Studies on efficiency and toxicity of in vivo delivery systems for siRNA and plasmid DNA [Elektronische Ressource] / vorgelegt von Nicole Tietze

Nicole Tietze

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 36
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und
Pharmazie der Ludwig-Maximilians-Universität München


Studies on Efficiency and Toxicity of in vivo Delivery
Systems for siRNA and plasmid DNA



vorgelegt von
Nicole Tietze
aus Hannover
2008
Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der
Promotionsordnung vom 29. Januar 1998 von Prof. Ernst Wagner betreut.





Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig und ohne unerlaubte Hilfe erarbeitet.

München, 19.02.2009





Dissertation eingereicht am 08. Dezember 2008
1. Gutacher: Prof. Dr. Ernst Wagner
2. Guta. Christian Wahl-Schott
Mündliche Prüfung am 19. Februar 2009
Für meine Eltern
Brigitte und Joachim Tietze
Table of Contents
1  Introduction............................................................................... 5 
1.1  Gene Therapy ................................................................................................ 6 
1.2  Synthetic vectors for gene therapy............................................................. 7 
1.3  Decreasing immidiate vector toxicity ......................................................... 9 
1.4  Decreasing intermediate vector toxicity..................................................... 9 
1.5  Targeted gene therapy................................................................................ 12 
1.6  Intracellular pathway of complexes .......................................................... 12 
1.7  RNA Interference......................................................................................... 14 
1.8  Ran protein .................................................................................................. 15 
1.9  Plasmid DNA - design and its impact on gene expression ..................... 17 
1.10  Specific aims of this work.......................................................................... 19 
2  Materials and Methods........................................................... 20 
2.1  Reagents and chemicals ............................................................................ 21 
2.2  Plasmid design............................................................................................ 22 
2.3  Formation of transfection complexes....................................................... 22 
2.4  Measurement of particle size and zeta-potential...................................... 23 
2.5  Cell culture experiments ............................................................................ 23 
2.6  Transfection in vitro.................................................................................... 23 
2.7  Cytotoxicity assay ...................................................................................... 23 
2.8  Luciferase reporter gene expression........................................................ 24 
2.9  Hemolysis of erythrocytes ......................................................................... 24 
2.10  Polymer induced erythrocyte aggregation............................................... 25 
2.11  Ethidium bromide exclusion assay........................................................... 25 
2.12  Polyplex stability in heparin....................................................................... 25 
2.13  Animals ........................................................................................................ 25 
2.13.1  In vivo applications for RNAi experiments ........................................... 26 
2.13.2  In vivo applications of plasmid DNA..................................................... 26 
2.13.2.1  Low pressure tail vein injections.............................................. 26 
2.13.2.2  High pressure pDNA tail vein injections................................... 27 
2.13.3  In vivo applications for polymer comparison....................................... 27 
2.14  In vivo Imaging............................................................................................ 28 
2.15  Luciferase gene expression detection in situ .......................................... 28 
2.16  Blood analysis............................................................................................. 28 
2.17  Cytokine antibody array ............................................................................. 29 
2.18  Histopathological examination.................................................................. 29 
2.19  TUNEL staining of tissue sections ............................................................ 29 
2.20  Western Blot Analysis ................................................................................ 30 
2.21  RNA isolation............................................................................................... 30 
2.22  Reverse transcription of RNA to cDNA..................................................... 31 
2.23  Real-time polymerase chain reaction (RT-PCR)....................................... 31 
2.24  Statistical analysis ...................................................................................... 32 
3  Results..................................................................................... 33 
3.1  Therapy with siRNA .................................................................................... 34 
3.1.1  OEI-HD as a delivery vector ................................................................... 34 
3.1.1.1  Biophysical properties of OEI-HD/siRNA polyplexes ............... 34 
3.1.1.2  Preliminary in vivo studies....................................................... 35 
3.1.1.3  Tumor growth reduction in vivo ............................................... 36 
3.1.1.4  Blood cell and liver enzyme levels in treated mice .................. 37 
3.1.1.5  Reduction of RAN protein expression in Neuro2A tumors....... 38 
3.1.1.6  Reduction of RAN mRNA ex tumors ....... 40 
3.1.1.7  Apoptosis in tumors of RAN siRNA treated mice..................... 41 
3.1.2  OEI-HA10 as a delivery vector ............................................................... 43 
3.1.2.1  Acute toxicity of free OEI-HA10 polymer ................................. 43 
3.1.2.2  Lytic activity of frlymer................................... 43 
3.1.2.3  Erythrocyte aggregation induced by free OEI-HA10 polymer.. 44 
3.1.2.4  Acute toxicity of OEI-HA10 / Ran siRNA polyplexes................ 45 
3.1.2.5  Tumor growth reduction after polyplex application in vivo ....... 46 
3.1.2.6  Liver enzyme levels in treated mice ........................................ 47 
3.1.2.7  RAN protein expression in Neuro2A tumors............................ 48 
3.1.2.8  Reduction of RAN mRNA expression in Neuro2A tumors ....... 49 
3.1.2.9  Histopathological analysis of tumors ....................................... 50 
3.1.2.10  OEI-HA10 / siRNA complexes formed with helper lipids ......... 51 
3.1.2.11  Acute toxicity of OEI-HA10 / DOPE......................................... 52 
3.1.2.12  Acute toxicity of OEI-HA10 / DOPE / siRNA complexes.......... 52 
3.2  Gene Delivery with pDNA........................................................................... 53 
3.2.1  Influence of delivery vectors on toxicity and transfection efficiency 53 
3.2.1.1  DNA condensation................................................................... 53 
3.2.1.2  Stability against Heparin Displacement ................................... 54 
3.2.1.3  Size und Zetapotential............................................................. 54 
3.2.1.4  Transfection efficiency in vitro ................................................. 55 
3.2.1.5  Acute toxicity of polymers and polyplexes in vivo.................... 56 
3.2.1.6  Transfection efficiency in vivo.................................................. 58 
3.2.2  Influence of CpG motifs in pDNA on transfection efficiency.............. 60 
3.2.2.1  Biophysical properties of L-PEI plasmid complexes................ 60 
3.2.2.2  L-PEI based pDNA delivery in Neuro2A cells .......................... 61 
3.2.2.3  In vivo transfection efficiency after standard i.v. application .... 62 
3.2.2.4  Cytokine expression after i.v. polyplex application .................. 64 
3.2.2.5  In vivo transfection efficiency after hydrodynamic tail vein
application ............................................................................................. 65 
4  Discussion .............................................................................. 67 
4.1  Therapy with siRNA .............