Thesis about animals and its' phisiology

Thesis

Dana Moldabay

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5 February 2017

DEVELOPMENT gene therapy method for killing Cancer cells by gene expression KILLER AND TK HSV IMMUNOMODULATOR GM-CSF

As you know, cancer is second in cause of death of the adult population after cardiovascular diseases. One of the main goals of cancer therapy is to selectively killing cancer cells. This work aims to develop gene based drugs against one of the most malignant and types of cancer - melanoma. Among the various genetically therapeutic strategies kill cancer cells most It is a universal approach using gene-killers. Gene-murderer herpes simplex virus thymidine kinase (HSV TK) converts nucleoside analogue ganciclovir into toxic metabolites that results in suppression of the synthesis of DNA and RNA polymerase activity in tumor cells and their death. Also TK HSV peculiar manifestation of the "presence" (bystander effect), It manifested in the fact that the treatment of non-toxic substrate causes death not only cell expressing HSV TK, but also neighboring tumor cells lacking containing the enzyme.

Granulocyte-macrophage colony stimulating factor (GMCSF) - Representative of the family of cytokines capable of stimulating growth, maturation, differentiation of granulocytes and antigen-presenting cells (APC), allowing the induction of the immune response.

We believe that co-expression of cDNA TK HSV and GM-CSF in tumor cells would lead to the death of cancer cells and the release of them tumor antigens that will effectively represent GM-CSF- activated T cell-APC cells of the immune system, providing activation of specific anti-tumor immunity, whereby significantly increase mortality of tumor cells and reduce the chance occurrence of metastasis.

In this paper we were obtained bicistronic expression design based on a retroviral vector (pQCXIX), containing the cDNA gena-killer HSV TK in conjunction with mouse GM-CSF (m GM-CSF) or human (h GMCSF) under the control of the CMV promoter, and the expression constructs containing these genes by promoter gene BIRC5 and melanoma-specific promoters (promoter of the gene MIA human and mouse tyrosinase gene promoter).

The resulting construct was demonstrated by transient transfection of cells lines HEK293, Calu I, A549, A375. Expression and bioactivity TK

HSV in the composition of the resulting construct was confirmed by vestern-blot analysis and functional test of cytotoxicity in HSV TK followed by treatment with ganciclovir. The expression of h GM-CSF and m GM-CSF confirmed by ELISA. Functional activity of the h GM-CSF was confirmed by a functional test of the hGM-CSF - dependent cell line TF1. Thus, it was shown that the introduction of the obtained expression constructs into cultured tumor cells leading to the appearance of apoptotic cells and their death.

Animal Physiology and Biomedicine

Evaluating the effectiveness of the resulting construct carrying TK HSV and mGM-CSF on panel of melanoma cell lines and in vivo in mice inoculated with murine melanoma.

Obtaining positive results in in vivo experiments in mice, give reason to believe that the result of the work will be to develop specific fashion effectively destroy malignant cells melanoma gene by the TK HSV-killer and a powerful immune stimulator of human GM-CSF. The developed method specific destruction of melanoma cells must provide reduced resistance melanoma to standard therapy, and increased life expectancy patients. Methods of destruction of tumor cells to be developed in this study, we focused on the widespread use in scientific research organizations and global competitiveness.