transcription factor family that is specific to neurons. For example, the human Brn2 molecule has GenBank ID# 5454, and its protein sequence is shown in SEQ ID NO. 5; NCBI Reference Sequence: NM_005604.4, and its CDS sequence is shown in SEQ ID NO. 7.

[0080] The functional fragment of Ascl1 is a polynucleotide or protein fragment thereof derived from a mammal and encoding an Achaete-scute homolog 1 transcription factor. Ascl1 is a bHLH (basic helix-loop-helix) transcription factor. For example, the Ascl1 molecule from human has GenBank ID# 429, its protein sequence is shown in SEQ ID NO. 9; NCBI Reference Sequence: NM_004316.4, and its CDS sequence is shown in SEQ ID NO. 11.

[0081] The functional fragment of Ngn2, also known as Neurog2, is a polynucleotide or protein fragment derived from mammals that encodes the Neurogenin-2 transcription factor. Ngn2 is a bHLH (basic helix-loop-helix) transcription factor. For example, the human Ngn2 molecule has an ID# of 63973 in GenBank, and its protein sequence is shown in SEQ ID NO. 13; NCBI Reference Sequence: NM_024019.4, and its CDS sequence is shown in SEQ ID NO. 15.

[0082] Any method that can increase the expression of transcription factors that promote glial cell transdifferentiation, including but not limited to directly contacting or introducing an inducing factor or a functional fragment that promotes the expression of a transcription factor into the tumor cells, thereby promoting the expression of any of NeuroD1, Brn2, Ascl1, and Ngn2 transcription factors in glial cells and promoting the transdifferentiation of the tumor cells into non-tumorigenic cells. The method of increasing the expression of the functional fragment of the above-mentioned transcription factor can also be achieved by targeting the DNA of the relevant transcription factor to activate the expression of the gene, or by targeting the RNA of the relevant transcription factor by CRISPR / Cas13 to increase the expression of the functional protein of the transcription factor.

[0083] Those skilled in the art can screen for methods to promote the aforementioned transcription factors based on existing databases. It should be understood that based on the functions of the transcription factors disclosed herein on tumor cell transdifferentiation, those skilled in the art can reasonably foresee that any substance that promotes the aforementioned transcription factors will also be functional in promoting tumor cell transdifferentiation.

[0084] Preferably, the reprogramming transcription factor of the present invention can be used in conjunction with a modified expression element to further enhance the expression of the transcription factor of the present invention.

[0085] Oncolytic viruses

[0086] The oncolytic virus described in the present invention is a replicative recombinant oncolytic virus derived from adenovirus, poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, vesicular stomatitis virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, Ekovirus, Coxsackievirus, Newcastle disease virus and Maraba virus with oncolytic effect.

[0087] The oncolytic virus that can be used in the present invention is not particularly limited, but is preferably derived from adenovirus type 5, poxvirus, and herpes simplex virus type 1 or type 2.

[0088] tumor cells

[0089] As used herein, tumor cells are any glioblastoma, glioma, astrocytoma, astroblastoma, medulloblastoma, schwannoma, brain metastasis, 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, or any other human or non-human mammal. , 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, stomach cancer, thymic cancer, trophoblastic carcinoma, endometrial cancer, vaginal cancer, vulvar cancer, insulinoma, blood cancer, peritoneal cancer, or pleural cancer.

[0090] The tumor cells that can be used in the present invention are not particularly limited. Preferably, they are derived from various gliomas including those from the central nervous system of mammals, such as glioblastoma, astrocytoma, oligodendroglioma, ependymoma or neuroblastoma.

[0091] Glioma, also known as glioma or glioma of the glioma, broadly refers to all tumors of neuroepithelial origin, and narrowly refers to tumors arising from various types of glial cells. Glioma is 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 scheme, gliomas are categorized as astrocytomas, oligodendrogliomas, ependymomas, mixed gliomas, choroid plexus tumors, neuroepithelial tumors, neuronal and mixed neuronal-glial tumors, pineal parenchymal tumors, embryonal tumors, and neuroblastomas.

[0092] Pharmaceutical composition and administration method

[0093] The present invention also provides a pharmaceutical composition, which is a pharmaceutical composition for treating cancer, comprising a recombinant oncolytic virus containing a combination of functional fragments that promote the expression of tumor cell transdifferentiation transcription factors and a pharmaceutically acceptable carrier.

[0094] Pharmaceutically acceptable carriers refer to carriers used for administration of therapeutic agents, including various excipients and diluents.

[0095] The pharmaceutical composition of the present invention generally contains 10 6 -10 9 PFU / g of adenovirus type 5 particles, preferably 10 6 -108 PFU / g of adenovirus type 5 particles, preferably 10 7 -10 8 PFU / g of adenovirus type 5 particles.

[0096] The pharmaceutical composition of the present invention generally contains 10 5 -10 8 PFU / g of herpes simplex virus type 1 particles, preferably 10 6 -10 8 PFU / g of herpes simplex virus type 1 particles, preferably 10 6 -10 7 PFU / g of herpes simplex virus type 1 particles.

[0097] Pharmaceutically acceptable carriers may include any one or a combination of at least two of a coating layer, a capsule, a microcapsule or a nanocapsule. It should be noted that the carrier needs to be non-toxic and may have no significant effect on the activity of the key components in the composition (e.g., the above-mentioned oncolytic virus, the anti-cancer promoting molecules expressed by the oncolytic virus). In some embodiments, the carrier can protect the key components in the composition and reduce or avoid the phenomenon of inactivation or decomposition of the key components under some negative conditions (e.g., oxidation, denaturation caused by strong acid or strong base, etc.). For example, enzymes in gastric juice or relatively low pH values ​​may cause the key components to decompose or inactivate. The carrier can help maintain or improve the efficacy of the pharmaceutical composition by protecting the key components in the composition.

[0098] In some embodiments, the carrier can be used for controlled release of key components (e.g., oncolytic viruses). Controlled release may include, but is not limited to, slow release, sustained release, or targeted release. For example, the carrier may include hydrogel capsules, microcapsules, or nanocapsules made of any one or a combination of at least two of collagen, gelatin, chitosan, alginate, polyvinyl alcohol, polyethylene oxide, starch, or cross-linked starch.

[0099] In some embodiments, pharmaceutically acceptable carriers may include dispersion media (e.g., solvents), coatings, buffers, stabilizers, isotonic or absorption delaying agents, etc. Exemplary pharmaceutically acceptable carriers may include phosphate-buffered saline solutions, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, gels, or bioabsorbable matrix materials, etc., or other suitable materials, or any combination thereof.

[0100] In some embodiments, the excipient may include, but is not limited to, water, saline, polyethylene glycol, hyaluronic acid, ethanol, a pharmaceutically acceptable salt, for example, a salt of an inorganic acid (e.g., hydrochloric acid, hydrobromide, phosphate or sulfate, etc.) or a salt of an organic acid (e.g., acetate, propionate or benzoate, etc.).

[0101] Generally, the pharmaceutical composition of the present invention can be obtained by mixing the expression vector and a pharmaceutically acceptable carrier.

[0102] There is no particular limitation on the administration method of the composition of the present invention. Representative examples include (but are not limited to): intratumoral or near-tumor injection, hydrogel administration, convection-enhanced delivery (CED), Ommaya capsule administration, intraperitoneal administration, subarachnoid administration, or intravenous administration.

[0103] The pharmaceutical composition can be administered to a subject suffering from cancer, for example, a human or an animal. In some embodiments, the pharmaceutical composition can be administered to a subject via one or more modes of administration. The one or more modes of administration may include, but are not limited to, oral administration, injection, or topical administration. The form of a composition suitable for oral administration may include, but is not limited to, tablets, liposome preparations, sustained-release capsules, microparticles, microspheres, or any other suitable form. The form of a composition suitable for injectable administration may include, but is not limited to, sterile aqueous solutions or oily preparations, etc. The form of a composition suitable for topical administration may include, but is not limited to, sterile aqueous solutions or non-aqueous solutions, suspensions, or emulsions. Taking nasal administration as an example, the form of the composition may include aerosols, mists, powders, solutions, suspensions, or gels, etc.

[0104] In some embodiments, the pharmaceutical composition can be stored at a suitable temperature, which may include room temperature (about 20°C), 40°C, -20°C, and -80°C, etc. The composition can also be made into various forms that are conducive to storage and transportation, such as powders. The powder can be a sterile powder, and a solvent can be added to the sterile powder and mixed evenly before use to prepare a solution for oral, injection, or topical administration. In some embodiments, the pharmaceutical composition may also include a component that has an antibacterial effect but has no significant negative impact on the survival of the oncolytic virus, so 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).

[0105] Therapeutic applications

[0106] The recombinant oncolytic virus of the present invention contains any molecular entity that promotes the expression or activity enhancement of the functional fragment of the transcription factor, or a delivery system containing the functional fragment of the transcription factor or the activity-enhanced molecular entity can be used to prepare anti-tumor drugs.

[0107] The composition of the present application can be used before or after administering other pharmaceutical compositions for treating cancer. Alternatively, the composition disclosed in the present application and other treatment modalities can be combined to treat the cancer suffered by the subject. For example, other treatment modalities include but are not limited to administering other pharmaceutical compositions that can treat cancer to the subject, surgically removing the subject's tumor, radiotherapy or electric field therapy, etc. Specifically, pharmaceutical compositions that can be used to treat cancer include but are not limited to cytotoxic anticancer drugs and / or non-cytotoxic anticancer drugs. Non-cytotoxic anticancer drugs can include hormone drugs, targeted drugs (e.g., bevacizumab) or immunotherapy drugs (e.g., monoclonal antibodies and / or tumor vaccines), etc.

[0108] Beneficial effects of the present invention

[0109] After extensive and in-depth research, the inventors found that the use of oncolytic virus expression vectors, carrying a group of transcription factors or combinations of transcription factors with transdifferentiation and reprogramming functions, can differentiate tumor cells into non-tumorigenic cells that no longer divide in vitro or in vivo. Based on this discovery, the inventors further explored the application of this method in exploring the development of tumor drugs, especially in glioma animal models, and observed that oncolytic virus expression vectors can achieve effective synergy between oncolytic therapy and reprogramming, enhance the anti-tumor effect, significantly reduce the size of the tumor in animals, and significantly prolong the survival time. Therefore, this batch of transcription factors or combinations of transcription factors with transdifferentiation / reprogramming functions using oncolytic virus expression vectors are expected to be applied to the development of tumor drugs, especially glioma drugs.

[0110] Compared with the existing technology, the present invention has the following main competitive advantages: innovative use of oncolytic viruses combined with reprogramming effects, obtaining a group of transcription factors or combinations with reprogramming functions expressed in oncolytic viruses and recombinant oncolytic viruses, and exploring the ability of transcription factors and their combinations to transdifferentiate tumor cells into non-tumorigenic cells. Such recombinant oncolytic viruses have both efficient tumor killing and tumor inhibition effects, which can better treat tumors and prevent tumor recurrence.

[0111] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0112] The experimental methods in the following examples where specific conditions are not specified were generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0113] General Methods

[0114] Materials and Methods

[0115] Amino acid sequence

[0116] SEQ ID NO.1 (hNeuroD1 amino acid sequence):

[0117] SEQ ID NO.2 (mNeuroD1 amino acid sequence):

[0118] SEQ ID NO.3 (hNeuroD1 nucleotide sequence):

[0119] SEQ ID NO.4 (mNeuroD1 nucleotide sequence):

[0120] SEQ ID NO.5 (hBrn2 amino acid sequence):

[0121] SEQ ID NO.6 (mBrn2 amino acid sequence):

[0122] SEQ ID NO.7 (hBrn2 nucleotide sequence):

[0123] SEQ ID NO.8 (mBrn2 nucleotide sequence):

[0124] SEQ ID NO.9 (hAscl1 amino acid sequence):

[0125] SEQ ID NO.10 (mAscl1 amino acid sequence):

[0126] SEQ ID NO.11 (hAscl1 nucleotide sequence):

[0127] SEQ ID NO.12 (mAscl1 nucleotide sequence):

[0128] SEQ ID NO.13 (hNgn2 amino acid sequence):

[0129] SEQ ID NO.14 (mNgn2 amino acid sequence):

[0130] SEQ ID NO.15 (hNgn2 nucleotide sequence):

[0131] SEQ ID NO.16 (mNgn2 nucleotide sequence):

[0132] SEQ ID NO.17:

[0133] SEQ ID NO.18 (h-SA-Ascl1 amino acid sequence):

[0134] SEQ ID NO.19 (h-SA-Ascl1 nucleotide sequence):

[0135] Cell culture

[0136] For human glioma cell lines U251 and U87 cells (purchased from the cell bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), as well as normal human astrocyte HA cells (purchased from Sciencell), the culture medium was DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin in an incubator containing 5% CO2 at 37°C. After the addition of viral infection, the medium was completely replaced with induction medium (DMEM, 2% B-27, 1% PS) 12 hours after infection, and again with neural culture medium (DMEM / F-12, 2% B-27, 1% PS, 20ng / mL BDNF, 20ng / mL GDNF) 48 hours later. Half of the culture medium was subsequently replaced every three days.

[0137] Immunochromogenic

[0138] Immunofluorescence 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)). Immunofluorescence of tissue sections was performed according to published methods. Primary antibodies used for immunofluorescence 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.

[0139] MTT assay method

[0140] On the first day, the cell density was adjusted to 5 × 10 4 100 μL / well of the 96-well plate was plated. The next day, the culture medium was removed and 5×10 3 Count / well. Serially dilute the corresponding virus at a multiplicity of infection (MOI) of ten and add it to each well to infect the cells, with three replicates per MOI. On the seventh day, gently remove the supernatant and add 50 μL of culture medium to each well, followed by 20 μL of 5 mg / mL MTT solution. After 3 hours, add 100 μL of dissolution buffer and incubate at 37°C overnight to dissolve the formed formazan crystals. Measure the OD at 570 nm.

[0141] Glioma model

[0142] Glioma subcutaneous model: culture human U87 human glioma cells, in the logarithmic growth phase, inoculated into the axilla of nude mice, and after two passages. The tumor mass was removed from the tumor-bearing mice under sterile conditions, cut into small pieces of uniform size, and inoculated into the nude mouse axilla using an insert inoculation needle. When the tumor grew to 100mm, 3Nude mice with appropriate tumor masses were randomly divided into groups. After grouping, drug administration began. All samples were dissolved in PBS and the intratumoral injection volume was 50 μL / tumor. The length and width of the tumor masses were measured every 3 days, and the tumor volume was calculated using the following formula:

[0143] Volume = (length × width 2 ) / 2

[0144] The tumor inhibition rate was calculated according to the following formula:

[0145] Tumor inhibition rate % = (V model group - V drug group) / V model group × 100%

[0146] The animals were sacrificed at a later stage, and the tumor masses were weighed and subjected to biochemical and molecular tests.

[0147] The mice used for the orthotopic glioma model were seven-week-old NOD-scid mice. Human glioma cells that had been induced for 3 days or not were digested with 0.25% trypsin. The supernatant was removed by centrifugation and the cell density was approximately 2.5×10 5 cells / μL, 2 μL was transplanted into the striatum of each mouse, i.e., a total of 5×10 5 Cells were transplanted and histochemistry was performed 3 weeks after transplantation or one week after virus injection, followed by immunohistochemistry.

[0148] Example 1 Vector Construction and Packaging of Recombinant Oncolytic Virus (i.e., Oncolytic Virus Containing Recombinant Nucleic Acid) Strain

[0149] Among the recombinant oncolytic viruses involved 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 is shown in SEQ ID NO.17) replaces the endogenous promoter of the wild-type E1A gene, and a fragment derived from human Ascl1 (SEQ ID No.11) and a CDS (SEQ ID No.15) fragment of the human Ngn2 gene are inserted into the vector. P2A is a self-cleaving polypeptide that achieves efficient co-expression of hAscl1 and Ngn2 to obtain Ad5-AN. The empty Ad5-Vector is used as the experimental control vector.

[0150] For adenovirus packaging and purification, HEK-293 cells are co-transfected with the adenovirus packaging backbone plasmid and the shuttle plasmid. After the cells develop plaques, the supernatant and cell lysate are collected and the virus is purified by concentration or cesium chloride density gradient centrifugation. Adenovirus titer is determined by enzyme-linked immunosorbent assay (ELISA), and the virus titer is calculated by counting the number of positive cells that stain brown after infection.

[0151] Example 2 Selective killing effect of recombinant oncolytic virus on cancer cells

[0152] In this example, human glioblastoma cells were used as an example. Glioma cell lines U251 and U87, as well as normal human astrocytes HA, were seeded into T25 tissue culture flasks. The complete culture medium was DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. The cell density was adjusted to 5×10 on the first day. 4 100 μL / well of the 96-well plate was plated. The next day, the culture medium was removed and 5×10 3 100 μL of virus (Ad5-vector, Ad5-AN) was serially diluted at an MOI of ten and added to each well to infect the cells, with three replicates per MOI. On the seventh day, the supernatant was gently removed and 50 μL of culture medium was added to each well, followed by 20 μL of 5 mg / mL MTT solution. After 3 hours, 100 μL of dissolution buffer was added. The cells were incubated at 37°C overnight to dissolve the formed formazan crystals, and the OD value was measured at 570 nm.

[0153] The results are shown in Figures 1A and 1B, which show the killing curves of Ad5-AN oncolytic adenovirus at different infection multiplicities for U87 and U118 cells. The IC50 of Ad5-AN oncolytic adenovirus for U87 and U118 cells were 0.9 and 0.6, respectively, showing good tumor killing ability, while the IC50 for normal human astrocyte HA was 136, indicating that Ad5-AN oncolytic adenovirus has good specificity and safety, providing a larger spatial range for subsequent dosing. The IC50 of Ad5-vector oncolytic adenovirus for U87 and U118 cells were 0.8 and 0.7, respectively, while the IC50 for normal human astrocyte HA was 116, which also has specific tumor killing ability.

[0154] Example 3 Reprogramming effect of recombinant oncolytic virus on cancer cells

[0155] This example explores the application implementation plan of recombinant oncolytic viruses carrying transcription factors or transcription factor combinations with high conversion efficiency to promote the transdifferentiation of glioma cells into non-tumorigenic cells, taking the Ad5-AN oncolytic adenovirus with the factor combination of Ascl1 and Ngn2 as an example.

[0156] Human glioma cells were plated and cultured for 24 hours before adenovirus was added. To observe the effect of adenovirus on the transdifferentiation of glioma cells into non-tumorigenic cells, a low-titer infection model (MOI of 0.05) was used. To better indicate infected cells, tumor cells were co-infected with the green fluorescent protein-carrying lentivirus FUGW-IRES-EGFP. After 24 hours of 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 culture medium every three days. Ten days after infection of cultured human glioma U251 cells, immunofluorescence analysis revealed the emergence of some Tuj1-positive cells (a neural marker) exhibiting neuronal morphology, as shown in Figures 2A and 2B. These results demonstrate that Ad5-AN can transdifferentiate U251 glioma cells into non-tumorigenic neurons, with a conversion efficiency of 75.2%. In Ad5-vector infected glioma cells, no Tuj1-positive cells were found by cell immunofluorescence, indicating that the promotion of glioma cell transdifferentiation into non-tumorigenic neuronal cells is specifically mediated by the combination of Ascl1 and Ngn2 factors.

[0157] Example 4: Oncolytic adenovirus type 5 vector expressing reprogramming factors inhibits the growth of tumor cells in a mouse ectopic inoculation model of glioma

[0158] In order to verify the combined effect of oncolytic virus and redifferentiation therapy, oncolytic virus (for example, type 5 oncolytic adenovirus) vectors were used to express reprogramming factors. With the help of type 5 oncolytic adenovirus specificity amplification in tumor cells, the oncolytic effect and in vivo transdifferentiation therapy were used to synergistically inhibit gliomas.

[0159] This example uses a human glioma U87BALB / CA-nu mouse ectopic inoculation model. Cultured U87 human glioma cells are inoculated into the axilla of nude mice during the logarithmic growth phase. 3 The nude mice with appropriate tumor masses were randomly divided into groups. After grouping, drug administration was started. The control group was the PBS group, and the Ad5-AN-low group was given a dose of 3×10 8 PFU, the dose of Ad5-vector-high and Ad5-AN-high groups was 1×10 9 PFU were administered once every two days for 5 times. The volume of the tumor was measured and calculated every 3 days. At the end of the study, the animals were sacrificed, and the tumors were weighed and subjected to biochemical and molecular testing.

[0160] The results are shown in Figures 3A, 3B and 3C. Figures 3A, 3B and 3C show that the type 5 oncolytic adenovirus vector expressing reprogramming factors inhibited the growth of tumor cells in the ectopic inoculation model of glioma mice. The results showed that compared with 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). It can be seen that the addition of reprogramming factors significantly enhanced the inhibitory ability of oncolytic adenovirus on glioma and significantly reduced the growth of tumor cells. Realtime-PCR analysis also found that the expression of early neuronal marker molecule DCX in cells in the Ad5-AN-high group was significantly increased (Figure 3B), and HE staining also found that the growth of glioma was inhibited (Figure 3C). When the mouse tumor grew to nearly 2000mm 3 The animals were then sacrificed, with an average survival time of 21.3 days in the PBS control group, 24.5 days in the Ad5 empty vector group, 23.6 days in the Ad5-AN-low group, and 35.4 days in the Ad5-AN-high group (Figure 3A-2). These results suggest that oncolysis and in vivo reprogramming therapy synergize to achieve a more pronounced inhibition of glioma growth.

[0161] In addition, for the human Ascl1 protein, if the 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) are mutated to alanine-proline (AP) (enhanced type, protein sequence SEQ ID NO: 18, nucleotide sequence SEQ ID NO: 19), the inhibitory ability of Ad5-AN on glioma can be further improved.

[0162] Example 5 Experiment on combined use of oncolytic viruses expressing reprogramming factors

[0163] In order to explore the effect of combining oncolytic viruses expressing reprogramming factors with existing therapies, this example uses human glioblastoma cells as an example, uses oncolytic virus (type 5 oncolytic adenovirus as an example) vectors to express reprogramming factors, and uses type 5 oncolytic adenovirus to specifically amplify them in tumor cells, combined with temozolomide.

[0164] This example uses a human glioma U87BALB / CA-nu mouse ectopic inoculation model. Cultured U87 human glioma cells are inoculated into the axilla of nude mice during the logarithmic growth phase. 3 The nude mice with appropriate tumor masses were randomly divided into groups. After grouping, drug administration was started. The control group was the PBS group, and the Ad5-AN group was given 1×10 9PFU, temozolomide (TMZ) gavage group (15 mg / kg, once daily, 5 doses, 2 rests), and Ad5-AN group and temozolomide combination group (Ad5-AN + TMZ), Ad5-AN was administered every two days for 5 consecutive doses. Tumor volume was measured and calculated every 3 days. Later, the animals were sacrificed, and tumors were weighed and subjected to biochemical and molecular analysis.

[0165] The results are shown in Figure 4, which shows an experiment on the combined use of oncolytic viruses expressing reprogramming factors. The results showed that compared with the control PBS group (control group), the tumor volume of the Ad5-AN group was significantly reduced, the tumor volume of the temozolomide group was significantly reduced, and the tumor volume of the Ad5-AN and temozolomide combination group was significantly greater than that of the Ad5-AN group and the temozolomide group alone, and the tumor basically disappeared (4 / 6); in addition, after an extended experiment (Figure 4-2), it was found that the tumor in the temozolomide group gradually recurred and grew back about 35 days after administration, especially after 56 days of administration, the tumors of the mice in the seven parallel experiments all recurred (Figure 5, 7 / 7), and the tumor grew to nearly 2000mm around 60 days. 3 This is consistent with the high recurrence of glioma after temozolomide chemotherapy in clinical practice. The tumor volume of the Ad5-AN and temozolomide combination group was significantly higher than that of the Ad5-AN group and the temozolomide group alone, and the tumor basically disappeared (7 / 7). More importantly, after 93 days, the tumors of the mice in the seven parallel experiments did not recur (Figure 6, 0 / 7). The results show that oncolytic viruses expressing reprogramming factors can be used in combination with existing therapies to achieve better anti-tumor effects.

[0166] Example 6 Tumor in situ model of oncolytic virus expressing reprogramming factors

[0167] To further confirm the therapeutic ability of oncolytic viruses expressing reprogramming factors for tumors, this example uses an in situ human glioblastoma cell brain model as an example. Oncolytic virus (type 5 oncolytic adenovirus is used as an example) vector is used to express reprogramming factors. With the help of type 5 oncolytic adenovirus specificity amplification in tumor cells, the effect of oncolysis and in vivo transdifferentiation therapy on synergistically inhibiting glioma is verified.

[0168] In this example, glioma cells (U87-luc) were transplanted into the brain (5×10 5 ). 7 days after transplantation, the patients were divided into groups and given drugs. The control group was the PBS group, and the Ad5-AN group was given 1×10 9PFU, temozolomide gavage group (15 mg / kg, once a day, 5 times and 2 times), and Ad5-AN group and temozolomide combination group, Ad5-AN was administered once every 4 days for 3 consecutive times. 30 days after virus injection, immunohistochemical analysis of some brain tissues of the Ad5-AN group showed that cells infected with the virus expressed the neural marker molecule Tuj1, showing the morphology of neurons. 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). At the same time, the combination of Ad5-AN and temozolomide was significantly longer than that of the temozolomide group alone (average 97.5 days vs 56.8 days). The results also showed that in the case of in situ tumors, oncolytic viruses expressing reprogramming factors can be combined with existing therapies to achieve better anti-tumor and long-term anti-recurrence effects.

[0169] Example 7 Oncolytic Virus Vector Expressing Reprogramming Factors Inhibits the Growth of Tumor Cells in Glioma PDX Models

[0170] To further verify the combined effect 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 axilla of nude mice for passage, and then inoculated into the axilla of nude mice again during the logarithmic growth phase. When the tumor grew to 100 mm, the PDX tissue was inoculated into the axilla of nude mice for passage. 3 The nude mice with appropriate tumor masses were randomly divided into groups. After grouping, drug administration was started. The control group was the PBS group, and the Ad5-AN group was given 1×10 9 PFU, temozolomide gavage group (15 mg / kg, once a day, 5 times and 2 times), and Ad5-AN group and temozolomide combination group, Ad5-AN was administered once every two days, for 5 consecutive times. The volume of the tumor mass was measured and calculated every 3 days, and the animals were killed later, the tumor mass was weighed, and biochemical and molecular tests were performed. The results showed that, similar to the subcutaneous model of glioma cell lines, compared with the control PBS group, the tumor volume of the Ad5-AN group was significantly reduced, and the tumor volume of the temozolomide group was also significantly reduced. However, the tumor gradually recurred about 45 days after administration and grew back (6 / 6). By about 72 days, the tumor had grown to nearly 2000mm. 3 The tumor volume in the Ad5-AN and temozolomide combination group was significantly lower than that in the Ad5-AN group and the temozolomide group alone, with the tumors essentially disappearing (72 days, 5 / 6). More importantly, the anti-tumor effect was maintained for a long time (102 days, 4 / 6 without recurrence). These results suggest that oncolytic viruses expressing reprogramming factors can be combined with existing therapies to achieve better anti-tumor and long-term anti-recurrence effects in in vivo models of patient-derived tissue.

[0171] Example 8 Recombinant Herpes Simplex Virus Expressing Reprogramming Factors Inhibits Glioma Growth

[0172] In this example, herpes simplex virus type 1 was used as an oncolytic virus vector, and a fragment derived from the human Ascl1 (SEQ ID No. 11) and the CDS (SEQ ID No. 15) fragment of the human Ngn2 gene were inserted to achieve efficient co-expression of Ascl1 and Ngn2. After the recombinant herpes simplex virus was packaged and purified, the human brain glioma U87BALB / CA-nu mouse ectopic inoculation model was tested. When the tumor grew to 100 mm 3 The nude mice with appropriate tumor masses were randomly divided into groups and drug administration was started after grouping. The dose of the control PBS group, HSV (empty vector without reprogramming factors) and HSV-AN group was 2×10 6 PFU, temozolomide gavage group (15 mg / kg, once a day, 5 times and 2 times off), and HSV-AN and temozolomide combination group. HSV was administered once every two days for 5 consecutive times. The volume of the tumor mass was measured and calculated every 3 days. The animals were sacrificed later, the tumor mass was weighed, and biochemical and molecular tests were performed. The results showed that compared with the control PBS group, the tumor volume of the HSV empty group decreased by 25.6% after 10 days of administration, and the tumor volume of the HSV-AN group decreased by 58.9%. It can be seen that the addition of reprogramming factors significantly enhanced the inhibitory ability of oncolytic herpes virus on glioma. The tumor volume of the temozolomide group decreased by 73.6%, and the recurrence rate was (6 / 6) 2 months after administration. The tumor volume of the HSV-AN and temozolomide combination group decreased by 92.5%, and the recurrence rate was (1 / 6) 2 months after administration. These results indicate that the oncolytic effect of oncolytic herpes virus drugs and the synergistic effect of tumor cell transdifferentiation in the body have achieved a more significant ability to inhibit glioma growth. When used in combination with the existing therapy TMZ, it has a better anti-tumor and long-term anti-recurrence effect.

[0173] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

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

2. The recombinant oncolytic virus according to claim 1, characterized in that The recombinant nucleic acid includes a group of functional fragments that synergistically promote the reprogramming / transdifferentiation of tumor cells into non-tumorigenic cells; The functional fragment contains 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.

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

4. The recombinant oncolytic virus according to claim 1, characterized in that The functional fragment is a polynucleotide encoding a functional protein, and the functional fragment is selected from a polynucleotide encoding a transcription factor having a sequence homology of not less than 75% 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 a transcription factor functional protein having a sequence homology of not less than 85% 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 the functional fragment is selected from a polynucleotide encoding a transcription factor having a sequence homology of not less than 85% 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 homology of not less than 95% 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 recombinant oncolytic virus according to claim 4, characterized in that The functional fragment is a polynucleotide encoding a functional protein, and the functional fragment is selected from polynucleotides encoding transcription factors having a sequence homology of not less than 95% 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 functional protein is selected from transcription factor functional proteins having a sequence homology of not less than 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, and SEQ ID NO.

14.

6. The recombinant oncolytic virus according to claim 1, characterized in that The functional fragment is a polynucleotide encoding a functional protein, and the functional fragment is a polynucleotide encoding a transcription factor having a sequence homology of not less than 75% with SEQ ID NO.19; or The functional protein is a transcription factor functional protein having a sequence homology of not less than 85% with SEQ ID NO.18; Preferably, The functional fragment is a polynucleotide encoding a functional protein, and the functional fragment is a polynucleotide encoding a transcription factor having a sequence homology of not less than 85% with SEQ ID NO.19; or The functional protein is a transcription factor functional protein having a sequence homology of not less than 95% with SEQ ID NO.18; More preferably, The functional fragment is a polynucleotide encoding a functional protein, and the functional fragment is a polynucleotide encoding a transcription factor having a sequence homology of not less than 95% with SEQ ID NO.19; or The functional protein is a transcription factor functional protein having a sequence homology of not less than 99% with that of SEQ ID NO.

18.

7. The recombinant oncolytic virus according to claim 1, characterized in that The expression system of the functional fragments that promote the expression of transcription factors is constructed under the same expression vector, or 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 includes a selectively replicative recombinant oncolytic virus.

9. The recombinant oncolytic virus according to claim 8, characterized in that The selective replicative 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, Ekko virus, Coxsackie virus, Newcastle disease virus or Maraba virus with oncolytic effect.

10. A method for promoting the reprogramming / transdifferentiation of tumor cells into non-tumorigenic cells by oncolytic viruses, characterized in that: The method comprises the following steps: The recombinant oncolytic virus according to any one of claims 1 to 9 is contacted with tumor cells, thereby reprogramming / transdifferentiating and reprogramming the tumor cells into non-tumorigenic cells.

11. A composition for treating cancer, characterized in that: The composition comprises: (A) the recombinant oncolytic virus according to any one of claims 1 to 9; (B) Pharmaceutically acceptable excipients.

12. The composition according to claim 11, characterized in that The composition further comprises: (C) Anti-tumor drugs; The anti-tumor drugs include one or both of temozolomide and bevacizumab.

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

14. The use according to claim 13, characterized in that In the use, the recombinant oncolytic virus is formulated into a therapeutic agent for administration into the tumor or near the tumor, and the administration of the therapeutic agent includes any one of injection, intraperitoneal administration, subarachnoid administration or intravenous administration.

15. The use according to claim 13, characterized in that In the use, the recombinant oncolytic virus is formulated into a therapeutic agent that is administered through the tumor or near the tumor, and the administration method of the therapeutic agent includes one or more of hydrogel administration, convection-enhanced administration, and Ommaya capsule administration.

16. The use according to claim 13, 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, 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 carcinoma, endometrial cancer, vaginal cancer, vulvar cancer, insulinoma, blood cancer, peritoneal cancer or pleural cancer.

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

Images