DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Andreas Albers :
Development and characterization of a method for direct ballistic transfer of genes into tumor cells, for production of autologous anti-tumor vaccines
Entwicklung und Charakterisierung einer Methode zum direkten ballistischen Transfer von Genen in Tumorzellen zur Herstellung von autologen Anti-Tumorvakzinen

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Abstract

The weak point of all gene therapy projects is still the gene transfer procedure. For clinical applications, a transfection method would be optimal, by which with small expenditure and without safety risk, an efficient transfection of only the desired cell type, in sufficient number, could be achieved. The cells should be changed exclusively in the necessary way, without modifying other characteristics. The method of the ballistomagnetic gene transfer represents a beginning for the solution of this problem (134). It is limited however by the fact that only adherent cells can be effectively transfected. Thus, before transfection, an adherent cell culture must be made from a patients? tumor cells. For our gene therapy studies at Centrum Somatische Gentherapie, Berlin, this represents a considerable restriction, since it comes with the cultivation of the cells won from tumor material to a selection of the adherent cell clones and therefore all nonadherent tumor cells are removed from the culture. Thereby the tumor cell pool, of which the vaccine is subsequently made, is limited. In addition, the portion of the fast-growing tumor cells overgrow with increased cultivation duration, the slowly growing cells in the culture. At the time of transfection the tumor cells used for the production of the vaccine thus only partly represent the original tumor, from which they were won. The long-term cultivation of the tumor cells is limited to approximately 30% of the operated patients tumor cells, since only this portion of tumor derived cells can be kept successfully in culture. In my study, the ballistomagnetic gene transfer was modified for application in clinical gene therapy studies. The work routine was optimized and its effectiveness was increased. In order to be able to transfect cells, independently of their adherence characteristics, the cells are now briefly plated in a monolayer on mircoporous polycarbonate supports. This method is called in the following MIP-method (microporous polycarbonate membrane method). By combination of the ballistomagnetic gene transfer with the MIP-method, it is now possible to transfect directly the autologous won tumor cells of all patients without previous cultivation. This permits the production of therapeutic vaccines from tumor cells with immunologically unchanged cell surface and thereby increased probability, that the structures relevant for the immunisation e.g. tumor antigens are presented to the immune system of the patient. On average 89% of the used cell lines could be recovered after ballistic gene transfer if the MIP-Method was used. With application of the comparison method developed by Burkholder et al. (123) 58% of the cells could be recovered. Also the magnetic separation was more efficient, if the cells were plated before with the MIP method. Thus one received on average 1.7 times more cells over the used cell lines after ballistic gene transfer and altogether 3.7 times more cells after magnetic separation than with the comparison method. The relative number of transfected cells as determined by flow cytometric evaluation, did not differ significantly for both methods. If one regards however the absolute number of the transfected cells, then the application of the MIP method is clearly more favourable, since here after the ballistic gene transfer more cells are at disposal for the magnetic separation. Moreover the magnetic enrichment by itself is more efficient. With a computer simulation, the optimal conditions for the ballistic gene transfer were determined. With the received data, concrete suggestions for a practical application in the laboratory could be made. These realizations can be used directly to further optimize the production of gene-therapeutic vaccines for future studies and clinical applications.

Table of Contents

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Titelblatt, Inhaltsverzeichnis
1. Zusammenfassung 8
2. Einleitung 10
2.1 Hintergrund: Karzinogenese 10
2.2 Einführung zum Thema Gentherapie 13
2.3 Übersicht über Gentherapiestrategien und Beispiele für Kandidatengene 14
2.4 Suizidgentherapie 15
2.5 Tumorsuppressor- und Anti-Onkogentherapie 16
2.6 Supportive Gentherapiemaßnahmen 16
2.7 Aktive Krebs-Immungentherapie 17
2.8 Gentransfer in der Gentherapie 32
2.9 Zielsetzung der Arbeit 43
3. Materialien 44
3.1 Verwendete Geräte 44
3.2 Verwendete Materialien und Chemikalien 45
3.3 Bakterienstämme und Zellinien 46
3.4 Zellkulturmedien 46
3.5 Plasmidkonstrukte und Oligdesoxyribonukleotide 47
3.6 Puffer, Lösungen und Medien 47
4. Methoden 49
4.1 DNA-Präparation und Aufreinigung 49
4.2 Zellkulturarbeiten 53
4.3 Ballistomagnetischer Gentransfer 59
4.4 Antikörperfärbung zum Nachweis der CD40L-Expression im Durchflußzytometer 67
4.5 Messungen mit dem Durchflußzytometer 68
4.6 ELISA zur in vitro Quantifizierung der Zytokin-Expression 74
4.7 Näherungsformel zur Bestimmung der bei der MIP-Methode maximal einsetzbaren Zellzahl 76
4.8 Computerprogramm zur Simulation des Beschießens von Zellen 80
4.9 Fehlerbetrachtung der Versuchsergebnisse 83
5. Ergebnisse 84
5.1 Begriffsdefinitionen 84
5.2 Die MIP-Methode ermöglicht ballistischen Gentransfer in Zellen unabhängig von ihrer Fähigkeit zu adhärieren 84
5.3 Kotransfektion von Primärkulturen mit GM-CSF und IL-7 98
5.4 Computersimulation des ballistischen Gentransfers 100
6. Diskussion 106
6.1 Statistische Auswertung der Ergebnisse 106
6.2 Auswertung der durchflußzytometrischen Messungen 106
6.3 MIP-Methode 108
6.4 Berechnung der auszusäenden Zellzahl 109
6.5 Vergleich der MIP-Methode mit der Burkholder-Methode 109
6.6 Ballistischer Gentransfer 111
6.7 Computersimulation des ballistischen Gentransfers 114
7. Literaturverzeichnis 116
8. Anhang 128
8.1 Abkürzungsverzeichnis 128
8.2 Protokoll: Ballistomagnetischer Gentransfer mit der MIP Methode 129
9. Curriculum Vitae 132
10. Publikationsliste 135

More Information:

Online available: http://www.diss.fu-berlin.de/2002/177/indexe.html
Language of PhDThesis: german
Keywords: genetherapy, zytokines, tumor vaccine, gene transfer, interleukin-7, GM-CSF, ballistic, transfection
DNB-Sachgruppe: 33 Medizin
Date of disputation: 25-Jun-2002
PhDThesis from: Fachbereich Humanmedizin, Freie Universität Berlin
First Referee: Professor Dr. Burghardt Wittig
Second Referee: Professor Dr. Werner Rosenthal
Contact (Author): andreasalbers@planet-interkom.de
Contact (Advisor): bw@zedat.fu-berlin.de
Date created:03-Sep-2002
Date available:06-Sep-2002

 

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