Restoration of Apoptosis Propensity of Tumor Cells
Restoration of Apoptosis Propensity of Tumor Cells by Transfection of the Apoptosis-Promoting Gene Cassettes Containing Translational Enhancing Elements: Potential Application for Targeting Gene Therapy
Tech Area / Field
- MED-DRG/Drug Discovery/Medicine
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
Moscow State University / A.N. Belozersky Institute of Physical and Chemical Biology, Russia, Moscow
- Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- US Department of Health & Human Services / National Institute of Health / Institute of Child Health and Human Development / Section of Intercellular Interactions, USA, MD, Bethesda\nKarolinska Institute, Sweden, Stockholm\nWadsworth Center / Oncofetal Antigen Laboratories, USA, NY, Albany\nBoston University / School of Medicine, USA, MA, Boston\nIcon Genetics AG, Germany, Munich\nUniversity of Turku / Finnish-Russian Joint Biotechnology Laboratory, Finland, Turku\nMax Planck Institute of Molecular Cell Biology and Genetics, Germany, Dresden
Project summaryCancer is a disease of genes. Many of the genetic aberrations in cancer cells target the apoptotic signalling pathways. Resistance of tumor cells to the cytotoxic effects of chemotherapeutic drugs and radiation limits the chemo- and radiocurability of many types of cancer. Advances in molecular biology have enabled gene delivery into human somatic cells and given the possibility of repairing of genetic mutations in cancer cells by introducing the corrected genes responsible for apoptosis signalling. The major goal of anti-cancer gene therapy – destruction of cancer cells – can be achieved by the targeted expression of apoptosis activating genes (AAGs) regulated on levels of transcription and translation.
Traditionally gene therapy includes the cap-dependent translation stage during the expression of genetic material transferred into a cancer cell. It has been shown that cap-dependent translation of vast majority of eukaryotic mRNAs including AAG is suppressed during apoptosis. Thus, inefficiency of many gene therapy approaches can be explained by the inefficient cap-dependent translation of anticancer AAGs during early stages of programmed cell death.
In this project, we propose to elaborate a technology that allows to perform targeted and effective expression of AAGs in tumour cells using gene cassettes carrying the AAGs under the control of the cancer-specific promoters and elements providing cap-independent translation – internal ribosome entry sequences (IRES).
Competence of the project team. Results of experiments and studies performed by groups of MSU (study of apoptosis and initiation of translation) and IIE (study and monitoring of apoptosis processes) were published in more than 50 articles in international journals (see supplement 1). Previous IIE group studies were supported by ISTC projects #401-98 and 1878-01. Research carried out by MSU group was performed within a number of international and Russian grants. Data and experience obtained by both groups could be combined to develop a novel technology of targeted activation of apoptosis in cancer cells.
Main objectives of the project. The main goal of the project is to develop the novel targeted gene therapy method for the restoration of the apoptosis propensity of tumor cells to enhance chemo- and radiocurability of various types of cancer. The key approach of the proposed method is the employment of cap-independent translation and polycistronic cassettes for targeted expression of AAG in tumor cells.
Targeting and specificity of the therapy can be provided by employment of cancer-activated promoters and translational regulatory elements. The following genes can be used as promising candidates for activation of apoptosis: c-myc, AFP, p53 and Smac/DIABLO. AFP and hTERT promoters can be employed as cancer-specific promoters. Regulation at the translational level can be provided by previously described IRES elements. It is also planned to perform a separate study to find novel IRES elements that provide effective translation in cancer cells. As another approach to restore apoptosis propensity in tumor cells a posttranscriptional silencing of anti-apoptotic genes (e.g. bcl-2) can be used. Inactivation of bcl-2 gene can be performed using short interfering RNA technology.
Major research tasks:
- Theoretical prediction and synthesis of the novel IRES elements for their application in the tumor-specific targeting of apoptosis-activating gene therapy.
- Use of AFP transcriptional promoter for transcription and cap-independent translation-targeted cancer gene therapy: IRES-mediated expression of AAG in tumor cells.
- Use of hTERT transcriptional promoter for transcription and cap-independent translation-targeted cancer gene therapy: IRES-mediated expression of AAG in tumor cells.
- Comparative analysis of the functional activity of the transfected apoptosis-activating genes in tumor and normal cells.
- Development of the experimental approach to the transcriptional regulation of the apoptosis-inhibitory gene expression by the transfection of short interfering RNA sequences.
- Development of the business strategy for the novel cancer gene therapy technology obtained during the project implementation.
Expected Results and their applications
- Identification and characterization of novel IRES elements that overcome cap-dependent translation blockage during apoptosis induction.
- The experimental data on mechanism of apoptosis induction in normal and tumor cells transfected with cassettes providing cap-independent expression of Smac/DIABLO, c-myc, AFP and p53 genes.
- Estimation of efficiency of AFP and hTERT promoters for targeted expression of apoptosis-associated genes in tumor cells.
- Study of the influence of inhibition of bcl-2 expression by short iRNA on the development of apoptosis in cancer cells.
- Development of the novel targeted anticancer gene therapy approach based cap-independent translation of AAG in tumor cells under control of IRES-elements.
- Design of the cDNA cassettes providing cap-independent expression of the AAGs under transcriptional control of AFP and hTERT.
- Design of the dicistronic cassettes expressing various AAGs under control of IRES-elements for their use in targeted cancer gene therapy.
- Development of the short interfering RNA sequence technology to induce the targeting suppression of the apoptosis inhibitory bcl-2 gene in tumor cells.
Execution of this project will contribute into the civil conversion of the scientists from the Institute of Immunological Engineering, who fulfilled earlier the State orders of the Ministry of Defence. It would also provide scientists involved with some financial independence by realization of licensing of developed technology.
Consistent with the scope of activities of the project proposal the collaboration with the foreign scientists will conclude: (i) information exchange in the course of project implementation; (ii) discussions of the experimental data obtained in the course of the project execution; (iii) collaborative research works.
To perform the tasks set up in the project modern techniques of molecular and cell biology will be employed, including gene synthesis and cloning, site-directed mutagenesis, flow cytometry, confocal and phase contrast microscopy, assays for cell proliferation and cytotoxicity.