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Projects / Programmes source: ARIS

Preparation and validation of therapeutic plasmids without selection gene for antibiotic resistance for cancer gene therapy using inducible and tissue-specific promoters

Research activity

Code Science Field Subfield
3.04.00  Medical sciences  Oncology   

Code Science Field
B200  Biomedical sciences  Cytology, oncology, cancerology 

Code Science Field
3.01  Medical and Health Sciences  Basic medicine 
Keywords
gene therapy. cancer, plasmid DNA, Antibiotic resistance gene, tissue-specific promotors, inducible promotors, horizontal gene transfer, electroporation
Evaluation (rules)
source: COBISS
Researchers (27)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  13654  PhD Jerneja Ambrožič Avguštin  Biochemistry and molecular biology  Researcher  2011 - 2014  258 
2.  11308  PhD Andrej Cör  Oncology  Researcher  2011 - 2014  420 
3.  14575  PhD Maja Čemažar  Oncology  Head  2011 - 2014  1,438 
4.  22616  PhD Tina Eleršek  Biology  Researcher  2011 - 2014  264 
5.  09892  PhD Metka Filipič  Biology  Researcher  2011 - 2014  587 
6.  33895  PhD Tadeja Jakus  Public health (occupational safety)  Researcher  2011 - 2014  104 
7.  33227  PhD Tanja Jesenko  Oncology  Junior researcher  2011 - 2014  170 
8.  28387  PhD Urška Kamenšek  Oncology  Researcher  2011 - 2014  190 
9.  21756  PhD Ulrika Klopčič  Oncology  Researcher  2011 - 2014  62 
10.  29297  PhD Katja Kološa  Biology  Technical associate  2011 - 2014  42 
11.  19058  PhD Simona Kranjc Brezar  Medical sciences  Researcher  2011 - 2014  321 
12.  15819  PhD Jaka Lavrenčak  Oncology  Researcher  2011 - 2014  74 
13.  32175  PhD Boštjan Markelc  Medical sciences  Junior researcher  2011 - 2012  227 
14.  31305  PhD Cecil J.W. Meulenberg  Neurobiology  Researcher  2011 - 2014  76 
15.  28330  PhD Jana Nunić  Biochemistry and molecular biology  Researcher  2011 - 2012  56 
16.  20052  PhD Irena Oblak  Oncology  Researcher  2011 - 2014  305 
17.  32084  PhD Mitja Rak  Microbiology and immunology  Researcher  2011 - 2013  47 
18.  32176  PhD Aleš Sedlar  Plant production  Junior researcher  2011 - 2012  54 
19.  08800  PhD Gregor Serša  Oncology  Researcher  2011 - 2014  1,520 
20.  12250  PhD Marko Snoj  Oncology  Researcher  2011 - 2014  196 
21.  14576  PhD Primož Strojan  Oncology  Researcher  2011 - 2014  809 
22.  32094  PhD Alja Štern  Control and care of the environment  Junior researcher  2011 - 2013  81 
23.  29469  PhD Vesna Todorović  Medical sciences  Researcher  2011 - 2014  57 
24.  10974  PhD Irena Zajc  Biochemistry and molecular biology  Researcher  2012  134 
25.  07750  PhD Matjaž Zwitter  Oncology  Researcher  2011 - 2014  386 
26.  20767  PhD Bojana Žegura  Biology  Researcher  2012 - 2014  353 
27.  24930  PhD Boštjan Žvanut  Computer science and informatics  Researcher  2011 - 2014  279 
Organisations (4)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0105  National Institute of Biology  Ljubljana  5055784  13,476 
2.  0302  Institute of Oncology Ljubljana  Ljubljana  5055733000  15,790 
3.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  67,273 
4.  2413  Universita del Litorale, Facolta di Scienze della Salute  Izola  1810014005  9,346 
Abstract
Background. Gene therapy is becoming increasingly more important in cancer therapy. In recent years, non-viral approaches for transfection have become very attractive. One of the goals of researchers dealing with gene therapy or DNA vaccination is to develop safe and efficient systems for gene transfer into the target cells. Namely, this topic is still one the major hurdles in the progress of different approaches in gene therapy and is crucial factor for success of the therapy. Other factors that should be considered for development of gene therapy are the choice of appropriate therapeutic gene, regulation of its expression and administration route. In the proposed project, two of these factors will be addresses for the use in cancer gene therapy: safety of plasmid DNA by preparation of plasmid DNA without marker for antibiotic resistance and regulation of therapeutic gene expression by linking the therapeutic gene to cellular promoters to achieve either stress induced (p21 and radiation) or physiological regulation (tissues specific promoters for endothelial and muscle cells and fibroblsats) of expression.  In addition, possible horizontal gene transfer between recombinant plasmid DNA or transgenic bacteria and commensal naturally occuring bacteria will be evaluated. Therefore, our hypothesis is that prepared plasmids encoding therapeutic genes under the control of tissue specific and inducible promoters without gene for antibiotic resistance are safe vectors for gene therapy with also regulated expression. Methods. Plasmids encoding reporter or therapeutic genes under the control of tissue specific or inducible promoters will be prepared by standard molecular biology techniques and according to the instructions of the producer of ORT® plasmid in order to prepare plasmids without gene for antibiotic resistance. Appropriate bacterial strains will be transformed with the plasmids to produce larger quantities of endotoxin free plasmid to be used in further experiments. The correctness of plasmids will be evaluated by gel electrophoresis and sequencing. To evaluate horizontal gene transfer, PCR assays and other standard laboratory procedures used for studying horizontal gene transfer as well as standard laboratory procedures used for preparing biofilm cultures will be used. To determine the effects of prepared plasmids on cell in cultures, standard test for proliferation, antiangiogenic and antimetastatic potential will be used. Antitumor effectiveness and histological analysis will be determined on tumor models in vivo by tumor growth delay assay, tumor control probability assay and immunohistochemistry. Execution and management of the project. The experiments will be performed at the laboratories of 5 partners participating in the project. All necessary equipment for molecular biology, cell cultures, immunocytochemistry, and animals experiments is available. The researchers involved in the project have vast experience in the field of plasmid preparation, electroporation as a drug and gene delivery method and radiobiology. The management and coordination of the project will be assured by regular meetings and constant communication with partners on the project. Relevance and potential impact of the results. The proposed project is designed as a preclinical project that addresses important issues regarding safe use of cancer gene therapy in clinical setting. The results of the project, if successful, may have impact on design and execution of cancer gene therapy trials in Slovenia and worldwide.
Significance for science
One of the major focuses of researchers on field of cancer gene therapy is development of efficient and safe systems for transfer of transgens in target cells. An ideal gene transfer method would allow introduction of sufficinet concentration of DNA into the desired target cells with minimal side effect. Gene therapy using electroporation has proved to be a good alternative to viral methods. The research that we carried out within the project is the continuation of our work and has addressed important issues regarding safe use of cancer gene therapy in clinical setting. We sucessfully prepared therapeutic plasmids with tissue specific promoters without antibiotic resistance gene. Prepared plasmids are in accordance with regulatory agencies, European Medicines Agency and Food and Drug Administration, and are suitable for use in cancer gene therapy clinical trails. Additionally, the results of our study on the horizontal gene transfer into naturally occurring commensal bacteria contributes to the risk assessment for currently used plasmid DNA in clinical studies. These results are also very important for different authorities that are involved in risk assessment as well as granting permission for clinical trails.
Significance for the country
Preparation of therapeutic plasmids without antibiotic resistance gene has proven to be successful, therefore we intend to continue with clinical studies also with other therapeutic genes. The preparation of plasmid for clinical studies must be in accordance with the strict regulations and has to be prepared according to the GMP. This preparation of plasmids involves also substantial administrative work, therefore a spin-off company could be started in Slovenia to produce GMP grade plasmid preparation and to help all potental customers with administrative help in preparation application for clinical studies. Additionally, the results of the project can be also interesting for pharmaceutical and biotechnological companies. The patentability of new knowledge and product, as the result of the project, will be evaluated and if patentable, patent applications will be prepared, Intellectual property protected, and contacts established with potential users. Project results were presented on international conferences, summer schools and in the form of scientific papers, which is promotion of Slovenia as a high technological society with innovative knowledge.
Most important scientific results Annual report 2011, 2012, 2013, final report, complete report on dLib.si
Most important socioeconomically and culturally relevant results Annual report 2011, 2012, 2013, final report, complete report on dLib.si
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