GBM targeting oncolytic adenovirus vector as well as construction method and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a brain glioblastoma (GBM) targeting oncolytic adenovirus vector as well as a construction method and application thereof. The human type 5 oncolytic adenovirus vector for treating malignant tumors, especially treating GBM, is constructed by using human type 5 adenovirus, replacing an E1 promoter with a tumor specific promoter through gene modification and inserting a CD47 / PDL1 bispecific antibody and an immunomodulatory factor as therapeutic genes. In-vivo and in-vitro experiment results prove that the human type 5 oncolytic adenovirus vector prepared by the invention can target a tumor through a tumor specific promoter, can efficiently express an exogenous gene anti-CD47 / PDL1 and a therapeutic gene IL12 in GBM cells, can remarkably increase infiltration of macrophages and T cells in tumor tissues, and has a remarkable anti-tumor effect. The compound is expected to be used as a tumor targeting gene engineering drug for clinical treatment of malignant tumors, especially GBM.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a brain glioblastoma (GBM) targeted oncolytic adenovirus vector and a construction method and application thereof. BACKGROUND

[0002] Glioblastoma (GBM) is the most malignant glioma among astrocytic tumors, and is the most common primary malignant brain tumor in adults. Even if surgery, radiotherapy, chemotherapy or combined therapy is used, the prognosis of the vast majority of patients is still very poor. Therefore, GBM is one of the major challenges in tumor treatment today, and it is urgent to develop new therapies to improve the prognosis of GBM tumor patients.

[0003] In view of the malignant nature of brain glioma, various preclinical and clinical trials are constantly exploring new treatment methods, such as gene therapy, immunotherapy and viral therapy. In recent years, the field of tumor immunotherapy has made a number of breakthroughs, and the FDA has approved a number of new immunotherapy drugs for the treatment of various tumors, such as immune checkpoint inhibitors and CAR-T cell therapy. Previous studies have shown that the brain has an immune privilege, but recent studies have found that the central nervous system has an appropriate immune response and is linked to the peripheral immune system. It was found that there are functional lymphatic vessels near the dural venous sinuses, which absorb the cerebrospinal fluid and brain interstitial fluid adjacent to the subarachnoid space, and immune cells can be seen in the lymphatic vessels, and the lymphatic vessels communicate with the deep cervical lymph nodes through the skull base holes, which indicates that there are immune cells in the brain, including T lymphocytes that mainly play a role in killing tumor cells, providing a theoretical basis for the immunotherapy of GBM.

[0004] Oncolytic adenovirus (OAd) is a new immunotherapy for glioma, which is a purposeful modification of the adenovirus gene structure, so that the virus can selectively infect and promote the lysis of glioma cells, while not damaging normal tissues. The antitumor effect of oncolytic virus is divided into direct effect and indirect effect. The direct effect is the activation of the innate systemic immune response in tumor cells, which causes the termination of protein synthesis, the stop of proliferation and division, and the rapid death of cells. The indirect effect is the release of tumor-associated antigens, virus pathogen-associated molecular patterns and cell danger-associated molecular pattern signals after the lysis of tumor cells, which induce the body to produce an adaptive immune response to the tumor, so that tumor cells that are not directly infected with the virus are also recognized by the antitumor immune system, which is one of the bases for the combination of oncolytic virus therapy with other immunotherapy.

[0005] Oncolytic adenovirus Onyx-015 was the first genetically engineered adenovirus used to treat high-grade gliomas, but the median survival time for patients was only 6.2 months, indicating that prognostic survival needs further improvement. While oncolytic adenovirus DNX-2401 improved patient survival, it still has some adverse reactions, such as headache, nausea, altered mental status, vomiting, and fever. Therefore, further development of oncolytic adenoviruses for treating high-grade gliomas such as GBM is needed, aiming to significantly enhance the activity of immune cells in the tumor microenvironment while reducing side effects and improving safety. Summary of the Invention

[0006] To further improve the immunotherapeutic efficacy of oncolytic adenovirus in treating high-grade gliomas such as GBM, while reducing toxic side effects, this invention provides a novel GBM-targeting oncolytic adenovirus vector, its construction method, and its applications. This vector exerts its anti-tumor effect by killing tumor cells through the virus itself and by activating macrophages and T cells through CD47 / PD-L1 bispecific antibodies and immune cytokines, exhibiting targeted, safe, and highly effective characteristics.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0008] In a first aspect, the present invention provides a human oncolytic adenovirus type 5 vector, which is a genetically modified human adenovirus type 5 vector, wherein the viral vector gene contains gene sequences of a bispecific antibody against CD47 / PDL1 regulated by a tumor-specific promoter and an immunomodulatory factor.

[0009] Further, the immunomodulatory factor is selected from at least one of IL12, GM-CSF, IL-15, or IFN-γ. Preferably, the immunomodulatory factor is selected from IL12, and its nucleotide sequence is shown in SEQ ID NO:3 or SEQ ID NO:4.

[0010] Furthermore, the tumor-specific promoter is a promoter that replaces the E1A of human adenovirus type 5, selected from at least one of survivin or hTERT promoters. Preferably, the tumor-specific promoter is a survivin promoter, and its survivin-E1 nucleotide sequence is shown in SEQ ID NO:5.

[0011] Furthermore, the nucleotide sequence of the anti-CD47 / PDL1 bispecific antibody is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0012] More preferably, the nucleotide sequence of the human oncolytic adenovirus type 5 vector is as shown in SEQ ID NO:15-SEQ ID NO:18 or SEQ ID NO:19-SEQ ID NO:22.

[0013] Secondly, the present invention provides a method for constructing the above-mentioned human type 5 oncolytic adenovirus vector, which includes the following steps:

[0014] S1: Insert the IL12 fragment into the human adenovirus type 5 backbone plasmid Ad5-ATTR to construct plasmid Ad5-ATTR-IL12;

[0015] S2: The shuttle plasmid pENTR-ATTL-survivin-E1-antiCD47 / PDL1 was constructed using an enzyme digestion and ligation method.

[0016] S3: Co-transform plasmid Ad5-ATTR-IL12 with pENTR-ATTL-survivin-E1-antiCD47 / PDL1, then transfect and package the mixture. After viral amplification and purification, the recombinant oncolytic adenovirus vector Ad5-survivin-antiCD47 / PDL1-IL12 can be obtained.

[0017] Furthermore, the nucleotide sequence of the plasmid Ad5-ATTR-IL12 is shown in SEQ ID NO:9 or SEQ ID NO:10.

[0018] Furthermore, the shuttle plasmid pENTR-ATTL-survivin-E1-antiCD47 / PDL1 is shown in SEQ ID NO:13 or SEQ ID NO:14.

[0019] Furthermore, the nucleotide sequence of the Ad5-survivin-antiCD47 / PDL1-IL12 is shown in SEQ ID NO:15-SEQ ID NO:18 or SEQ ID NO:19-SEQ ID NO:22.

[0020] Thirdly, the present invention provides a pharmaceutical composition containing the above-mentioned human oncolytic adenovirus type 5 vector.

[0021] Fourthly, the present invention provides the use of the above-mentioned human type 5 oncolytic adenovirus vector or pharmaceutical composition in the preparation of drugs for treating malignant tumors.

[0022] Furthermore, the tumor is selected from at least one of glioma, melanoma, osteosarcoma, colon cancer, rectal cancer, breast cancer, uveal melanoma, ovarian cancer, liver cancer, bladder cancer, cervical cancer, sarcoma, skin cancer, prostate cancer, pancreatic cancer, nasopharyngeal carcinoma, lung cancer, gastric cancer, head and neck cancer, hypopharyngeal cancer, kidney cancer, laryngeal cancer, oral cancer, malignant mesothelioma, neuroblastoma, ovarian cancer, papillomavirus, and retinoblastoma.

[0023] Preferably, the glioma is an astrocytic tumor. More preferably, it is glioblastoma (GBM).

[0024] Furthermore, the dosage form of the drug is an injection, and the injection method is at least one of intratumoral injection, intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intramyocardial injection, or intraperitoneal injection.

[0025] Beneficial Effects: This invention utilizes genetic engineering to modify adenoviruses, preparing a human type 5 oncolytic adenovirus vector, Ad5-survivin-antiCD47 / PDL1-IL12, targeting GBM. This vector employs a tumor-specific survivin promoter, combined with intratumoral injection, enhancing its tumor-targeting specificity, safety, and efficacy. Simultaneously, using antiCD47 / PDL1 and IL12 as therapeutic genes, under the guidance of the survivin promoter, it efficiently expresses the antiCD47 / PDL1 bispecific antibody and the cytokine IL12 at the tumor site, blocking CD47 and PDL1, enhancing the phagocytic capacity of macrophages against tumor cells, and activating T cells, promoting T cell infiltration in tumor tissue, thus exerting a good anti-tumor effect. Therefore, the human type 5 oncolytic adenovirus vector provided by this invention can serve as an effective and safe gene immunotherapy vector system for treating tumors, and this virus holds promise as a tumor-targeting gene-engineered drug for the clinical treatment of malignant tumors, especially GBM. Attached Figure Description

[0026] Figure 1 This is a structural diagram of Ad5-survivin, Ad5-survivin-antihCD47 / PDL1-hIL12 and Ad5-survivin-antimCD47 / PDL1-mIL12 in Embodiment 1 of the present invention.

[0027] Figure 2 This is a diagram showing the specific expression results of the bispecific antibody antiCD47 / PDL1 and cytokine IL12 in GBM cells after infection with Ad5-survivin-antiCD47 / PDL1-IL12 in Example 2 of the present invention.

[0028] Figure 3This is a graph showing the replication ability test results of Ad5-survivin-antiCD47 / PDL1-IL12 in GBM cell line in Example 3 of the present invention.

[0029] Figure 4 This is a graph showing the detection results of the killing ability of Ad5-survivin-antiCD47 / PDL1-IL12 against GBM cell lines in Example 3 of the present invention.

[0030] Figure 5 This is a diagram showing the effect of Ad5-survivin-antiCD47 / PDL1-IL12 on the phagocytic function of macrophages in the GBM cell-macrophage co-culture system in Example 4 of the present invention.

[0031] Figure 6 This is a diagram showing the activation effect of Ad5-survivin-antiCD47 / PDL1-IL12 on T cells in a GBM cell and T cell co-culture system in Example 5 of the present invention.

[0032] Figure 7 This image shows the therapeutic effect of Ad5-survivin-antimCD47 / PDL1-mIL12 on GL261 GBM tumor-bearing mice and the activation results of immune cells in Example 6 of this invention. Detailed Implementation

[0033] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0034] The term "glioma" is a general term for a group of primary intracranial tumors originating from glial cells in the brain and spinal cord. Glial cells are the supporting cells of the nervous system and include astrocytes, oligodendrogliocytes, and ependymal cells, thus gliomas are correspondingly classified into astrocytomas, oligodendrogliomas, and ependymomas. The classification and grading of gliomas determine their treatment plan and prognosis. The World Health Organization (WHO) classifies them into grades I-IV based on the malignancy of the tumor cells.

[0035] The term "glioblastoma (GBM)" refers to the most malignant (grade IV) glioma among astrocytic tumors. It grows extremely rapidly, has indistinct borders, and infiltrates normal brain tissue like tree roots. Complete resection is difficult, recurrence rates are high, and the prognosis is the worst. Therefore, developing methods to treat advanced gliomas, represented by GBM, is of great significance to the field of clinical medicine.

[0036] The term "human adenovirus" encompasses six different subgenera: A, B, C, D, E, and F. These subgenera differ in their tropism, tumorigenicity, and disease history. Ad5 adenovirus belongs to type 5 within subgenera C. It enters cells via the Coxsackievirus receptor (CAR), and since many cancer cells highly express the CAR receptor, Ad5 is currently the most common viral vector and serotype used in tumor gene therapy. It also possesses the following advantages: a known genome sequence, facilitating gene manipulation; a wide range of susceptible cells, enabling efficient infection of different types of human tissue cells; high viral titers; a large genome, facilitating the insertion of large exogenous gene fragments; and high safety and non-carcinogenicity after infecting host cells, as its genome does not integrate into the host genome. Therefore, this invention utilizes Ad5 adenovirus for oncolytic virus modification and gene therapy vectors.

[0037] The term "survivin" refers to an important inhibitor of apoptosis (IAP) protein that is active during fetal development but is silenced or not expressed in most terminally differentiated normal adult tissues. However, survivin is efficiently and extensively re-expressed in almost all malignant tumors (such as lung cancer, liver cancer, breast cancer, and colorectal cancer). Therefore, survivin is an ideal tumor-specific promoter, enabling selective and efficient viral replication within tumors.

[0038] The term "CD47 / PD-L1," referring to bispecific antibodies, represents an emerging strategy in tumor immunotherapy. It aims to synergistically activate both the innate and adaptive immune systems by simultaneously blocking two key immunosuppressive pathways: the "don't eat me" signal of CD47 and the T-cell inhibitory signal of PD-L1. Its core mechanisms of action include: relieving the "don't eat me" signal on the surface of tumor cells (through binding to macrophage SIRPα), enhancing macrophage phagocytosis of tumors, and promoting antigen presentation; relieving PD-1 / PD-L1-mediated T-cell exhaustion, restoring the tumor-killing function of cytotoxic T cells; and significantly enhancing the anti-tumor response through the dual activation of innate immunity (macrophages) and adaptive immunity (T cells). However, traditional CD47 monoclonal antibodies readily bind to normal erythrocytes, leading to anemia and thrombocytopenia. The bispecific antibody design, guided by PD-L1, enriches the drug in the tumor microenvironment (TME), reducing off-target toxicity to normal tissues. Therefore, this invention uses CD47 / PD-L1 as a therapeutic gene, which can be efficiently expressed at the tumor site under the guidance of the survivin promoter, thereby blocking CD47 and PDL1, improving the ability of macrophages to phagocytose tumor cells, promoting the infiltration of T cells in tumor tissue, and exerting a good anti-tumor effect.

[0039] The term "interleukin-12 (IL-12)" refers to a pleiotropic cytokine with potent immune-activating effects. It can stimulate NK cells to secrete IFN-γ, directly killing tumor cells and promoting CD8 activation. + T cells differentiate into effector phenotypes, enhancing cytotoxicity; IL-12 can enhance tumor antigen presentation efficiency by upregulating MHC-I / II expression in APC cells; it induces IFN-γ release, further activating chemokines such as CXCL10, recruiting more immune cells to infiltrate "cold tumors," and promoting their transformation into "hot tumors."

[0040] Based on the above analysis, in one embodiment of the present invention, a novel replication-selective oncolytic adenovirus, Ad5-survivin-antiCD47 / PDL1-IL12, is constructed using human adenovirus type 5 as a base. The antiCD47 / PDL1 bispecific antibody and the cytokine IL12 are inserted as payload genes into the genome of the type 5 adenovirus to increase the phagocytic capacity of macrophages and the activation of T cells, thereby stimulating an adaptive anti-tumor immune response and enhancing the tumor-killing effect. Furthermore, the E1A promoter of Ad5 is replaced with a survivin promoter to restrict the virus's selective replication and proliferation in GBM cells, exerting its oncolytic effect without affecting normal cells.

[0041] In a specific embodiment of the present invention, a bispecific antibody sequence against human or mouse CD47 / PDL1 is inserted after the E1 region of Ad5, and a human IL12 or mouse IL12 gene sequence is inserted after the E3 region.

[0042] hTERT expression is a nearly universal marker for cancer cells to maintain their malignant proliferative capacity, making its promoter a perfect switch to drive gene-specific expression in tumors. Therefore, in some embodiments of the present invention, the hTERT promoter can be used to replace the Ad5 adenovirus E1A promoter or used in conjunction with the survivin promoter as a specific promoter for selective and efficient viral replication in tumors.

[0043] CD47 / PD-L1 and IL12 are both tumor therapeutic genes that can be efficiently expressed at the tumor site under the guidance of promoters. Therefore, in some embodiments of the present invention, IL12 can be replaced or added to other immune factors that activate and enhance the anti-glial tumor immune response, such as GM-CSF, IL-15 and IFN-β.

[0044] In another embodiment of the present invention, in vitro and in vivo experiments have confirmed that the constructed oncolytic adenovirus vector Ad5-survivin-antiCD47 / PDL1-IL12 has significant anti-tumor effects in both in vivo and in vitro studies on GBM, thus providing a new treatment method for the immunotherapy of GBM.

[0045] In a specific embodiment of the present invention, the constructed GBM-targeting human oncolytic adenovirus type 5 Ad5-survivin-antiCD47 / PDL1-IL12 was used to evaluate the in vitro and in vivo antitumor effects of GBM, including the following methods:

[0046] (1) Replication and killing of Ad5-survivin-antiCD47 / PDL1-IL12 virus in GBM cells: 293A cells were infected with adenovirus Ad5-survivin-antiCD47 / PDL1-IL12. After large-scale proliferation, purification and identification, different GBM cell lines were seeded into 96-well plates and infected with different tumor cells at a virus titer of MOI=0.1 or 1. Supernatant and cells were collected at 24h, 48h, 72h and 96h, respectively. Three replicates were set at each time point. After repeated freeze-thaw cycles, the replication ability of oncolytic adenovirus in different tumor cell lines was evaluated by TCID50 method.

[0047] The different tumor cells were seeded into 96-well plates with 8000 cells per well. The virus was serially diluted with an MOI of 100 as the starting concentration. The killing ability of the virus on different tumor cells was detected by MTT assay 48 h after infection.

[0048] (2) Evaluation of the antitumor effect of Ad5-survivin-antiCD47 / PDL1-IL12 virus in GBM mouse tumor model:

[0049] 1×10 5 GL261-Luc cells were orally injected into the brains of C57BL / 6 mice. Three days later, 3 μL of 1×10⁻⁶ cells were injected into the brains of the mice. 9 PFU-based treatments, including Ad5-survivin-antiCD47 / PDL1-IL12, Ad5-survivin, and Saline, were administered via intratumoral injection. Tumor growth was monitored using in vivo imaging on days 10, 20, and 30 post-inoculation, and mouse survival was recorded. Tumor growth curves and mouse survival curves were plotted to evaluate the therapeutic effect of the virus.

[0050] In some embodiments of the present invention, the viral vector is administered via intravenous injection (iv), intraarterial injection (ia), intratumoral injection (it), and inhalation.

[0051] In a specific embodiment of the present invention, Ad5-survivin-antiCD47 / PDL1-IL12 is administered via intratumoral injection, for example, by being formulated into an intratumoral injection solution before administration to the subject. Intratumoral injection is the most widely used method of drug delivery, being a local administration method that can directly reach the tumor lesion without requiring the blood-brain barrier to be breached. In contrast, intravenous injection, arterial injection, and inhalation are all systemic administration methods that require blood circulation to reach the intracranial lesion. The adenovirus vector of the present invention does not have blood-brain barrier penetration capability; therefore, intratumoral injection is preferred for drug delivery.

[0052] In some practical applications of the oncolytic adenovirus of this invention, suitable excipients may be added when preparing the injection. These include osmotic pressure regulators, pH adjusters, solubilizers, co-solvents, antioxidants, antibacterial agents, emulsifiers, and suspending agents. The excipients used should not affect the efficacy of the drug, and the concentration used should not cause toxicity or significant irritation. Antioxidants may include sodium sulfite, sodium bisulfite, and sodium metabisulfite, with a concentration of 0.1%-0.2%; antibacterial agents may include 0.5% phenol, 0.3% cresol, and 0.5% chlorobutanol, etc.

[0053] In some practical applications of the oncolytic adenovirus of the present invention, the effective amount of the viral vector of the present invention can be any amount used to treat cancer, alleviate cancer symptoms, or inhibit the growth of cancer cells, calculated based on the viral titer.

[0054] In some practical applications of the oncolytic adenovirus of the present invention, the drug of the present invention can be administered to the subject at any effective dose. Preferably, the drug of the present invention can be administered in multiple doses, and a preferred embodiment is: treatment begins on the day after surgery, with one injection per day (10... 6 The dose was administered continuously for 6 days (PFU / mL), followed by a 15-day observation period, for a total of 21 days as one cycle. At the start of the second cycle, the dose was increased and maintained at 10 PFU / mL. 8 PFU / mL; at least 2 cycles of treatment should be received, but no more than 5 cycles should be received.

[0055] It should be understood that the medicament of the present invention can be formulated in any suitable manner for administration via any suitable route, and the administration method can be adjusted according to the actual situation of different subjects.

[0056] The following specific embodiments will be provided to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0057] Example 1: Construction of GBM-targeted human oncolytic adenovirus type 5 vector Ad5-survivin-antiCD47 / PDL1-IL12

[0058] (1) A type 5 adenovirus backbone plasmid (Ad5-ATTR, SEQ ID NO:6-SEQ ID NO:8) was synthesized by Yunzhou Biotechnology (Guangzhou) Co., Ltd. The plasmid lacks the E1 and E3 regions of adenovirus and has Gateway LR reaction sequences before and after the E1 region, as shown in SEQ ID NO:23 and SEQ ID NO:24.

[0059] (2) The survivin-E1 fragment sequence (SEQ ID NO:5), the human or mouse antiCD47 / PDL1 fragment sequence (SEQ ID NO:1 or SEQ ID NO:2), and the human or mouse IL12 (SEQ ID NO:3 or SEQ ID NO:4) were amplified by PCR.

[0060] (3) Using the restriction enzyme sites (SPEI / BSTBI) in the E3 backbone region of the Ad5-ATTR adenovirus vector, human or mouse Ad5-ATTR-IL12 (SEQ ID NO:9 or SEQ ID NO:10) was constructed in the E3 region of the adenovirus vector Ad5-ATTR by using the restriction enzyme ligation method.

[0061] (4) Amplify human or mouse survivin-E1-antiCD47 / PDL1 (including survivin-E1+CMV promoter+antiCD47 / PDL1 sequence, as shown in SEQ ID NO:11 or SEQ ID NO:12) by overlapping PCR, and construct human or mouse shuttle plasmid pENTR-ATTL-survivin-E1-antiCD47 / PDL1 (SEQ ID NO:13 or SEQ ID NO:14) by enzyme digestion and ligation.

[0062] (5) Using the Gateway LR reaction system, construct human or mouse recombinant oncolytic adenovirus vector Ad5-survivin-antiCD47 / PDL1-IL12 (SEQ ID NO:15-SEQ ID NO:18 or SEQ ID NO:19-SEQ ID NO:22).

[0063] The specific experimental procedure is as follows:

[0064] Construction of recombinant oncolytic adenovirus plasmid Ad5-survivin-antiCD47 / PDL1-IL12:

[0065] The IL12 fragment (SEQ ID NO:3 or SEQ ID NO:4) was inserted into the E3 region of the Ad5-ATTR (SEQ ID NO:6-SEQ ID NO:8) vector using enzyme digestion and ligation. This was followed by transformation of *E. coli*, and positive clones were screened in ampicillin-resistant strains to construct the adenovirus vector plasmid Ad5-ATTR-IL12 (SEQ ID NO:9 or SEQ ID NO:10). Similarly, the survivin-E1-antiCD47 / PDL1 (SEQ ID NO:11 or SEQ ID NO:12) was inserted into the pENTR-ATTL shuttle plasmid using enzyme digestion and ligation. This was followed by transformation of *E. coli*, and positive clones were screened in kanamycin-resistant strains to construct the shuttle plasmid pENTR-ATTL-survivin-E1-antiCD47 / PDL1 (SEQ ID NO:13 or SEQ ID NO:10). NO:14); The adenovirus vector plasmid Ad5-ATTR-IL12 and pENTR-ATTL-survivin-E1-antiCD47 / PDL1 shuttle plasmid were reacted with Gateway LRClonase II enzyme (Thermo Fisher Scientific, REF, 11791020) and transformed into E. coli. Positive clones were screened in ampicillin-resistant strains to construct the recombinant oncolytic adenovirus plasmid Ad5-survivin-antiCD47 / PDL1-IL12.

[0066] Construction of recombinant oncolytic adenovirus vector Ad5-survivin-antiCD47 / PDL1-IL12:

[0067] The constructed recombinant oncolytic adenovirus Ad5-survivin-antiCD47 / PDL1-IL12 plasmid was linearized by PacI (NEB, REF, R0547L) restriction endonuclease. After digestion, the plasmid was purified using the conventional phenol-chloroform-isopropanol method. Simultaneously, 293A cells for virus packaging were prepared, and 5 μg of the linearized Ad5-survivin-antiCD47 / PDL1-IL12 plasmid was transfected into each 6 cm diameter culture dish. After virus collection, amplification, and purification, GBM-targeted human type 5 oncolytic adenovirus Ad5-survivin-antihCD47 / PDL1-hIL12 (SEQ ID NO:15-SEQ ID NO:18) or mouse Ad5-survivin-antimCD47 / PDL1-mIL12 (or SEQ ID NO:19-SEQ ID NO:22) were obtained. Their structures are shown below. Figure 1 As shown. In the above process of constructing recombinant oncolytic adenovirus vectors for humans and mice, the nucleotide gene sequences used were all constructed using the corresponding human or mouse sequences, and the human and mouse gene sequences were not used interchangeably.

[0068] Example 2: Specific expression of bispecific antibody antiCD47 / PDL1 and cytokine IL12 in GBM cells after infection with GBM-targeting human oncolytic adenovirus type 5 vector Ad5-survivin-antiCD47 / PDL1-IL12.

[0069] Human GBM cells U87 and U251 were infected with an Ad5-survivin-antihCD47 / PDL1-hIL12 viral titer at MOI=1. Supernatant from virus-infected tumor cells was collected 48 h post-infection, and the expression of antihCD47 / PDL1 and the cytokine hIL12 in the human GBM cell lines was detected using ELISA. Results are as follows: Figure 2 As shown in a, the expression levels of antihCD47 / PDL1 and cytokine hIL12 were significantly increased in the human GBM cell line.

[0070] Mouse GBM cells GL261 were infected with an Ad5-survivin-antimCD47 / PDL1-mIL12 viral titer at an MOI of 100. Supernatant of virus-infected tumor cells was collected 48 h post-infection, and the expression of antimCD47 / PDL1 and the cytokine mIL12 in the human GBM cell line was detected by ELISA. Results are attached. Figure 2As shown in b, the expression levels of antihCD47 / PDL1 and the cytokine hIL12 were also significantly increased in the mouse GBM cell line. This indicates that after GBM cells were infected with the human type 5 oncolytic adenovirus vector Ad5-survivin-antiCD47 / PDL1-IL12 constructed in this invention, the two antibodies, antiCD47 / PDL1 and cytokine IL12, could be efficiently and specifically expressed in the cells.

[0071] Example 3: Detection of the replication and killing ability of GBM-targeting human oncolytic adenovirus type 5 vector Ad5-survivin-antiCD47 / PDL1-IL12 in GBM cell lines

[0072] Oncolytic adenovirus Ad5-survivin-antiCD47 / PDL1-IL12 was used to infect 293A cells. After large-scale proliferation, purification, and identification, different GBM cell lines, including human U87 and U251 cells, and mouse GL261 cells, were seeded into 96-well plates. Different tumor cell lines were infected with virus titers of MOI = 0.1, 1, or 100. Supernatant and cells were collected at 12h, 24h, 48h, 60h, and 72h, with three replicates at each time point. After three freeze-thaw cycles, the replication capacity of oncolytic adenovirus in different tumor cell lines was assessed using the TCID50 method. Results were presented by... Figure 3 It can be seen that both the oncolytic adenovirus Ad5-survivin-antiCD47 / PDL1-IL12 and the control adenovirus Ad5-survivin can replicate extensively in tumor cell lines at different MOI titers.

[0073] Different tumor cell lines were seeded into 96-well plates with 8000 cells per well. The virus was serially diluted with an initial titer of MOI = 100. After 48 hours of infection, the MTT assay was used to assess the virus's killing effect on different tumor cells. Figure 4 It can be seen that, compared with the control virus, the killing ability of Ad5-survivin-antiCD47 / PDL1-IL12 against tumor cells was not affected.

[0074] Example 4: Effect of GBM-targeting human oncolytic adenovirus type 5 vector Ad5-survivin-antiCD47 / PDL1-IL12 on macrophage phagocytic function in a GBM cell-macrophage co-culture system.

[0075] Human GBM cells U87 and U251 were infected with an Ad5-survivin-antihCD47 / PDL1-hIL12 viral titer at an MOI of 1. Mouse GBM cells GL261 were infected with an Ad5-survivin-antimCD47 / PDL1-mIL12 viral titer at an MOI of 1. Forty-eight hours later, macrophages were added at an effector-to-target ratio of 10. Twelve hours later, tumor cells and macrophages were labeled using tumor cell markers (fluorescein diacetate succinimide (CFSE, green dye)) and macrophage markers (APC anti-mouse / human CD11b Antibody flow cytometry antibody). Cells exhibiting both macrophage and tumor cell markers were detected by flow cytometry; these were identified as macrophages that had engulfed tumor cells. Figure 5 It can be seen that, compared with the control virus, Ad5-survivin-antiCD47 / PDL1-IL12 infection of tumor cells increased the phagocytic capacity of macrophages against tumor cells.

[0076] Example 5: Activation effect of GBM-targeted human oncolytic adenovirus type 5 vector Ad5-survivin-antiCD47 / PDL1-IL12 on T cells in a GBM cell-T cell co-culture system.

[0077] Human GBM cells U87 and U251 were infected with an Ad5-survivin-antihCD47 / PDL1-hIL12 viral titer at an MOI of 1. Mouse GBM cells GL261 were infected with an Ad5-survivin-antimCD47 / PDL1-mIL12 viral titer at an MOI of 1. Forty-eight hours later, T cells were added at an effector-to-target ratio of 10. Twelve hours later, T cells were labeled with T cell activation-related markers (PE anti-human / mouse CD107a Antibody flow cytometry antibody for staining human / mouse T cells) and detected by flow cytometry. Figure 6 It can be seen that, compared with the control virus, Ad5-survivin-antiCD47 / PDL1-IL12 can promote T cell activation after infecting tumor cells.

[0078] Example 6: Therapeutic effect of GBM-targeting human oncolytic adenovirus type 5 vector Ad5-survivin-antiCD47 / PDL1-IL12 on GBM tumor-bearing mice

[0079] 1×10 5GL261-GFP cells were orally inoculated into the brains of C57BL / 6 mice. Three days later, 3 μL of 1×10⁻⁶ cells were injected into the brains of the mice. 9 PFU-based treatments, including Ad5-survivin-antimCD47 / PDL1-mIL12, Ad5-survivin, and Saline, were administered via intratumoral injection. Tumor growth was monitored using in vivo imaging on days 10, 20, and 30 post-inoculation, and mouse survival was recorded. Tumor growth curves and mouse survival curves were plotted to evaluate the therapeutic effect of the virus. Experimental results are as follows: Figure 7 As shown.

[0080] Depend on Figure 7 As can be seen, Ad5-survivin-antimCD47 / PDL1-mIL12 significantly prolonged the survival time of mice compared with the Ad5-survivin or Saline control group.

[0081] Depend on Figure 7 b indicates that Ad5-survivin-antimCD47 / PDL1-mIL12 has a stronger anti-tumor effect compared to the Ad5-survivin or Saline control group.

[0082] Depend on Figure 7 c indicates that Ad5-survivin-antimCD47 / PDL1-mIL12 significantly increased the infiltration of CD3+ T cells in tumors compared to the Ad5-survivin or Saline control group.

[0083] Depend on cgggctattctaattcaggtttctctagaa As shown in d, Ad5-survivin-antimCD47 / PDL1-mIL12 significantly increased the ability of macrophages within the tumor to phagocytose tumor cells compared to the Ad5-survivin or Saline control group.

[0084] The following is the sequence information involved in this invention.

[0085] SEQ ID NO:1: antihCD47 / PDL1 nucleotide sequence

[0086]

[0087] SEQ ID NO:2: antimCD47 / PDL1 nucleotide sequence

[0088]

[0089] SEQ ID NO:3: hIL12 nucleotide sequence

[0090]

[0091] SEQ ID NO:4: mIL12 nucleotide sequence

[0092]

[0093] SEQ ID NO:5: Nucleotide sequence of survivin-E1 fragment

[0094]

[0095] SEQ ID NO:6-SEQ ID NO:8: The backbone plasmid Ad5-ATTR lacking the E1 and E3 regions of adenovirus is obtained by sequentially linking the bases of SEQ ID NO:6-SEQ ID NO:8 end to end.

[0096] SEQ ID NO:9, the Ad5-ATTR-hIL12 nucleotide sequence, is a CMV promoter + hIL12 nucleotide sequence inserted into the E3 region of the Ad5-ATTR vector sequence. The following sequence is a truncated version of SEQ ID NO:9, specifically composed of the start position (single underline) of the E3 region of the Ad5-ATTR vector sequence (SEQ ID NO:6-SEQ ID NO:8) + CMV promoter + hIL12 nucleotide sequence + the end position (double underline) of the E3 region of the Ad5-ATTR vector sequence.

[0097] atggacggaattattacagagcagcgcctg cgggctattctaattcaggtttctctagaa SEQ ID NO:10: Ad5-ATTR-mIL12 nucleotide sequence. Simply replace the hIL12 nucleotide sequence in SEQ ID NO:9 with the mIL12 nucleotide sequence.

[0098] tggacggaattattacagagcagcgcctga ​

[0099] SEQ ID NO:11: survivin-E1-antihCD47 / PDL1, consisting of the survivin-E1+mCMV promoter+antihCD47 / PDL1 nucleotide sequence.

[0100] SEQ ID NO:12: survivin-E1-antimCD47 / PDL1, consisting of the survivin-E1+mCMV promoter+antimCD47 / PDL1 nucleotide sequence.

[0101] SEQ ID NO:13: pENTR-ATTL-survivin-E1-antihCD47 / PDL1 nucleotide sequence.

[0102] SEQ ID NO:14: pENTR-ATTL-survivin-E1-antimCD47 / PDL1 nucleotide sequence.

[0103] SEQ ID NO:15-SEQ ID NO:18: Recombinant oncolytic adenovirus vector Ad5-survivin-antihCD47 / PDL1-hIL12 nucleotide sequence (its complete sequence is obtained by sequentially linking the bases of SEQ ID NO:15-SEQ ID NO:18 in the sequence listing, as shown below)

[0104] catcatcaataatataccttattttggattgaagccaatatgataatgagggggtggagtttgtgacgtggcgcggggcgtgggaacggggcg

[0105] ggtgacgtagtagtgtggcggaagtgtgatgttgcaagtgtggcggaacacatgtaagcgacggatgtggcaaaagtgacgtttttggtgtgcg

[0106] ccggtgtacacaggaagtgacaattttcgcgcggttttaggcggatgttgtagtaaatttgggcgtaaccgagtaagatttggccattttcgcggga

[0107] aaactgaataagaggaagtgaaatctgaataattttgtgttactcatagcgcgtaatatttgtctagggccgcggggactttgaccgtttacgtgga

[0108] gactcgcccaggtgtttttctcaggtgttttccgcgttccgggtcaaagttggcgttttattattatagtcagtcgaagcttggatccggtacctctaga

[0109] attctcgagcggccgctagcgacatcgatcaactttgtatagaaaagttggctaactataacggtcctaaggtagcgacgtcgacgtggcaaaca

[0110] gctattatgggtattatgggtacaagtttgtacaaaaaagcaggctttaaaggaaccaattcagtcgactaactataacggtcctaaggtagcgaa

[0111] gatctctaggtgtgggcagggacgagctggcgcggcgtcgctgggtgcaccgcgaccacgggcagagccacgcggcgggaggactacaa

[0112] ctcccggcacaccccgcgccgccccgcctctactcccagaaggccgcggggggtggaccgcctaagagggcgtgcgctcccgacatgccc

[0113] cgcggcgcgccattaaccgccagatttgaatcgcgggacccgttggcagaggtggcggcggcggcgaattcgccaccatgagacatattatct

[0114] gccacggaggtgttattaccgaagaaatggccgccagtcttttggaccagctgatcgaagaggtactggctgataatcttccacctcctagccatt

[0115] ttgaaccacctacccttcacgaactgtatgatttagacgtgacggcccccgaagatcccaacgaggaggcggtttcgcagatttttcccgactctg

[0116] taatgttggcggtgcaggaagggattgacttactcacttttccgccggcgcccggttctccggagccgcctcacctttcccggcagcccgagca

[0117] gccggagcagagagccttgggtccggtttctatgccaaaccttgtaccggaggtgatcgatccacccagtgacgacgaggatgaagagggtg

[0118] aggagtttgtgttagattatgtggagcaccccgggcacggttgcaggtcttgtcattatcaccggaggaatacgggggacccagatattatgtgtt

[0119] cgctttgctatatgaggacctgtggcatgtttgtctacagtaagtgaaaattatgggcagtgggtgatagagtggtgggtttggtgtggtaatttttttt

[0120] ttaatttttacagttttgtggtttaaagaattttgtattgtgatttttttaaaaggtcctgtgtctgaacctgagcctgagcccgagccagaaccggagcc

[0121] tgcaagacctacccgccgtcctaaaatggcgcctgctatcctgagacgcccgacatcacctgtgtcaagagaatgcaatagtagtacggatagc

[0122] tgtgactccggtccttctaacacacctcctgagatacacccggtggtcccgctgtgccccattaaaccagttgccgtgagagttggtgggcgtcg

[0123] ccaggctgtggaatgtatcgaggacttgcttaacgagcctgggcaacctttggacttgagctgtaaacgccccaggccataaggtgtaaacctgt

[0124] gattgcgtgtgtggttaacgcctttgtttgctgaatgagttgatgtaagtttaataaagggtgagataatgtttaacttgcatggcgtgttaaatggggc

[0125] ggggcttaaagggtatataatgcgccgtgggctaatcttggttacatctgacctcatggaggcttgggagtgtttggaagatttttctgctgtgcgta

[0126] acttgctggaacagagctctaacagtacctcttggttttggaggtttctgtggggctcatcccaggcaaagttagtctgcagaattaaggaggatta

[0127] caagtgggaatttgaagagcttttgaaatcctgtggtgagctgtttgattctttgaatctgggtcaccaggcgcttttccaagagaaggtcatcaaga

[0128] ctttggatttttccacaccggggcgcgctgcggctgctgttgcttttttgagttttataaaggataaatggagcgaagaaacccatctgagcggggg

[0129] gtacctgctggattttctggccatgcatctgtggagagcggttgtgagacacaagaatcgcctgctactgttgtcttccgtccgcccggcgataata

[0130] ccgacggaggagcagcagcagcagcaggaggaagccaggcggcggcggcaggagcagagcccatggaacccgagagccggcctgga

[0131] ccctcgggaatgaatgttgtacaggtggctgaactgtatccagaactgagacgcattttgacaattacagaggatgggcaggggctaaagggg

[0132] gtaaagagggagcggggggcttgtgaggctacagaggaggctaggaatctagcttttagcttaatgaccagacaccgtcctgagtgtattacttt

[0133] tcaacagatcaaggataattgcgctaatgagcttgatctgctggcgcagaagtattccatagagcagctgaccacttactggctgcagccagggg

[0134] atgattttgaggaggctattagggtatatgcaaaggtggcacttaggccagattgcaagtacaagatcagcaaacttgtaaatatcaggaattgttg

[0135] ctacatttctgggaacggggccgaggtggagatagatacggaggatagggtggcctttagatgtagcatgataaatatgtggccgggggtgctt

[0136] ggcatggacggggtggttattatgaatgtaaggtttactggccccaattttagcggtacggttttcctggccaataccaaccttatcctacacggtgt

[0137] aagcttctatgggtttaacaatacctgtgtggaagcctggaccgatgtaagggttcggggctgtgccttttactgctgctggaagggggtggtgtg

[0138] tcgccccaaaagcagggcttcaattaagaaatgcctctttgaaaggtgtaccttgggtatcctgtctgagggtaactccagggtgcgccacaatgt

[0139] ggcctccgactgtggttgcttcatgctagtgaaaagcgtggctgtgattaagcataacatggtatgtggcaactgcgaggacagggcctctcaga

[0140] tgctgacctgctcggacggcaactgtcacctgctgaagaccattcacgtagccagccactctcgcaaggcctggccagtgtttgagcataacata

[0141] ctgacccgctgttccttgcatttgggtaacaggaggggggtgttcctaccttaccaatgcaatttgagtcacactaagatattgcttgagcccgaga

[0142] gcatgtccaaggtgaacctgaacggggtgtttgacatgaccatgaagatctggaaggtgctgaggtacgatgagacccgcaccaggtgcaga

[0143] ccctgcgagtgtggcggtaaacatattaggaaccagcctgtgatgctggatgtgaccgaggagctgaggcccgatcacttggtgctggcctgc

[0144] acccgcgctgagtttggctctagcgatgaagatacagattgaggatccgagtcattagggactttccaatgggttttgcccagtacataaggtcaa

[0145] taggggtgaatcaacaggaaagtcccattggagccaagtacactgagtcaatagggactttccattgggttttgcccagtacaaaaggtcaatag

[0146] ggggtgagtcaatgggtttttcccattattggcacgtacataaggtcaataggggtgagtcattgggtttttccagccaatttaattaaaacgccatgt

[0147] actttcccaccattgacgtcaatgggctattgaaactaatgcaacgtgacctttaaacggtactttcccatagctgattaatgggaaagtaccgttctc

[0148] gagccaatacacgtcaatgggaagtgaaagggcagccaaaacgtaacaccgccccggttttcccctggaaattccatattggcacgcattctatt

[0149] ggctgagctgcgttctacgtgggtataagaggcgcgaccagcgtcggtaccgtcgcagtcttcggtctgaccaccgtagaacgcagatcgcaa

[0150] gttgtacaaaagagctcaagcaggctacgcgtgccaccatggagtttgggctgagctgggttttcctcgttgctctttttagaggtgtccagtgtac

[0151] cggtcaggtgcagctgcaggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtacagcctctggaatcacctacag

[0152] taggtactggatgggctggttccgccaggctccagggaaggagcgcgagggggtcgctagtattggtagtgatggtaggacaagctatgcag

[0153] actccgtgaagggccgattcaccatctccaaagacaacgccaagatcacgctgtttctccaaatgaacagcctgaaacctgaggacactgccat

[0154] gtactactgtgcggctggtgttagcgcttggaaaagtgccgctacaaactggggccaggggacccaggtcaccgtctcctcagaattcggtgga

[0155] ggcggttcacctccttgtccaccttgtccagctccagaactgctgggtgggccttctgtgtttctgttcccacctaagccaaaggataccctgatgat

[0156] ctctaggaccccagaagtgacctgtgtggtcgtcgatgtgtctcatgaagaccctgaagtgaagttcaactggtacgtggacggggtggaagtg

[0157] cataacgcaaagaccaagcccagggaagagcaatacaactccacctacagggtggtctccgtcctgacagtcctgcatcaggattggctgaac

[0158] ggcaaggagtacaagtgcaaggtctccaataaagccctgcctgcccctatcgagaaaaccattagcaaagccaaaggccagcccagggagc

[0159] cccaggtctatacactgccccccagcagggaggagatgacaaaaaatcaggtcagcctgacatgcctggtcaaaggcttttatcccagcgacat

[0160] tgccgtcgagtgggagtccaatggccagcccgagaataattataaaacaacaccccccgtcctggacagcgacggcagcttttttctgtatagca

[0161] aactgacagtcgataaaagcaggtggcagcagggcaatgtcttttcctgcagcgtcatgcacgaggccctgcacaatcactatactcagaaaag

[0162] cctgagcctgtcccccgggaaagtcgacggcggtggcagcaatattgtgatgacccagagtcccaaattcctgcttgtatcagcaggagacag

[0163] ggttaccataacctgcaaggccagtcagagtgtgagtaatggtttagcttggtaccaacagaagccaggtcagtctcctgaactgctgatatacta

[0164] tgcatccaatcgctatagtggagtccctgatcgcttcactggcagtggatatgggacggatttcactctcaccatcagcactgtgcaggctgaaga

[0165] cctggcagtttatttctgtcagcaggattatagctctccattcacgttcggctcggggacaaagttggaaataaaaggtggtggcggttcaggcgg

[0166] aggtggctctggcggtggcggatcggaggtgcagctgcaggagtctggacctgacctggtgaaaccttctcagtcactttcactcacctgcact

[0167] gtcactggctactccatcagcagtggttataactggcactggatccggcagtttccaggcgacaaactggaatggatgggctacatacactacag

[0168] tggtatcactaattccaacccatctctcaaaagtcgaatctctatcactcgcgacacatccaagagccagttcttcctgcacttgagttctgtgactac

[0169] tgaggacacagccacatattactgtgcaagagaagggcgtacctatgaggggtttgcttactggggccaagggactctggtcactgtctctgcat

[0170] aatagacccagctttcttgtacaaagtggttagggataacagggtaatatctatgtcgggtgcggagaaagaggtaatgaaatggaaaaatcgat

[0171] aaatacccagctttcttgtacaaagtggtggggaggctaactgaaacacggaaggagacaataccggaaggaacccgcgctatgacggcaa

[0172] taaaaagacagaataaaacgcacgggtgttgggtcgtttgttcataaacgcggggttcggtcccagggctggcactctgtcgataccccaccga

[0173] gaccccattggggccaatacgcccgcgtttcttccttttccccaccccacccccaagttcgggtgaaggcccagggctcgcagccaacgtcg

[0174] gggcggcaggccctgccatagcgatcgattcgacagatcactgaaatgtgtgggcgtggcttaagggtgggaaagaatatataaggtgggggt

[0175] cttatgtagttttgtatctgttttgcagcagccgccgccgccatgagcaccaactcgtttgatggaagcattgtgagctnotttgacaacgcgcat

[0176] gcccccatgggccggggtgcgtcagaatgtgatgggctccagcattgatggtcgccccgtcctgcccgcaaactctactaccttgacctacgag

[0177] accgtgtctggaacgccgttggagactgcagcctccgccgccgcttcagccgctgcagccaccgcccgcgggattgtgactgactttgctttcc

[0178] tgagcccgcttgcaagcagtgcagcttcccgttcatccgcccgcgatgacaagttgacggctcttttggcacaattggattctttgacccgggaac

[0179] ttaatgtcgtttctcagcagctgttggatctgcgccagcaggtttctgccctgaaggcttcctcccctcccaatgcggtttaaaacataaataaaaaa

[0180] ccagactctgtttggatttggatcaagcaagtgtcttgctgtctttatttaggggttttgcgcgcgcggtaggcccgggaccagcggtctcggtcgtt

[0181] gagggtcctgtgtattttttccaggacgtggtaaaggtgactctggatgttcagatacatgggcataagcccgtctctggggtggaggtagcacca

[0182] ctgcagagcttcatgctgcggggtggtgttgtagatgatccagtcgtagcaggagcgctgggcgtggtgcctaaaaatgtctttcagtagcaagc

[0183] tgattgccaggggcaggcccttggtgtaagtgtttacaaagcggttaagctgggatgggtgcatacgtggggatatgagatgcatcttggactgt

[0184] atttttaggttggctatgttcccagccatatccctccggggattcatgttgtgcagaaccaccagcacagtgtatccggtgcacttgggaaatttgtc

[0185] atgtagcttagaaggaaatgcgtggaagaacttggagacgcccttgtgacctccaagattttccatgcattcgtccataatgatggcaatgggccc

[0186] acgggcggcggcctgggcgaagatatttctgggatcactaacgtcatagttgtgttccaggatgagatcgtcataggccatttttacaaagcgcg

[0187] ggcggagggtgccagactgcggtataatggttccatccggcccaggggcgtagttaccctcacagatttgcatttcccacgctttgagttcagat

[0188] ggggggatcatgtctacctgcggggcgaatgaagaaaacggtttccggggtaggggagatcagctgggaagaaagcaggttcctgagcagct

[0189] gcgacttaccgcagccggtgggcccgtaaatcacacctattaccggctgcaactggtagttaagagagctgcagctgccgtcatccctgagca

[0190] ggggggccacttcgttaagcatgtccctgactcgcatgttttccctgaccaaatccgccagaaggcgctcgccgcccagcgatagcagttcttgc

[0191] aaagcaaagtttttcaacggtttgagaccgtccgccgtaggcatgcttttgagcgtttgaccaagcagttccaggcggtcccacagctcggt

[0192] cacctgctctacggcatctcgatccagcatatctcctcgtttcgcgggttggggcggctttcgctgtacggcagtagtcggtgctcgtccagacgg

[0193] gccagggtcatgtctttccacgggcgcagggtcctcgtcagcgtagtctgggtcacggtgaaggggtgcgctccgggctgcgcgctgccag

[0194] ggtgcgcttgaggctggtcctgctggtgctgaagcgctgccggtcttcgccctgcgcgtcggccaggtagcatttgaccatggtgtcatagtcca

[0195] gcccctccgcggcgtggcccttggcgcgcagcttgcccttggaggaggcgccgcacgaggggcagtgcagacttttgagggcgtagagctt

[0196] gggcgcgagaaataccgattccggggagtaggcatccgcgccgcaggccccgcagacggtctcgcattccacgagccaggtgagctctgg

[0197] ccgttcggggtcaaaaaccaggtttcccccatgctttttgatgcgtttcttacctctggtttccatgagccggtgtccacgctcggtgacgaaaagg

[0198] ctgtccgtgtccccgtatacagacttgagaggcctgtcctcgagcggtgttccgcggtcctcctcgtatagaaactcggaccactctgagacaaa

[0199] ggctcgcgtccaggccagcacgaaggaggctaagtgggaggggtagcggtcgttgtccactagggggtccactcgctccagggtgtgaaga

[0200] cacatgtcgccctcttcggcatcaaggaaggtgattggtttgtaggtgtaggccacgtgaccgggtgttcctgaaggggggctataaaaggggg

[0201] tgggggcgcgttcgtcctcactctcttccgcatcgctgtctgcgagggccagctgttggggtgagtactccctctgaaaagcgggcatgacttct

[0202] gcgctaagattgtcagtttccaaaaacgaggaggatttgatattcacctggcccgcggtgatgcctttgagggtggccgcatccatctggtcaga

[0203] aaagacaatctttttgttgtcaagcttggtggcaaacgacccgtagagggcgttggacagcaacttggcgatggagcgcagggtttggtttttgtc

[0204] gcgatcggcgcgctccttggccgcgatgtttagctgcacgtattcgcgcgcaacgcaccgccattcgggaaagacggtggtgcgctcgtcggg

[0205] caccaggtgcacgcgccaaccgcggttgtgcagggtgacaaggtcaacgctggtggctacctctccgcgtaggcgctcgttggtccagcaga

[0206] ggcggccgcccttgcgcgagcagaatggcggtagggggtctagctgcgtctcgtccggggggtctgcgtccacggtaaagaccccgggca

[0207] gcaggcgcgcgtcgaagtagtctatcttgcatccttgcaagtctagcgcctgctgccatgcgcgggcggcaagcgcgcgctcgtatgggttga

[0208] gtgggggaccccatggcatggggtgggtgagcgcggaggcgtacatgccgcaaatgtcgtaaacgtagaggggctctctgagtattccaaga

[0209] tatgtagggtagcatcttccaccgcggatgctggcgcgcacgtaatcgtatagttcgtgcgagggagcgaggaggtcgggaccgaggttgcta

[0210] cgggcgggctgctctgctcggaagactatctgcctgaagatggcatgtgagttggatgatatggttggacgctggaagacgttgaagctggcgt

[0211] ctgtgagacctaccgcgtcacgcacgaaggaggcgtaggagtcgcgcagcttgttgaccagctcggcggtgacctgcacgtctagggcgca

[0212] gtagtccagggtttccttgatgatgtcatacttatcctgtcccttttttttccacagctcgcggttgaggacaaactcttcgcggtctttccagtactcttg

[0213] gatcggaaacccgtcggcctccgaacggtaagagcctagcatgtagaactggttgacggcctggtaggcgcagcatcccttttctacgggtag

[0214] cgcgtatgcctgcgcggccttccggagcgaggtgtgggtgagcgcaaaggtgtccctgaccatgactttgaggtactggtatttgaagtcagtgt

[0215] cgtcgcatccgccctgctcccagagcaaaaagtccgtgcgctttttggaacgcggatttggcagggcgaaggtgacatcgttgaagagtatcttt

[0216] cccgcgcgaggcataaagttgcgtgtgatgcggaagggtcccggcacctcggaacggttgttaattacctgggcggcgagcacgatctcgtca

[0217] aagccgttgatgttgtggcccacaatgtaaagttccaagaagcgcgggatgcccttgatggaaggcaattttttaagttcctcgtaggtgagctctt

[0218] caggggagctgagcccgtgctctgaaagggcccagtctgcaagatgagggttggaagcgacgaatgagctccacaggtcacgggccattag

[0219] catttgcaggtggtcgcgaaaggtcctaaactggcgacctatggccattttttctggggtgatgcagtagaaggtaagcgggtcttgttcccagcg

[0220] gtcccatccaaggttcgcggctaggtctcgcgcggcagtcactagaggctcatctccgccgaacttcatgaccagcatgaagggcacgagctg

[0221] cttcccaaaggcccccatccaagtataggtctctacatcgtaggtgacaaagagacgctcggtgcgaggatgcgagccgatcgggaagaactg

[0222] gatctcccgccaccaattggaggagtggctattgatgtggtgaaagtagaagtccctgcgacgggccgaacactcgtgctggcttttgtaaaaac

[0223] gtgcgcagtactggcagcggtgcacgggctgtacatcctgcacgaggttgacctgacgaccgcgcacaaggaagcagagtgggaatttgag

[0224] cccctcgcctggcgggtttggctggtggtcttctacttcggctgcttgtccttgaccgtctggctgctcgaggggagttacggtggatcggaccac

[0225] cacgccgcgcgagcccaaagtccagatgtccgcgcgcggcggtcggagcttgatgacaacatcgcgcagatgggagctgtccatggtctgg

[0226] agctcccgcggcgtcaggtcaggcgggagctcctgcaggtttacctcgcatagacgggtcagggcgcgggctagatccaggtgatacctaatt

[0227] tccaggggctggttggtggcggcgtcgatggcttgcaagaggccgcatccccgcggcgcgactacggtaccgcgcggcgggcggtgggcc

[0228] gcgggggtgtccttggatgatgcatctaaaagcggtgacgcgggcgagcccccggaggtagggggggctccggacccgccgggagaggg

[0229] ggcaggggcacgtcggcgccgcgcgcgggcaggagctggtgctgcgcgcgtaggttgctggcgaacgcgacgacgcggcggttgatctc

[0230] ctgaatctggcgcctctgcgtgaagacgacgggcccggtgagcttgaacctgaaagagagttcgacagaatcaatttcggtgtcgttgacggcg

[0231] gcctggcgcaaaatctcctgcacgtctcctgagttgtcttgataggcgatctcggccatgaactgctcgatctcttcctcctggagatctccgcgtc

[0232] cggctcgctccacggtggcggcgaggtcgttggaaatgcgggccatgagctgcgagaaggcgttgaggcctccctcgttccagacgcggct

[0233] gtagaccacgcccccttcggcatcgcgggcgcgcatgaccacctgcgcgagattgagctccacgtgccgggcgaagacggcgtagtttcgc

[0234] aggcgctgaaagaggtagttgagggtggtggcggtgtgttctgccacgaagaagtacataacccagcgtcgcaacgtggattcgttgatatccc

[0235] ccaaggcctcaaggcgctccatggcctcgtagaagtccacggcgaagttgaaaaactgggagttgcgcgccgacacggttaactcctcctcca

[0236] gaagacggatgagctcggcgacagtgtcgcgcacctcgcgctcaaaggctacaggggcctcttcttcttcttcaatctcctcttccataagggcct

[0237] ccccttcttcttcttctggcggcggtgggggaggggggacacggcggcgacgacggcgcaccgggaggcggtcgacaaagcgctcgatcat

[0238] ctccccgcggcgacggcgcatggtctcggtgacggcgcggccgttctcgcgggggcgcagttggaagacgccgcccgtcatgtcccggttat

[0239] gggttggcggggggctgccatgcggcagggatacggcgctaacgatgcatctcaacaattgttgtgtaggtactccgccgccgagggacctg

[0240] agcgagtccgcatcgaccggatcggaaaacctctcgagaaaggcgtctaaccagtcacagtcgcaaggtaggctgagcaccgtggcgggcg

[0241] gcagcgggcggcggtcggggttgtttctggcggaggtgctgctgatgatgtaattaaagtaggcggtcttgagacggcggatggtcgacagaa

[0242] gcaccatgtccttgggtccggcctgctgaatgcgcaggcggtcggccatgccccaggcttcgttttgacatcggcgcaggtctttgtagtagtctt

[0243] gcatgagcctttctaccggcacttcttcttctccttcctcttgtcctgcatctcttgcatctatcgctgcggcggcggcggagtttggccgtaggtggc

[0244] gccctcttcctcccatgcgtgtgaccccgaagcccctcatcggctgaagcagggctaggtcggcgacaacgcgctcggctaatatggcctgct

[0245] gcacctgcgtgagggtagactggaagtcatccatgtccacaaagcggtggtatgcgcccgtgttgatggtgtaagtgcagttggccataacgga

[0246] ccagttaacggtctggtgacccggctgcgagagctcggtgtacctgagacgcgagtaagccctcgagtcaaatacgtagtcgttgcaagtccg

[0247] caccaggtactggtatcccaccaaaaagtgcggcggcggctggcggtagaggggccagcgtagggtggccggggctccgggggcgagat

[0248] cttccaacataaggcgatgatatccgtagatgtacctggacatccaggtgatgccggcggcggtggtggaggcgcgcggaaagtcgcggacg

[0249] cggttccagatgttgcgcagcggcaaaaagtgctccatggtcgggacgctctggccggtcaggcgcgcgcaatcgttgacgctctagaccgtg

[0250] caaaaggagagcctgtaagcgggcactcttccgtggtctggtggataaattcgcaagggtatcatggcggacgaccggggttcgagccccgta

[0251] tccggccgtccgccgtgatccatgcggttaccgcccgcgtgtcgaacccaggtgtgcgacgtcagacaacgggggagtgctccttttggcttcc

[0252] ttccaggcgcggcggctgctgcgctagcttttttggccactggccgcgcgcagcgtaagcggttaggctggaaagcgaaagcattaagtggct

[0253] cgctccctgtagccggagggttattttccaagggttgagtcgcgggacccccggttcgagtctcggaccggccggactgcggcgaacggggg

[0254] tttgcctccccgtcatgcaagaccccgcttgcaaattcctccggaaacagggacgagccccttttttgcttttcccagatgcatccggtgctgcggc

[0255] agatgcgcccccctcctcagcagcggcaagagcaagagcagcggcagacatgcagggcaccctcccctcctcctaccgcgtcaggagggg

[0256] cgacatccgcggttgacgcggcagcagatggtgattacgaacccccgcggcgccgggcccggcactacctggacttggaggagggcgagg

[0257] gcctggcgcggctaggagcgccctctcctgagcggcacccaagggtgcagctgaagcgtgatacgcgtgaggcgtacgtgccgcggcaga

[0258] acctgtttcgcgaccgcgagggagaggagcccgaggagatgcgggatcgaaagttccacgcagggcgcgagctgcggcatggcctgaatc

[0259] gcgagcggttgctgcgcgaggaggactttgagcccgacgcgcgaaccgggattagtcccgcgcgcgcacacgtggcggccgccgacctgg

[0260] taaccgcatacgagcagacggtgaaccaggagattaactttcaaaaaagcttttaacaaccacgtgcgtacgcttgtggcgcgcgaggaggtg

[0261] gctataggactgatgcatctgtgggactttgtaagcgcgctggagcaaaacccaaatagcaagccgctcatggcgcagctgttccttatagtgca

[0262] gcacagcagggacaacgaggcattcagggatgcgctgctaaacatagtagagcccgagggccgctggctgctcgatttgataaacatcctgc

[0263] agagcatagtggtgcaggagcgcagcttgagcctggctgacaaggtggccgccatcaactattccatgcttagcctgggcaagttttacgcccg

[0264] caagatataccataccccttacgttcccatagacaaggaggtaaagatcgaggggttctacatgcgcatggcgctgaaggtgcttaccttgagcg

[0265] acgacctgggcgtttatcgcaacgagcgcatccacaaggccgtgagcgtgagccggcggcgcgagctcagcgaccgcgagctgatgcaca

[0266] gcctgcaaagggccctggctggcacgggcagcggcgatagagaggccgagtcctactttgacgcgggcgctgacctgcgctgggccccaa

[0267] gccgacgcgccctggaggcagctggggccggacctgggctggcggtggcacccgcgcgcgctggcaacgtcggcggcgtggaggaatat

[0268] gacgaggacgatgagtacgagccagaggacggcgagtactaagcggtgatgtttctgatcagatgatgcaagacgcaacggacccggcggt

[0269] gcgggcggcgctgcagagccagccgtccggccttaactccacggacgactggcgccaggtcatggaccgcatcatgtcgctgactgcgcgc

[0270] aatcctgacgcgttccggcagcagccgcaggccaaccggctctccgcaattctggaagcggtggtcccggcgcgcgcaaaccccacgcacg

[0271] agaaggtgctggcgatcgtaaacgcgctggccgaaaacagggccatccggcccgacgaggccggcctggtctacgacgcgctgcttcagc

[0272] gcgtggctcgttacaacagcggcaacgtgcagaccaacctggaccggctggtgggggatgtgcgcgaggccgtggcgcagcgtgagcgcg

[0273] cgcagcagcagggcaacctgggctccatggttgcactaaacgccttcctgagtacacagcccgccaacgtgccgcggggacaggaggacta

[0274] caccaactttgtgagcgcactgcggctaatggtgactgagacaccgcaaagtgaggtgtaccagtctgggccagactattttttccagaccagta

[0275] gacaaggcctgcagaccgtaaacctgagccaggctttcaaaaacttgcaggggctgtggggggtgcgggctcccacaggcgaccgcgcga

[0276] ccgtgtctagcttgctgacgcccaactcgcgcctgttgctgctgctaatagcgcccttcacggacagtggcagcgtgtcccgggacacataccta

[0277] ggtcacttgctgacactgtaccgcgaggccataggtcaggcgcatgtggacgagcatactttccaggagattacaagtgtcagccgcgcgctg

[0278] gggcaggaggacacgggcagcctggaggcaaccctaaactacctgctgaccaaccggcggcagaagatcccctcgttgcacagtttaaaca

[0279] gcgaggaggagcgcattttgcgctacgtgcagcagagcgtgagccttaacctgatgcgcgacggggtaacgcccagcgtggcgctggacat

[0280] gaccgcgcgcaacatggaaccgggcatgtatgcctcaaaccggccgtttatcaaccgcctaatggactacttgcatcgcgcggccgccgtgaa

[0281] ccccgagtatttcaccaatgccatcttgaacccgcactggctaccgccccctggtttctacaccgggggattcgaggtgcccgagggtaacgat

[0282] ggattcctctgggacgacatagacgacagcgtgttttccccgcaaccgcagaccctgctagagttgcaacagcgcgagcaggcagaggcggc

[0283] gctgcgaaaggaaagcttccgcaggccaagcagcttgtccgatctaggcgctgcggccccgcggtcagatgctagtagcccatttccaagctt

[0284] gatagggtctcttaccagcactcgcaccacccgcccgcgcctgctgggcgaggaggagtacctaaacaactcgctgctgcagccgcagcgc

[0285] gaaaaaaacctgcctccggcatttcccaacaacgggatagagagcctagtggacaagatgagtagatggaagacgtacgcgcaggagcaca

[0286] gggacgtgccaggcccgcgcccgcccacccgtcgtcaaaggcacgaccgtcagcggggtctggtgtgggaggacgatgactcggcagac

[0287] gacagcagcgtcctggatttgggagggagtggcaacccgtttgcgcaccttcgccccaggctggggagaatgttttaaaaaaaaaaaaagcat

[0288] gatgcaaaataaaaaactcaccaaggccatggcaccgagcgttggttttcttgtattccccttagtatgcggcgcgcggcgatgtatgaggaagg

[0289] tcctcctccctcctacgagagtgtggtgagcgcggcgccagtggcggcggcgctgggttctcccttcgatgctcccctggacccgccgtttgtg

[0290] cctccgcggtacctgcggcctaccggggggagaaacagcatccgttactctgagttggcacccctattcgacaccacccgtgtgtacctggtgg

[0291] acaacaagtcaacggatgtggcatccctgaactaccagaacgaccacagcaactttctgaccacggtcattcaaaacaatgactacagcccgg

[0292] gggaggcaagcacacagaccatcaatcttgacgaccggtcgcactggggcggcgacctgaaaaccatcctgcataccaacatgccaaatgtg

[0293] aacgagttcatgtttaccaataagtttaaggcgcgggtgatggtgtcgcgcttgcctactaaggacaatcaggtggagctgaaatacgagtgggt

[0294] ggagttcacgctgcccgagggcaactactccgagaccatgaccatagaccttatgaacaacgcgatcgtggagcactacttgaaagtgggcag

[0295] acagaacggggttctggaaagcgacatcggggtaaagtttgacacccgcaacttcagactggggtttgaccccgtcactggtcttgtcatgcctg

[0296] gggtatatacaaacgaagccttccatccagacatcattttgctgccaggatgcggggtggacttcacccacagccgcctgagcaacttgttgggc

[0297] atccgcaagcggcaacccttccaggagggctttaggatcacctacgatgatctggagggtggtaacattcccgcactgttggatgtggacgcct

[0298] accaggcgagcttgaaagatgacaccgaacagggcgggggtggcgcaggcggcagcaacagcagtggcagcggcgcggaagagaactc

[0299] caacgcggcagccgcggcaatgcagccggtggaggacatgaacgatcatgccattcgcggcgacacctttgccacacgggctgaggagaa

[0300] gcgcgctgaggccgaagcagcggccgaagctgccgcccccgctgcgcaacccgaggtcgagaagcctcagaagaaaccggtgatcaaac

[0301] ccctgacagaggacagcaagaaacgcagttacaacctaataagcaatgacagcaccttcacccagtaccgcagctggtaccttgcatacaact

[0302] acggcgaccctcagaccggaatccgctcatggaccctgctttgcactcctgacgtaacctgcggctcggagcaggtctactggtcgttgccaga

[0303] catgatgcaagaccccgtgaccttccgctccacgcgccagatcagcaactttccggtggtgggcgccgagctgttgcccgtgcactccaagag

[0304] cttctacaacgaccaggccgtctactcccaactcatccgccagtttacctctctgacccacgtgttcaatcgctttcccgagaaccagattttggcg

[0305] cgcccgccagcccccaccatcaccaccgtcagtgaaaacgttcctgctctcacagatcacgggacgctaccgctgcgcaacagcatcggagg

[0306] agtccagcgagtgaccattactgacgccagacgccgcacctgcccctacgtttacaaggccctgggcatagtctcgccgcgcgtcctatcgag

[0307] ccgcactttttgagcaagcatgtccatccttatatcgcccagcaataacacaggctggggcctgcgcttcccaagcaagatgtttggcggggcca

[0308] agaagcgctccgaccaacacccagtgcgcgtgcgcgggcactaccgcgcgccctggggcgcgcacaaacgcggccgcactgggcgcac

[0309] caccgtcgatgacgccatcgacgcggtggtggaggaggcgcgcaactacacgcccacgccgccaccagtgtccacagtggacgcggccat

[0310] tcagaccgtggtgcgcggagcccggcgctatgctaaaatgaagagacggcggaggcgcgtagcacgtcgccaccgccgccgacccggca

[0311] ctgccgcccaacgcgcggcggcggccctgcttaaccgcgcacgtcgcaccggccgacgggcggccatgcgggccgctcgaaggctggcc

[0312] gcgggtattgtcactgtgccccccaggtccaggcgacgagcggccgccgcagcagccgcggccattagtgctatgactcagggtcgcaggg

[0313] gcaacgtgtattgggtgcgcgactcggttagcggcctgcgcgtgcccgtgcgcacccgccccccgcgcaactagattgcaagaaaaaactact

[0314] tagactcgtactgttgtatgtatccagcggcggcggcgcgcaacgaagctatgtccaagcgcaaaatcaaagaagagatgctccaggtcatcg

[0315] cgccggagatctatggccccccgaagaaggaagagcaggattacaagccccgaaagctaaagcgggtcaaaaagaaaaagaaagatgatg

[0316] atgatgaacttgacgacgaggtggaactgctgcacgctaccgcgcccaggcgacgggtacagtggaaaggtcgacgcgtaaaacgtgttttg

[0317] cgacccggcaccaccgtagtctttacgcccggtgagcgctccacccgcacctacaagcgcgtgtatgatgaggtgtacggcgacgaggacct

[0318] gcttgagcaggccaacgagcgcctcggggagtttgcctacggaaagcggcataaggacatgctggcgttgccgctggacgagggcaaccca

[0319] acacctagcctaaagcccgtaacactgcagcaggtgctgcccgcgcttgcaccgtccgaagaaaagcgcggcctaaagcgcgagtctggtga

[0320] cttggcacccaccgtgcagctgatggtacccaagcgccagcgactggaagatgtcttggaaaaaatgaccgtggaacctgggctggagcccg

[0321] aggtccgcgtgcggccaatcaagcaggtggcgccgggactgggcgtgcagaccgtggacgttcagatacccactaccagtagcaccagtatt

[0322] gccaccgccacagagggcatggagacacaaacgtccccggttgcctcagcggtggcggatgccgcggtgcaggcggtcgctgcggccgc

[0323] gtccaagacctctacggaggtgcaaacggacccgtggatgtttcgcgtttcagccccccggcgcccgcgccgttcgaggaagtacggcgccg

[0324] ccagcgcgctactgcccgaatatgccctacatccttccattgcgcctacccccggctatcgtggctacacctaccgccccagaagacgagcaac

[0325] tacccgacgccgaaccaccactggaacccgccgccgccgtcgccgtcgccagcccgtgctggccccgatttccgtgcgcagggtggctcgc

[0326] gaaggaggcaggaccctggtgctgccaacagcgcgctaccaccccagcatcgtttaaaagccggtctttgtggttcttgcagatatggccctca

[0327] cctgccgcctccgtttcccggtgccgggattccgaggaagaatgcaccgtaggaggggcatggccggccacggcctgacgggcggcatgc

[0328] gtcgtgcgcaccaccggcggcggcgcgcgtcgcaccgtcgcatgcgcggcggtatcctgcccctccttattccactgatcgccgcggcgattg

[0329] gcgccgtgcccggaattgcatccgtggccttgcaggcgcagagacactgattaaaaacaagttgcatgtggaaaaatcaaaataaaaagtctgg

[0330] actctcacgctcgcttggtcctgtaactattttgtagaatggaagacatcaactttgcgtctctggccccgcgacacggctcgcgcccgttcatggg

[0331] aaactggcaagatatcggcaccagcaatatgagcggtggcgccttcagctggggctcgctgtggagcggcattaaaaatttcggttccaccgtt

[0332] aagaactatggcagcaaggcctggaacagcagcacaggccagatgctgagggataagttgaaagagcaaaatttccaacaaaaggtggtag

[0333] atggcctggcctctggcattagcggggtggtggacctggccaaccaggcagtgcaaaataagattaacagtaagcttgatccccgccctcccgt

[0334] agaggagcctccaccggccgtggagacagtgtctccagaggggcgtggcgaaaagcgtccgcgccccgacagggaagaaactctggtgac

[0335] gcaaatagacgagcctccctcgtacgaggaggcactaaagcaaggcctgcccaccacccgtcccatcgcgcccatggctaccggagtgctg

[0336] ggccagcacacacccgtaacgctggacctgcctccccccgccgacacccagcagaaacctgtgctgccaggcccgaccgccgttgttgtaac

[0337] ccgtcctagccgcgcgtccctgcgccgcgccgccagcggtccgcgatcgttgcggcccgtagccagtggcaactggcaaagcacactgaac

[0338] agcatcgtgggtctgggggtgcaatccctgaagcgccgacgatgcttctgatagctaacgtgtcgtatgtgtgtcatgtatgcgtccatgtcgccg

[0339] ccagaggagctgctgagccgccgcgcgcccgctttccaagatggctaccccttcgatgatgccgcagtggtcttacatgcacatctcgggcca

[0340] ggacgctcggagtacctgagccccgggctggtgcagtttgcccgcgccaccgagacgtacttcagcctgaataacaagtttagaaaccccac

[0341] ggtggcgcctacgcacgacgtgaccacagaccggtcccagcgtttgacgctgcggttcatccctgtggaccgtgaggatactgcgtactcgta

[0342] caaggcgcggttcacctagctgtgggtgataaccgtgtgctggacatggcttccacgtactttgacatccgcggcgtgctggcacaggggccct

[0343] acttttaagccctactctggcactgcctacaacgccctggctcccaagggtgccccaaatccttgcgaatgggatgaagctgctactgctcttgaa

[0344] ataacctagaaagagaggacgatgacaacgaagacgaagtagacgagcaagctgagcagcaaaaaactcacgtattgggcaggcgcctt

[0345] attctggtataaatattacaaaggagggtattcaaataggtgtcgaaggtcaaacacctaaatatgccgataaaacatttcaacctgaacctcaaat

[0346] aggagaatctcagtggtacgaaacagaaattaatcatgcagctgggagagtcctaaaaaagactacccaatgaaaaccatgttacggttcatatg

[0347] caaaacccacaaatgaaaatggagggcaaggcattcttgtaaagcaacaaaatggaaagctagaaagtcaagtggaaatgcaatttttctcaact

[0348] actgaggcagccgcaggcaatggtgataacttgactcctaaagtggtattgtacagtgaagatgtagatatagaaaccccagacactcatatttctt

[0349] acatgcccactattaaggaaggtaactcacgagaactaatgggccaacaatctatgcccaacaggcctaattacattgcttttagggacaattttatt

[0350] ggtctaatgtattacaacagcacgggtaatatgggtgttctggcgggccaagcatcgcagttgaatgctgttgtagatttgcaagacagaaacaca

[0351] gagctttcataccagcttttgcttgattccattggtgatagaaccaggtacttttctatgtggaatcaggctgttgacagctatgatccagatgttagaa

[0352] ttattgaaaatcatggaactgaagatgaacttccaaattactgctttccactgggaggtgtgattaatacagagactcttaccaaggtaaaacctaaa

[0353] acaggtcaggaaaatggatgggaaaaagatgctacagaattttcagataaaaatgaaataagagttggaaataattttgccatggaaatcaatcta

[0354] aatgccaacctgtggagaaatttcctgtactccaacatagcgctgtatttgcccgacaagctaaagtacagtccttccaacgtaaaaatttctgataa

[0355] cccaaacacctacgactacatgaacaagcgagtggtggctcccgggctagtggactgctacattaaccttggagcacgctggtcccttgactata

[0356] tggacaacgtcaacccatttaaccaccaccgcaatgctggcctgcgctaccgctcaatgttgctgggcaatggtcgctatgtgcccttccacatcc

[0357] aggtgcctcagaagttctttgccattaaaaacctccttctcctgccgggctcatacacctacgagtggaacttcaggaaggatgttaacatggttct

[0358] gcagagctccctaggaaatgacctaagggttgacggagccagcattaagtttgatagcatttgcctttacgccaccttcttccccatggcccacaa

[0359] caccgcctccacgcttgaggccatgcttagaaacgacaccaacgaccagtcctttaacgactatctctccgccgccaacatgctctaccctatac

[0360] ccgccaacgctaccaacgtgcccatatccatcccctcccgcaactgggcggctttccgcggctgggccttcacgcgccttaagactaaggaaa

[0361] ccccatcactgggctcgggctacgacccttattacacctactctggctctataccctacctagatggaaccttttacctcaaccacacctttaagaag

[0362] gtggccattacctttgactcttctgtcagctggcctggcaatgaccgcctgcttacccccaacgagtttgaaattaagcgctcagttgacggggag

[0363] ggttacaacgttgcccagtgtaacatgaccaaagactggttcctggtacaaatgctagctaactataacattggctaccagggcttctatatcccag

[0364] agagctacaaggaccgcatgtactccttctttagaaacttccagcccatgagccgtcaggtggtggatgatactaaatacaaggactaccaacag

[0365] gtgggcatcctacaccaacacaacaactctggatttgttggctaccttgcccccaccatgcgcgaaggacaggcctaccctgctaacttcccctat

[0366] ccgcttataggcaagaccgcagttgacagcattacccagaaaaagtttctttgcgatcgcaccctttggcgcatcccattctccagtaactttatgtc

[0367] catgggcgcactcacagacctgggccaaaaccttctctacgccaactccgcccacgcgctagacatgacttttgaggtggatcccatggacga

[0368] gcccacccttctttatgttttgtttgaagtctttgacgtggtccgtgtgcaccagccgcaccgcggcgtcatcgaaaccgtgtacctgcgcacgccc

[0369] ttctcggccggcaacgccacaacataaagaagcaagcaacatcaacaacagctgccgccatgggctccagtgagcaggaactgaaagccatt

[0370] gtcaaagatcttggttgtgggccatattttttgggcacctatgacaagcgctttccaggctttgtttctccacacaagctcgcctgcgccatagtcaat

[0371] acggccggtcgcgagactgggggcgtacactggatggcctttgcctggaacccgcactcaaaaacatgctacctctttgagccctttggcttttct

[0372] gaccagcgactcaagcaggtttaccagtttgagtacgagtcactcctgcgccgtagcgccattgcttcttcccccgaccgctgtataacgctgga

[0373] aaagtccacccaaagcgtacaggggcccaactcggccgcctgtggactattctgctgcatgtttctccacgcctttgccaactggccccaaactc

[0374] ccatggatcacaaccccaccatgaaccttattaccggggtacccaactccatgctcaacagtccccaggtacagcccaccctgcgtcgcaacca

[0375] ggaacagctctacagcttcctggagcgccactcgccctacttccgcagccacagtgcgcagattaggagcgccacttctttttgtcacttgaaaaa

[0376] catgtaaaaataatgtactagagacactttcaataaaggcaaatgcttttatttgtacactctcgggtgattatttacccccacccttgccgtctgcgcc

[0377] gtttaaaaatcaaaggggttctgccgcgcatcgctatgcgccactggcagggacacgttgcgatactggtgtttagtgctccacttaaactcaggc

[0378] acaaccatccgcggcagctcggtgaagttttcactccacaggctgcgcaccatcaccaacgcgtttagcaggtcgggcgccgatatcttgaagt

[0379] cgcagttggggcctccgccctgcgcgcgcgagttgcgatacacagggttgcagcactggaacactatcagcgccgggtggtgcacgctggc

[0380] cagcacgctcttgtcggagatcagatccgcgtccaggtcctccgcgttgctcagggcgaacggagtcaactttggtagctgccttcccaaaaag

[0381] ggcgcgtgcccaggctttgagttgcactcgcaccgtagtggcatcaaaaggtgaccgtgcccggtctgggcgttaggatacagcgcctgcata

[0382] aaagccttgatctgcttaaaagccacctgagcctttgcgccttcagagaagaacatgccgcaagacttgccggaaaactgattggccggacagg

[0383] ccgcgtcgtgcacgcagcaccttgcgtcggtgttggagatctgcaccacatttcggccccaccggttcttcacgatcttggccttgctagactgct

[0384] ccttcagcgcgcgctgcccgttttcgctcgtcacatccatttcaatcacgtgctccttatttatcataatgcttccgtgtagacacttaagctcgccttc

[0385] gatctcagcgcagcggtgcagccacaacgcgcagcccgtgggctcgtgatgcttgtaggtcacctctgcaaacgactgcaggtacgcctgca

[0386] ggaatcgccccatcatcgtcacaaaggtcttgttgctggtgaaggtcagctgcaacccgcggtgctcctcgttcagccaggtcttgcatacggcc

[0387] gccagagcttccacttggtcaggcagtagtttgaagttcgcctttagatcgttatccacgtggtacttgtccatcagcgcgcgcgcagcctccatgc

[0388] ccttctcccacgcagacacgatcggcacactcagcgggttcatcaccgtaatttcactttccgcttcgctgggctcttcctcttcctcttgcgtccgc

[0389] ataccacgcgccactgggtcgtcttcattcagccgccgcactgtgcgcttacctcctttgccatgcttgattagcaccggtgggttgctgaaaccc

[0390] accatttgtagcgccacatcttctctttcttcctcgctgtccacgattacctctggtgatggcgggcgctcgggcttgggagaagggcgcttctttttc

[0391] ttcttgggcgcaatggccaaatccgccgccgaggtcgatggccgcgggctgggtgtgcgcggcaccagcgcgtcttgtgatgagtcttcctcgt

[0392] cctcggactcgatacgccgcctcatccgcttttttgggggcgcccggggaggcggcggcgacggggacggggacgacacgtcctccatggtt

[0393] gggggacgtcgcgccgcaccgcgtccgcgctcgggggtggtttcgcgctgctcctcttcccgactggccatttccttctcctataggcagaaaa

[0394] agatcatggagtcagtcgagaagaaggacagcctaaccgccccctctgagttcgccaccaccgcctccaccgatgccgccaacgcgcctacc

[0395] accttccccgtcgaggcacccccgcttgaggaggaggaagtgattatcgagcaggacccaggttttgtaagcgaagacgacgaggaccgctc

[0396] agtaccaacagaggataaaaagcaagaccaggacaacgcagaggcaaacgaggaacaagtcgggcggggggacgaaaggcatggcgac

[0397] tacctagatgtgggagacgacgtgctgttgaagcatctgcagcgccagtgcgccattatctgcgacgcgttgcaagagcgcagcgatgtgccc

[0398] ctcgccatagcggatgtcagccttgcctacgaacgccacctattctcaccgcgcgtaccccccaaacgccaagaaaacggcacatgcgagcc

[0399] caacccgcgcctcaacttctaccccgtatttgccgtgccagaggtgcttgccacctatcacatctttttccaaaactgcaagatacccctatcctgcc

[0400] gtgccaaccgcagccgagcggacaagcagctggccttgcggcagggcgctgtcatacctgatatcgcctcgctcaacgaagtgccaaaaatc

[0401] tttgagggtcttggacgcgacgagaagcgcgcggcaaacgctctgcaacaggaaaacagcgaaaatgaaagtcactctggagtgttggtgga

[0402] actcgagggtgacaacgcgcgcctagccgtactaaaacgcagcatcgaggtcacccactttgcctacccggcacttaacctaccccccaaggt

[0403] catgagcacagtcatgagtgagctgatcgtgcgccgtgcgcagcccctggagagggatgcaaatttgcaagaacaaacagaggagggccta

[0404] cccgcagttggcgacgagcagctagcgcgctggcttcaaacgcgcgagcctgccgacttggaggagcgacgcaaactaatgatggccgcag

[0405] tgctcgttaccgtggagcttgagtgcatgcagcggttctttgctgacccggagatgcagcgcaagctagaggaaacattgcactacacctttcga

[0406] cagggctacgtacgccaggcctgcaagatctccaacgtggagctctgcaacctggtctcctaccttggaattttgcacgaaaaccgccttgggc

[0407] aaaacgtgcttcattccacgctcaagggcgaggcgcgccgcgactacgtccgcgactgcgtttacttatttctatgctacacctggcagacggcc

[0408] atgggcgtttggcagcagtgcttggaggagtgcaacctcaaggagctgcagaaactgctaaagcaaaacttgaaggacctatggacggccttc

[0409] aacgagcgctccgtggccgcgcacctggcggacatcattttccccgaacgcctgcttaaaaccctgcaacagggtctgccagacttcaccagtc

[0410] aaagcatgttgcagaactttaggaactttatcctagagcgctcaggaatcttgcccgccacctgctgtgcacttcctagcgactttgtgcccattaa

[0411] gtaccgcgaatgccctccgccgctttggggccactgctaccttctgcagctagccaactaccttgcctaccactctgacataatggaagacgtga

[0412] gcggtgacggtctactggagtgtcactgtcgctgcaacctatgcaccccgcaccgctccctggtttgcaattcgcagctgcttaacgaaagtcaa

[0413] attatcggtacctttgagctgcagggtccctcgcctgacgaaaagtccgcggctccggggttgaaactcactccggggctgtggacgtcggctt

[0414] accttcgcaaatttgtacctgaggactaccacgcccacgagattaggttctacgaagaccaatcccgcccgcctaatgcggagcttaccgcctgc

[0415] gtcattacccagggccacattcttggccaattgcaagccatcaacaaagcccgccaagagtttctgctacgaaagggacggggggtttacttgg

[0416] acccccagtccggcgaggagctcaacccaatccccccgccgccgcagccctatcagcagcagccgcgggcccttgcttcccaggatggcac

[0417] ccaaaaagaagctgcagctgccgccgccacccacggacgaggaggaatactgggacagtcaggcagaggaggttttggacgaggaggag

[0418] gaggacatgatggaagactgggagagcctagacgaggaagcttccgaggtcgaagaggtgtcagacgaaacaccgtcaccctcggtcgcat

[0419] tcccctcgccggcgccccagaaatcggcaaccggttccagcatggctacaacctccgctcctcaggcgccgccggcactgcccgttcgccga

[0420] cccaaccgtagatgggacaccactggaaccagggccggtaagtccaagcagccgccgccgttagcccaagagcaacaacagcgccaagg

[0421] ctaccgctcatggcgcgggcacaagaacgccatagttgcttgcttgcaagactgtgggggcaacatctccttcgcccgccgctttcttctctacca

[0422] tcacggcgtggccttcccccgtaacatcctgcattactaccgtcatctctacagcccatactgcaccggcggcagcggcagcaacagcagcgg

[0423] ccacacagaagcaaaggcgaccggatagcaagactctgacaaagcccaagaaatccacagcggcggcagcagcaggaggaggagcgct

[0424] gcgtctggcgcccaacgaacccgtatcgacccgcgagcttagaaacaggatttttcccactctgtatgctatatttcaacagagcaggggccaag

[0425] aacaagagctgaaaataaaaaacaggtctctgcgatccctcacccgcagctgcctgtatcacaaaagcgaagatcagcttcggcgcacgctgg

[0426] aagacgcggaggctctcttcagtaaatactgcgcgctgactcttaaggactagtttcgcgccctttctcaaatttaagcgcgaaaactacgtcatct

[0427] ccagcggccacacccggcgccagcacctgttgtcagcgccattatgagcaaggaaattcccacgccctacatgtggagttaccagccacaaat

[0428] gggacttgcggctggagctgcccaagactactcaacccgaataaactacatgagcgcgggaccccacatgatatcccgggtcaacggaatac

[0429] gcgcccaccgaaaccgaattctcctggaacaggcggctattaccaccacacctcgtaataaccttaatccccgtagttggcccgctgccctggtg

[0430] taccaggaaagtcccgctcccaccactgtggtacttcccagagacgcccaggccgaagttcagatgactaactcaggggcgcagcttgcggg

[0431] cggctttcgtcacagggtgcggtcgcccgggcagggtataactcacctgacaatcagagggcgaggtattcagctcaacgacgagtcggtga

[0432] gctcctcgcttggtctccgtccggacgggacatttcagatcggcggcgccggccgctcttcattcacgcctcgtcaggcaatcctaactctgcag

[0433] acctcgtcctctgagccgcgctctggaggcattggaactctgcaatttattgaggagtttgtgccatcggtctactttaaccccttctcgggacctcc

[0434] cggccactatccggatcaatttattcctaactttgacgcggtaaaggactcggcggacggctacgactgaatgttaagtggagaggcagagcaa

[0435] ctgcgcctgaaacacctggtccactgtcgccgccacaagtgctttgcccgcgactccggtgagttttgctactttgaattgcccgaggatcatatc

[0436] gagggcccggcgcacggcgtccggcttaccgcccagggagagcttgcccgtagcctgattcgggagtttacccagcgccccctgctagttga

[0437] gcgggacaggggaccctgtgttctcactgtgatttgcaactgtcctaaccctggattacatcaagatctttgttgccatctctgtgctgagtataataa

[0438] atacagaaattaaaatatactggggctcctatcgccatcctgtaaacgccaccgtcttcacccgcccaagcaaaccaaggcgaaccttacctggt

[0439] acttttaacatctctccctctgtgatttacaacagtttcaacccagacggagtgagtctacgagagaacctctccgagctcagctactccatcagaa

[0440] aaaacaccaccctccttacctgccgggaacgtacgagtgcgtcaccggccgctgcaccacacctaccgcctgaccgtaaaccagactttttccg

[0441] gacagacctcaataactctgtttaccagaacaggaggtgagcttagaaaacccttagggtattaggccaaaggcgcagctactgtggggtttatg

[0442] aacaattcaagcaactctacgggctattctaattcaggtttctctagaacgttacataacttacggtaaatggcccgcctggctgaccgcccaacga

[0443] cccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaact

[0444] gcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtac

[0445] atgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgga

[0446] tagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtc

[0447] gtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcaggctagcgccaccatgtgtcaccagca

[0448] gttggtcatctcttggttttccctggtttttctggcatctcccctcgtggccatatgggaactgaagaaagatgtttatgtcgtagaattggattggtatc

[0449] cggatgcccctggagaaatggtggtcctcacctgtgacacccctgaagaagatggtatcacctggaccttggaccagagcagtgaggtcttag

[0450] gctctggcaaaaccctgaccatccaagtcaaagagtttggagatgctggccagtacacctgtcacaaaggaggcgaggttctaagccattcgct

[0451] cctgctgcttcacaaaaaggaagatggaatttggtccactgatattttaaaggaccagaaagaacccaaaaataagacctttctaagatgcgagg

[0452] ccaagaattattctggacgtttcacctgctggtggctgacgacaatcagtactgatttgacattcagtgtcaaaagcagcagaggctcttctgaccc

[0453] ccaaggggtgacgtgcggagctgctacactctctgcagagagagtcagaggggacaacaaggagtatgagtactcagtggagtgccaggag

[0454] gacagtgcctgcccagctgctgaggagagtctgcccattgaggtcatggtggatgccgttcacaagctcaagtatgaaaactacaccagcagct

[0455] tcttcatcagggacatcatcaaacctgacccacccaagaacttgcagctgaagccattaaagaattctcggcaggtggaggtcagctgggagta

[0456] ccctgacacctggagtactccacattcctacttctccctgacattctgcgttcaggtccagggcaagagcaagagagaaaagaaagatagagtct

[0457] tcacggacaagacctcagccacggtcatctgccgcaaaaatgccagcattagcgtgcgggcccaggaccgctactatagctcatcttggagcg

[0458] aatgggcatctgtgccctgcagtggcggcggcggctccggaggaggaggaagcggaggaggcggcagcagaaacctccccgtggccact

[0459] ccagacccaggaatgttcccatgccttcaccactcccaaaacctgctgagggccgtcagcaacatgctccagaaggccagacaaactctagaa

[0460] ttttacccttgcacttctgaagagattgatcatgaagatatcacaaaagataaaaccagcacagtggaggcctgtttaccattggaattaaccaaga

[0461] atgagagttgcctaaattccagagagacctctttcataactaatgggagttgcctggcctccagaaagacctcttttatgatggccctgtgccttagt

[0462] agtatttatgaagacttgaagatgtaccaggtggagttcaagaccatgaatgcaaagcttctgatggatcctaagaggcagatctttctagatcaaa

[0463] acatgctggcagttattgatgagctgatgcaggccctgaatttcaacagtgagactgtgccacaaaaatcctcccttgaagaaccggatttttataa

[0464] aactaaaatcaagctctgcatacttcttcatgctttcagaattcgggcagtgactattgatagagtgatgagctatctgaatgcttcctgaacgcgtaa

[0465] gcttaagtttaaaccgctgatcagcctcgaaataaaatatctttattttcattacatctgtgtgttggttttttgtgtgtctagaatggacggaattattaca

[0466] gagcagcgcctgctagaaagacgcagggcagcggccgagcaacagcgcatgaatcaagagctccaagacatggttaacttgcaccagtgca

[0467] aaaggggtatcttttgtctggtaaagcaggccaaagtcacctacgacagtaataccaccggacaccgccttagctacaagttgccaaccaagcg

[0468] tcagaaattggtggtcatggtgggagaaaagcccattaccataactcagcactcggtagaaaccgaaggctgcattcactcaccttgtcaaggac

[0469] ctgaggatctctgcacccttattaagaccctgtgcggtctcaaagatcttattccctttaactaataaaaaaaaataataaagcatcacttacttaaaat

[0470] cagttagcaaatttctgtccagtttattcagcagcacctccttgccctcctcccagctctggtattgcagcttcctcctggctgcaaactttctccacaa

[0471] tctaaatggaatgtcagtttcctcctgttcctgtccatccgcacccactatcttcatgttgttgcagatgaagcgcgcaagaccgtctgaagatacctt

[0472] caaccccgtgtatccatatgacacggaaaccggtcctccaactgtgccttttcttactcctccctttgtatcccccaatgggtttcaagagagtcccc

[0473] ctggggtactctctttgcgcctatccgaacctctagttacctccaatggcatgcttgcgctcaaaatgggcaacggcctctctctggacgaggccg

[0474] gcaaccttacctcccaaaatgtaaccactgtgagcccacctctcaaaaaaaccaagtcaaacataaacctggaaatatctgcacccctcacagtt

[0475] acctcagaagccctaactgtggctgccgccgcacctctaatggtcgcgggcaacacactcaccatgcaatcacaggccccgctaaccgtgcac

[0476] gactccaaacttagcattgccacccaaggacccctcacagtgtcagaaggaaagctagccctgcaaacatcaggccccctcaccaccaccgat

[0477] agcagtacccttactatcactgcctcaccccctctaactactgccactggtagcttgggcattgacttgaaagagcccatttatacacaaaatggaa

[0478] aactaggactaaagtacggggctcctttgcatgtaacagacgacctaaacactttgaccgtagcaactggtccaggtgtgactattaataatacttc

[0479] cttgcaaactaaagttactggagccttgggttttgattcacaaggcaatatgcaacttaatgtagcaggaggactaaggattgattctcaaaacaga

[0480] cgccttatacttgatgttagttatccgtttgatgctcaaaaccaactaaatctaagactaggacagggccctctttttataaactcagcccacaacttg

[0481] gatattaactacaacaaaggcctttacttgtttacagcttcaaacaattccaaaaagcttgaggttaacctaagcactgccaaggggttgatgtttga

[0482] cgctacagccatagccattaatgcaggagatgggcttgaatttggttcacctaatgcaccaaacacaaatcccctcaaaacaaaaattggccatg

[0483] gcctagaatttgattcaaacaaggctatggttcctaaactaggaactggccttagttttgacagcacaggtgccattacagtaggaaacaaaaata

[0484] atgataagctaactttgtggaccacaccagctccatctcctaactgtagactaaatgcagagaaagatgctaaactcactttggtcttaacaaaatgt

[0485] ggcagtcaaatacttgctacagtttcagttttggctgttaaaggcagtttggctccaatatctggaacagttcaaagtgctcatcttattataagatttg

[0486] acgaaaatggagtgctactaaacaattccttcctggacccagaatattggaactttagaaatggagatcttactgaaggcacagcctatacaaacg

[0487] ctgttggatttatgcctaacctatcagcttatccaaaatctcacggtaaaactgccaaaagtaacattgtcagtcaagtttacttaaacggagacaaa

[0488] actaaacctgtaacactaaccattacactaaacggtacacaggaaacaggagacacaactgcctgtgactgccgcggagactgtttctgcggcc

[0489] caagtgcatactctatgtcattttcatgggactggtctggccacaactacattaatgaaatatttgccacatcctcttacactttttcatacattgcccaa

[0490] gaataaagaatcgtttgtgttatgtttcaacgtgtttatttttcaattgcagaaaatttcgaatcatttttcattcagtagtatagccccaccaccacatag

[0491] cttatacagatcaccgtaccttaatcaaactcacagaaccctagtattcaacctgccacctccctcccaacacacagagtacacagtcctttctccc

[0492] cggctggccttaaaaagcatcatatcatgggtaacagacatattcttaggtgttatattccacacggtttcctgtcgagccaaacgctcatcagtgat

[0493] attaataaactccccgggcagctcacttaagttcatgtcgctgtccagctgctgagccacaggctgctgtccaacttgcggttgcttaacgggcgg

[0494] cgaaggagaagtccacgcctacatgggggtagagtcataatcgtgcatcaggatagggcggtggtgctgcagcagcgcgcgaataaactgct

[0495] gccgccgccgctccgtcctgcaggaatacaacatggcagtggtctcctcagcgatgattcgcaccgcccgcagcataaggcgccttgtcctcc

[0496] gggcacagcagcgcaccctgatctcacttaaatcagcacagtaactgcagcacagcaccacaatattgttcaaaatcccacagtgcaaggcgct

[0497] gtatccaaagctcatggcggggaccacagaacccacgtggccatcataccacaagcgcaggtagattaagtggcgacccctcataaacacgc

[0498] tggacataaacattacctcttttggcatgttgtaattcaccacctcccggtaccatataaacctctgattaaacatggcgccatccaccaccatcctaa

[0499] accagctggccaaaacctgcccgccggctatacactgcagggaaccgggactggaacaatgacagtggagagcccaggactcgtaaccatg

[0500] gatcatcatgctcgtcatgatatcaatgttggcacaacacaggcacacgtgcatacacttcctcaggattacaagctcctcccgcgttagaaccat

[0501] atcccagggaacaacccattcctgaatcagcgtaaatcccacactgcagggaagacctcgcacgtaactcacgttgtgcattgtcaaagtgttac

[0502] attcgggcagcagcggatgatcctccagtatggtagcgcgggtttctgtctcaaaaggaggtagacgatccctactgtacggagtgcgccgaga

[0503] caaccgagatcgtgttggtcgtagtgtcatgccaaatggaacgccggacgtagtcatatttcctgaagcaaaaccaggtgcgggcgtgacaaac

[0504] agatctgcgtctccggtctcgccgcttagatcgctctgtgtagtagttgtagtatatccactctctcaaagcatccaggcgccccctggcttcgggtt

[0505] ctatgtaaactccttcatgcgccgctgccctgataacatccaccaccgcagaataagccacacccagccaacctacacattcgttctgcgagtca

[0506] cacacgggaggagcgggaagagctggaagaaccatgtttttttttttattccaaaagattatccaaaacctcaaaatgaagatctattaagtgaacg

[0507] cgctcccctccggtggcgtggtcaaactctacagccaaagaacagataatggcatttgtaagatgttgcacaatggcttccaaaaggcaaacgg

[0508] ccctcacgtccaagtggacgtaaaggctaaacccttcagggtgaatctcctctataaacattccagcaccttcaaccatgcccaaataattctcatc

[0509] tcgccaccttctcaatatatctctaagcaaatcccgaatattaagtccggccattgtaaaaatctgctccagagcgccctccaccttcagcctcaag

[0510] cagcgaatcatgattgcaaaaattcaggttcctcacagacctgtataagattcaaaagcggaacattaacaaaaataccgcgatcccgtaggtcc

[0511] cttcgcagggccagctgaacataatcgtgcaggtctgcacggaccagcgcggccacttccccgccaggaaccatgacaaaagaacccacact

[0512] gattatgacacgcatactcggagctatgctaaccagcgtagccccgatgtaagcttgttgcatgggcggcgatataaaatgcaaggtgctgctca

[0513] aaaaatcaggcaaagcctcgcgcaaaaaagaaagcacatcgtagtcatgctcatgcagataaaggcaggtaagctccggaaccaccacagaa

[0514] aaagacaccatttttctctcaaacatgtctgcgggtttctgcataaacacaaaataaaataacaaaaaaacatttaaacattagaagcctgtcttaca

[0515] acaggaaaaacaacccttataagcataagacggactacggccatgccggcgtgaccgtaaaaaaactggtcaccgtgattaaaaagcaccac

[0516] cgacagctcctcggtcatgtccggagtcataatgtaagactcggtaaacacatcaggttgattcacatcggtcagtgctaaaaagcgaccgaaat

[0517] agcccgggggaatacatacccgcaggcgtagagacaacattacagcccccataggaggtataacaaaattaataggagagaaaaacacataa

[0518] acacctgaaaaaccctcctgcctaggcaaaatagcaccctcccgctccagaacaacatacagcgcttccacagcggcagccataacagtcagc

[0519] cttaccagtaaaaaagaaaacctattaaaaaaacaccactcgacacggcaccagctcaatcagtcacagtgtaaaaaagggccaagtgcagag

[0520] cgagtatatataggactaaaaaatgacgtaacggttaaagtccacaaaaaacacccagaaaaccgcacgcgaacctacgcccagaaacgaaa

[0521] gccaaaaaacccacaacttcctcaaatcgtcacttccgttttcccacgttacgtcacttcccattttaagaaaactacaattcccaacacatacaagtt

[0522] actccgccctaaaacctacgtcacccgccccgttcccacgccccgcgccacgtcacaaactccaccccctcattatcatattggcttcaatccaa

[0523] aataaggtatattattgatgatgttaattaatttaaatccgcatgcgatatcgagctctcccgggaattcggatctgcgacgcgaggctggatggcct

[0524] tccccattatgattcttctcgcttccggcggcatcgggatgcccgcgttgcaggccatgctgtccaggcaggtagatgacgaccatcagggaca

[0525] gcttcaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaa

[0526] tcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccg

[0527] accctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggt

[0528] cgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaa

[0529] gacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggc

[0530] ctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggca

[0531] aacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctac

[0532] ggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaatcaatcta

[0533] aagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctga

[0534] ctccccgtcgtgtagataactacgatacgggaggcttaccatctggccccagtgctgcaatgataccgcgagaccacgctcaccggctcca

[0535] gatttatcagcaataaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccg

[0536] ggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggctt

[0537] cattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcag

[0538] aagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtga

[0539] gtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaacacgggataataccgcgccacatagcaga

[0540] actttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtg

[0541] cacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggc

[0542] gacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttag

[0543] aaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaata

[0544] ggcgtatcacgaggccctttcgtcttcaagaattggatccgaattcccgggagagctcgatatcgcatgcggatttaaattaattaa

[0545] SEQ ID NO:19-SEQ ID NO:22: Nucleotide sequence of recombinant oncolytic adenovirus vector Ad5-survivin-antimCD47 / PDL1-mIL12 (its complete sequence is obtained by sequentially linking the bases of SEQ ID NO:19-SEQ ID NO:22 in the sequence listing, and its complete sequence is as follows)

[0546]

[0547] SEQ ID NO:23, ATTR1 nucleotide sequence.

[0548] SEQ ID NO:24, ATTR2 nucleotide sequence.

Claims

1. A human oncolytic adenovirus type 5 vector characterized in that: It is a genetically modified human adenovirus type 5 vector, and the viral vector gene contains the gene sequence of anti-CD47 / PDL1 bispecific antibody and immunomodulator regulated by tumor-specific promoter.

2. The human adenovirus type 5 oncolytic adenoviral vector of claim 1, wherein: The immunomodulator is at least one selected from IL12, GM-CSF, IL-15 or IFN-γ; preferably, the immunomodulator is selected from IL12, and the nucleotide sequence thereof is shown as SEQ ID NO: 3 or SEQ ID NO:

4.

3. The human adenovirus type 5 oncolytic adenoviral vector of claim 1 or 2, wherein: The tumor-specific promoter is the promoter replacing the E1A of human adenovirus type 5, and is at least one selected from survivin or hTERT promoter; preferably, the tumor-specific promoter is the survivin promoter, and the survivin-E1 nucleotide sequence thereof is shown as SEQ ID NO:

5.

4. The human adenovirus type 5 oncolytic adenoviral vector according to any one of claims 1 to 3, characterized in that: The nucleotide sequence of the anti-CD47 / PDL1 bispecific antibody is shown as SEQ ID NO: 1 or SEQ ID NO:

2.

5. The human type 5 oncolytic adenoviral vector according to any one of claims 1 to 4, characterized in that: The nucleotide sequence of the human adenovirus type 5 oncolytic adenovirus vector is shown as SEQ ID NO: 15-SEQ ID NO: 18 or SEQ ID NO: 19-SEQ ID NO:

22.

6. A method of constructing a human type 5 oncolytic adenoviral vector according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: S1: inserting an IL12 fragment into a human adenovirus type 5 backbone plasmid Ad5-ATTR to construct a plasmid Ad5-ATTR-IL12; S2: constructing a shuttle plasmid pENTR-ATTL-survivin-E1-antiCD47 / PDL1 by enzyme digestion and ligation; S3: co-transforming the plasmid Ad5-ATTR-IL12 and the pENTR-ATTL-survivin-E1-antiCD47 / PDL1, then transfecting a package, and obtaining a recombinant oncolytic adenovirus vector Ad5-survivin-antiCD47 / PDL1-IL12 through virus amplification and purification.

7. The construction method of claim 6, wherein, At least one of the following is met: The nucleotide sequence of the plasmid Ad5-ATTR-IL12 is shown as SEQ ID NO: 9 or SEQ ID NO: 10; The shuttle plasmid pENTR-ATTL-survivin-E1-antiCD47 / PDL1 is shown as SEQ ID NO: 13 or SEQ ID NO: 14; The nucleotide sequence of the Ad5-survivin-antiCD47 / PDL1-IL12 is shown as SEQ ID NO: 15-SEQ ID NO: 18 or SEQ ID NO: 19-SEQ ID NO:

22.

8. A pharmaceutical composition, characterized by: The human adenovirus type 5 oncolytic adenovirus vector of any one of claims 1-5.

9. Use of the human adenovirus type 5 oncolytic adenovirus vector of any one of claims 1-5 or the pharmaceutical composition of claim 8 in the preparation of a medicament for treating malignant tumors.

10. Use according to claim 9, characterized in that: The tumor is selected from at least one of glioma, melanoma, osteosarcoma, colon cancer, rectal cancer, breast cancer, uveal melanoma, ovarian cancer, liver cancer, bladder cancer, cervical cancer, sarcoma, skin cancer, prostate cancer, pancreatic cancer, nasopharyngeal cancer, lung cancer, gastric cancer, head and neck cancer, hypopharyngeal cancer, kidney cancer, laryngeal cancer, oral cancer, malignant mesothelioma, neuroblastoma, ovarian cancer, papillomatosis, retinoblastoma; Preferably, the glioma is astrocytic tumor; more preferably, it is glioblastoma GBM of the brain; Further, the dosage form of the drug is injection, and the injection mode is at least one of intratumoral injection, intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intramyocardial injection or intraperitoneal injection.

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