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Nucleic acids as terapeutic agent
Ráčková, Lucie ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
With the development of molecular biology development of oncotherapy proceeds. The major progress of modern medicine is gene therapy. In the gene therapy are two categeories, namely, viral vectors and nonviral vectors which are used mainly. Nonviral vectors include plasmids. Plasmid DNA used in medicine must be perfectly purified. Chromatographic methods are mainly used at present. Research and development deals with other methods for example two-phase aqueous systems and magnetic carriers. In experimental part of this thesis, isolation of pUC 19 plasmid DNA from Escherichia coli JM 109 (pUC 19) cell culture was performed via method of alkaline lysis. Quality of isolated plasmid DNA was verified spectrophotometrically and by agarose gel electrophoresis. Isolated plasmid DNA was purified using three methods: RNA in plasmid DNA was precipitated by lithium chloride, RNA was degraded by immobilized RNase A and plasmid DNA was purified using two-phase aqueous system.
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Novel methods of treatment of B cell malignancies based on immunotherapy with genetically modified T cells
Novotná, Natálie ; Otáhal, Pavel (advisor) ; Šmahel, Michal (referee)
CAR T cell therapy represents a promising method in treatment of hematological malignancies. Gene immunotherapy uses modified T cells that express a chimeric antigen receptor (CAR) on their surface. Modified T lymphocytes are able to recognize and destroy target cells based on specific surface markers. Although CAR T cell therapy is used in clinical practice, there is a number of limitations that reduce its effectiveness. The aim of this thesis is to explore new possibilities of making the entire therapy more efficient through endogenous secretion of interleukins (IL-7, IL-15, IL-21) under the control of inducible promoters, and thus to strengthen the persistence and expansion of CAR T cells in vivo. For this purpose, inducible expression systems containing the gene for CAR19 receptor specifically recognizing the CD19 molecule and the interleukin gene located under inducible NFAT or NR4A promoters, were constructed. The assembled vectors were electroporated into PBMC cells using the PiggyBac transposon system to achieve stable expression in T lymphocytes. After co-cultivation with RAMOS cell line, data were obtained by measurement on a flow cytometer and the ELISA method. Based on the results, it is evident that stimulated CAR T cells are able to generate higher concentrations of interleukins,...
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Translation potential of current preclinical techniques for gene therapy of neurological diseases in clinic. A critical review.
Žideková, Paulína ; Novák, Ondřej (advisor) ; Jendelová, Pavla (referee)
Research in the field of gene therapy has potential to become a revolutionary way to the existing treatment for a wide spectrum of neurological diseases. To treat these disorders causally, by specific substituting, deleting, silencing or editing faulty genes could be a privilege of gene therapy. The concept of translational medicine is to facilitate the transfer of working principles in preclinical research into treatment in humans. Its key issue is to overcome limitations associated with the gap between the tremendous variety molecular biology tools of preclinical research and the lack of simple corresponding options in humans. Clinical implementation of most of the preclinical approaches is still considered to be limited. The main focus of this thesis is to summarize latest advancements of molecular and genetic engineering tools that themselves or in combination have the potential to promote most preclinical gene therapy of neurological diseases to clinical use. Based on that, this study aims to suggest perspective methods of treatment for selected neurological diseases.
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The struggle for human dignity in the era of modern eugenics. Molecular genome editing tool CRISPR/CAS9 and its use in human genome therapy from the perspective of theological ethics
Auxt, Miroslav ; Štica, Petr (advisor) ; Fošum, Jan (referee)
The recent breakthrough discovery of the molecular genome editing tool CRISPR/CAS9 represents a complete revolution in the field of molecular biology, biomedicine and other related fields. It is a highly effective biomolecular tool, derived from the bacterial immune system, with which it is possible to introduce precise changes in the genomes of all organisms. The thesis is limited to the ethical evaluation of the use of CRISPR/CAS9 exclusively in human gene therapy. Thanks to its efficiency, simplicity, accuracy and low financial costs, the CRISPR/CAS9 editing tool, in compliance with ethical parameters, already has a broad spectrum of use in therapeutic procedures on somatic or body cells in the treatment of human genetically determined diseases without introducing a change into the future offspring of the given individual. In addition to great therapeutic potential, the application of CRISPR/CAS9 raises many ethical questions related to the possibilities of its further use, possibly misuse. Ethically problematic genetic procedures include: human hereditary genome editing, i.e. the targeted alteration of the genome of sex cells, progenitor cells and cells of early embryonic development stages with the therapeutic goal of eliminating a genetically determined disease associated with the...
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Current approaches to cystic fibrosis therapy
Chmelíková, Barbora ; Kubíčková, Božena (advisor) ; Marková, Vendula (referee)
This bachelor thesis is focused on possible herapeutic methods for cystic fibrosis with main focus on modulators. The individual modulators are described in terms of their function, their differences and application possibilities. The thesis are also discuss other methods of treatment of cystic fibrosis, both for the respiratory tract and gastrointestinal tract. The thesis itself will be divided into three complementary parts. The first theoretical part defines the basic concepts of terms related to cystic fibrosis, the second follow-up practical part is focused on the comparison of the various methods of treatment of cystic fibrosis and the third part in the form of detailed analysis of the individual modulators. As for the methodology of the work, a method of literature search using secondary verified sources will be used. Keywords: cysticfibrosis, gene therapy, therapy, modulator, potentiator, correctors
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Nanoparticle-Mediated Delivery System for Gene Therapy
Dvořáková, Nikola ; Ellederová, Zdeňka (advisor) ; Šálek, Petr (referee)
Gene editing with the CRISPR/Cas9 system is one of the options that sets a new trend in the development of gene therapy. The most commonly used delivery of DNA into the cells are via viruses. Nevertheless, they are often unable to take CRISPR/Cas9 system, which can be bigger than several kb. Nanoparticles (NPs), as non-viral transporters, seem to be a good alternative delivery system. For this work magnetic Fe3O4 NPs (MNPs) were selected, because of their excellent properties such as multifunctionality, biocompatibility, easy degradation and simple synthesis. The aim of this work was to synthesise MNPs and a complex of MNPs coated with PEI/CRISPR-Cas9 plasmid and to characterize them by physicochemical methods. The created complex MNPs/PEI/CRISPR-Cas9 was defined by exact parameters that are suitable for possible cell uptake. The hypothesis of stabilization of the MNPs/CRISPR-Cas9 plasmid complex by polyethylenimine (PEI), which can also protect plasmid DNA against restriction endonucleases, was verified. Next a stable modified cell line HEK293-TLR3, designed to evaluate the efficacy of double strand break (DSB) repair by nonhomologous end joining (NHEJ) or homologous recombination (HR) was, transfected with the synthesised MNPs/PEI/CRISPR-Cas9 complex. The results indicate a 25% transfection...
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Polymer systems for siRNA delivery
Blažková, Jana ; Pechar, Michal (advisor) ; Šťovíček, Vratislav (referee)
The process of RNA interference (RNAi) is a natural phenomenon posttranscriptionally controlling gene expression by means of small double-stranded RNA molecules (dsRNA). Small interfering RNA (siRNA) is a small dsRNA that can be used for targeted gene silencing as an alternative therapeutic treatment of genetic diseases. For in vivo administration, siRNA must be protected against degradation to ensure its efficient delivery to target cells using sophisticated vectors. This work is focused on description of non-viral vectors based on cationic polymers, forming polyelectrolyte complexes with siRNA (polyplexes), and surface-modifying hydrophilic polymers enabling protection of the vector during its transport in the bloodstream.
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Metodické přístupy v genové terapii na zvířecích modelech
Šubrtová, Hana
This bachelor thesis deals with gene therapy as a possible treatment for many diseases. It summarizes general information on gene therapy how it is divided and a few important milestones in history that have significantly contributed to its development. Subsequently, this thesis focuses on the most commonly used vectors. There are listed and described their main advantages and disadvantages as well as examples of specific diseases for which the given gene carriers can be used as a treatment. This thesis also deals with methods most commonly used to detect gene mutation and summarizes model organisms suitable for gene therapy research. Finally, problematic areas associated with the application of gene therapy are described here.
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Genová terapie, její principy, mechanizmy a typy vektorů
Dobešová, Lucie
In the beginning chapters, the thesis deals with general information about gene therapy and its history. The following chapters provide information on its principles (both classical and gene therapy in conjunction with CRISPR/Cas9) and the vectors that enable the gene therapy process to take place. In the final chapters, the thesis deals with model organisms that are used for clinical trials, the use of gene therapy in human patients and the ethical and safety circumstances associated with its use.
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Nanoparticle-Mediated Delivery System for Gene Therapy
Dvořáková, Nikola ; Ellederová, Zdeňka (advisor) ; Šálek, Petr (referee)
Gene editing with the CRISPR/Cas9 system is one of the options that sets a new trend in the development of gene therapy. The most commonly used delivery of DNA into the cells are via viruses. Nevertheless, they are often unable to take CRISPR/Cas9 system, which can be bigger than several kb. Nanoparticles (NPs), as non-viral transporters, seem to be a good alternative delivery system. For this work magnetic Fe3O4 NPs (MNPs) were selected, because of their excellent properties such as multifunctionality, biocompatibility, easy degradation and simple synthesis. The aim of this work was to synthesise MNPs and a complex of MNPs coated with PEI/CRISPR-Cas9 plasmid and to characterize them by physicochemical methods. The created complex MNPs/PEI/CRISPR-Cas9 was defined by exact parameters that are suitable for possible cell uptake. The hypothesis of stabilization of the MNPs/CRISPR-Cas9 plasmid complex by polyethylenimine (PEI), which can also protect plasmid DNA against restriction endonucleases, was verified. Next a stable modified cell line HEK293-TLR3, designed to evaluate the efficacy of double strand break (DSB) repair by nonhomologous end joining (NHEJ) or homologous recombination (HR) was, transfected with the synthesised MNPs/PEI/CRISPR-Cas9 complex. The results indicate a 25% transfection...
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