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A novel vascular targeted approach based on gene therapy silencing two independent signaling pathways in combination with radiotherapy

A novel vascular targeted approach based on gene therapy silencing two independent signaling pathways in combination with radiotherapy

Vsebinski opis projekta

Radiotherapy is one of the main tumor treatment modalities, which causes cell death due to DNA damage, and as a single primary treatment, it is effective in 40% of patients. Therefore, combinations with other treatment modalities are being extensively investigated. Vascular targeted therapies in combination with radiotherapy have been proven effective due to better oxygenation status of tumors, since oxygen is a key sensitizer and can promote tumor radiosensibilization. Vascular targeted therapies are divided into two subgroups; antiangiogenic therapy, targeting newly forming tumor vasculature, and vascular disrupting therapy, which disrupts existing tumor vasculature. Antiangiogenic therapy causes normalization of tumor vasculature, which leads to better tumor oxygenation, while vascular disrupting therapy causes central tumor necrosis, with the tumor rim still well oxygenated. The majority of these studies were combining antiangiogenic therapies and radiotherapy, nevertheless, the severe side effects and limited time of efficancy were the main disadvantage.

In the recent years, gene therapy has gained a lot of consideration since it can provide a new means for targeted and controlled treatment of tumors. As delivery system in gene therapy, mainly viral vectors are used, although electroporation holds a promise. Electric pulses generate structural changes of a cellular membrane, which allows molecules to enter the cells. Electroporation can be used for delivery of chemotherapeutic drugs (electrochemotherapy), as well as for plasmids (gene electrotransfer). Efficency of the latter has been demonstrated in a series of preclinical and clinical studies, by using immunomodulatory and antiangiogenic molecules.

By means of technology progress, a targeted and successful gene therapy can be achieved by gene electrotransfer of plasmid, targeting specific markers of a tumor vasculature. The aim of our study was to construct a plasmid that simultaneously targets two different signaling pathways, one TGF-β dependent, by silencing CD105 expression, and the other VEGFR2 dependent, by silencing CD146 expression. To maintain the safety of the patient and the environment (prevention of the possible horizontal gene transfer to commensal bacteria), the plasmid will devoid of gene encoding antibiotic resistance. We have already shown that targeting tumor vasculature can promote tumor radiosensibilization and can improve the therapeutic outcome; nevertheless, there were differences between tumor models. The reason is still unclear, although it is necessary for understanding the mechanistic events, which could further be useful as prognostic markers. Furthermore, proper monitoring and studying changes in the tumor microenvironment remains a challenge to be explored in order to obtain the best therapeutic results.

Detailed description of the work program

WP 1: Preparation and validation of the therapeutic plasmid devoid of the gene encoding antibiotic resistance

In this work package, we will construct a therapeutic plasmid devoid of the gene encoding antibiotic resistance, targeting two tumor vasculature signaling pathways (silencing of CD105 and CD146 expression). The expression cassette, containing the genes for the two targeted shRNA, will be made or ordered from artificial gene synthesis service. Thereafter, this cassette will be transferred into the new plasmid backbone, devoid of the antibiotic selective marker, with the appropriate techniques of molecular cloning. Furthermore, we will determine the effectiveness of the plasmid after gene electrotransfer alone and combined with irradiation. Based on our previous work, we selected a murine endothelial cell line, 2H-11, which expresses both targeted molecules and the murine TS/A mammary carcinoma cell line, whose cells do not express CD105 or CD146. On these cell, a functional tests for determination of antiangiogenic, cytotoxic and antimigratory activity of plasmids in cells after gene electrotransfer of plasmid alone or combined with irradiation will be performed.

Based on this in vitro data, we will be able to distinguish between effectiveness on the vasculature from the effectiveness on tumor cell. Furthermore, to elucidate the immune modulatory response the level of cytokines and STING protein will be determined.

ST 1.1: Construction of a therapeutic plasmid devoid of the gene encoding antibiotic resistance, targeting two tumor vasculature signaling pathways (silencing of CD105 and CD146 expression).

Objectives: Preparation of the plasmid encoding two shRNA important in tumor vasculature signaling pathways; CD105 and CD146, which is also devoid of the gene encoding antibiotic resistance.

Description: The expression cassette, containing the genes for the two targeted shRNA, will be made or ordered from artificial gene synthesis service. Thereafter, this cassette will be transferred into the new plasmid backbone, devoid of the antibiotic selective marker, with the appropriate techniques of molecular cloning.

Expected results: The successful preparation and validation of the plasmid encoding two shRNA important in tumor vasculature signaling pathways (CD105 and CD146), which is also devoid of the gene encoding antibiotic resistance.

ST 1.2: Functional tests for determination of antiangiogenic, cytotoxic and antimigratory activity of the plasmid in murine endothelial cells (2H-11) after gene electrotransfer alone and in combination with irradiation, in vitro.

Objectives: To determine the effect of plasmid devoid of the gene for antibiotic resistance and encoding two shRNA (CD105 and CD146) after gene electrotransfer alone or in combination with irradiation on tube formation of endothelial cells, cytotoxicity and migration of endothelial cells.

Description: Gene electrotransfer will be used for delivering the plasmid into the cells and its effectiveness of dual targeting will be monitored after the monotherapy or combined with irradiation. The tube formation of endothelial cells will be determined with tube formation assay on Matrigel. Cytotoxicity of endothelial cells will be determined by Presto Blue viability assay and it will be followed for several days. Migration of endothelial cells will be determined with xCELLigence system (Roche) or wound-healing assay and it will be followed for several hours.

Expected results: The evaluation of antiangiogenic, cytotoxic and antimigratory activity of plasmid with dual targeting after gene electrotransfer alone or in a combination with irradiation on endothelial cells.

ST 1.3: Functional tests for determination of cytotoxic and antimigratory activity of the plasmid in murine mammary adenocarcinoma tumor cells (TS/A) after gene electrotransfer alone and in combination with irradiation, in vitro.

Objectives: To determine the effect of plasmid devoid of the gene for antibiotic resistance and encoding two shRNA (CD105 and CD146) after gene electrotransfer alone or in combination with irradiation on cytotoxicity and migration of tumor cells.

Description: Gene electrotransfer will be used for delivering the plasmid into the cells and its effectiveness of dual targeting will be monitored after the monotherapy or combined with irradiation. Cytotoxicity of tumor cells will be determined by Presto Blue viability assay and it will be followed for several days. Migration of tumor cells will be determined with xCELLigence system (Roche) or wound-healing assay and it will be followed for several hours.

Expected results: The evaluation of antiangiogenic, cytotoxic and antimigratory activity of plasmid with dual targeting after gene electrotransfer alone or in a combination with irradiation on tumor cells.

ST 1.4: Evaluation of the expression of cytokines (TNFα, IFNβ, IL-1β) and protein STING after gene electrotransfer alone and in combination with irradiation in vitro.

Objectives: To determine the expression of the cytokines and STING protein on the cells after gene electrotransfer alone or combined with irradiation.

Description: To evaluate the immunomodulatory response of the therapies, the level of cytokines and STING protein will be determined in the treated cells. The cells will be harvested and lysed after the therapy, and the RNA will be isolated with the help of qPCR to determine the mRNA level. To determine the level of STING protein, the cell will be incubated with specific antibodies used for flow cytometry.

Expected results: Successful determination of the cytokines and STING protein on the cells after gene electrotransfer alone or combined with irradiation.

WP 2: Determination of antitumor effectiveness of gene electrotransfer of the plasmid alone or combined with irradiation, in vivo

In this work package, we will determine the antitumor effectiveness of plasmid encoding shRNA against CD105 and CD146, alone or combined with tumor irradiation in experimental tumors in mice. The antitumor effectiveness of gene electrotransfer of plasmid will be evaluated in vascular phase of tumor growth on TS/A mammary carcinoma, whose cells do not express CD105 nor CD146, whereas both are expressed in the endothelial tumor cells. Therefore, the effect on tumor vasculature could be distinguished from direct antitumor effect on tumor cells. The combination of gene electrotransfer and irradiation will be performed to determine radiosensitizing action of silencing two different signaling pathways of tumor vasculature. Furthermore, general systemic toxicity and acute skin damage will be evaluated after treatments.

ST 2.1: Antitumor action of gene electrotransfer alone in BALB/c mice bearing subcutaneous TS/A tumors

Objectives: To determine the antitumor action of gene electrotransfer of plasmid with dual targeting of tumor vasculature.

Description: In inbred laboratory mice, subcutaneous tumors will be transplanted by injection of viable tumor cells subcutaneously. When the tumor volume will reach 5-6 mm in the longest diameter, an intratumoral injection of the prepared plasmid will be performed and 10 min thereafter the tumors will be electroporated to perform gene electrotransfer. The antitumor effectiveness will be evaluated by tumor growth delay assay. In the case of complete responses mice will be followed 100 days after treatment.

Expected results: Determination of antitumor effectiveness of gene electrotransfer of plasmid targeting CD105 and CD146.

ST 2.2: Antitumor action of combined gene electrotransfer and irradiation (single and/or fractionated doses)

Objectives: To determine the antitumor action of gene electrotransfer of plasmid with dual targeting of tumor vasculature in combination with irradiation.

Description: In inbred laboratory mice, subcutaneous tumors will be transplanted by injection of viable tumor cells subcutaneously. When the tumor volume will reach 5-6 mm in the longest diameter, an intratumoral injection of the prepared plasmid will be performed and 10 min thereafter the tumors will be electroporated to perform gene electrotransfer. The next day the tumors will be irradiated with single or multiple doses. The antitumor effectiveness will be evaluated by tumor growth delay assay. In the case of complete responses mice will be followed 100 days after treatment.

Expected results: Determination of antitumor effectiveness of gene electrotransfer of plasmid targeting CD105 and CD146, in combination with irradiation.

ST 2.3: Determination of systemic toxicity and acute skin reaction in the irradiation field

Objectives: To evaluate the systemic toxicity and acute normal tissue damage in the irradiated filed.

Description: The skin reaction in the irradiated field around the tumor will be scored once a week until the disappearance of skin reactions in the animals. The weight of mice will be monitored as a general index of toxicity.

Expected results: Evaluation of normal tissue damage after combined treatment and determination of general systemic toxicity.

WP 3: Exploration of mechanisms of gene electrotransfer and irradiation

In this work package, we will explore the underlying mechanisms of action of gene electrotransfer of plasmid silencing two different signaling pathway of tumor vasculature. For this reason, histological changes after the therapies will be determined, as well as modulatory immune response after the therapies alone or combined in order to elucidate the importance of activated signaling pathways.

ST 3.1: Immunohistological analyses of tumors treated in WP 2 – staining for tumor vasculature, immune cell infiltration, cytokines and type of cell death

Objectives: To determine the histological changes in tumors after gene electrotransfer alone or combined with irradiation.

Description: Tumor samples will be collected from WP2. After gene electrotransfer of plasmid alone or combined with irradiation, the tumors will be excised, fixed in zinc fixative and embedded in paraffin blocks. Paraffin blocks will be cut with microtome to a specified thickness. Tumor sections will be stained with hematoxylin and eosin for determination of tumor necrosis and with immunohistochemical staining with specific primary antibodies for determination of blood vessels (CD31), infiltration of effector immune cells (Granzyme B), cytokines (TNFα and/or IFNβ) and for determination of cell death type (Giemsa, Calreticulin).

Expected results: Determination of the histological changes in tumors, which could identify the underlying mechanisms of action, and elucidate the antitumor effect.

ST 3.2: Determination of expression of cytokines (TNFα, IFNβ, IL-1β) and protein STING after gene electrotransfer alone and in combination with irradiation (single and/or fractionated doses)

Objectives: To determine the expression of the cytokines and STING protein on the tumors after gene electrotransfer alone or combined with irradiation (single or multiple doses).

Description: To evaluate the immunomodulatory response of the therapies, the level of cytokines and STING protein will be determined in the treated cells. The tumors will be macerated and the cells obtained will be harvested and lysed after the therapy. The RNA will be isolated with the help of qPCR to determine the mRNA level. To determine the level of STING protein, the obtained cells will be incubated with specific antibodies used for flow cytometry.

Expected results: Successful determination of the cytokines and STING protein on the tumors after gene electrotransfer alone or combined with irradiation.

 

 

© 2019 - Institute of Oncology Ljubljana
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