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Targeted delivery of nucleic acids to tumours

Targeted delivery of nucleic acids to tumours. Manfred Ogris Pharmaceutical Biotechnology Department of Phamacy University of Munich 05-09-2006. DNA Therapeutics.

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Targeted delivery of nucleic acids to tumours

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  1. Targeted delivery of nucleic acids to tumours Manfred Ogris Pharmaceutical Biotechnology Department of Phamacy University of Munich 05-09-2006

  2. DNA Therapeutics “... DNAacts as therapeutic prodrug which can be delivered by a variety of technologies. The body cells read the genetic information carried by the DNA and translate it into the actual drug (usually a protein) …. “

  3. Cancer Gene Therapy Therapeutic concepts: • Direct killingof tumor cells (e.g. enzyme/prodrug) • Block cell cycle/induceapoptosis • Inhibit tumor cellmetastasis/migration • Inhibit/revert tumorneoangiogenesis • Stimulate anti-tumorimmunity(cytokines, vaccines)

  4. Barriers for systemic gene delivery tight junction fenestration plasma proteins DNA blood cells extracellular matrix target cells

  5. Non-specific interactions: Non-target cells Blood Receptor mediated uptake into target cells * Bioresponsive Disassembly ++ ++ ++ * * * * * * * * * * * Targeting Ligand (Tf, EGF) Transport Domain (melittin) * * * * * * Shielding Agent PEG * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Endosomal release Polyplexes: Towards ‘Synthetic Viruses’ Compacting Polycation ++ ++ ++ ++ DNA Kursa 2003 ; Ogris 2003; Boeckle 2005; Walker2005

  6. Endocytosis * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * DNA siRNA Endosome NUCLEUS * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Nuclear Trafficking Endosomal Release Cellular Uptake of Synthetic Viruses

  7. + + + * + * * * + * * + * * * + + * * * * * * * * + + + * * * + * * * + * * * + + * * * * * * * + Single Particle MicroscopyDNA/PEI Polyplexes HUH-7 cells / GFP-tagged tubulin

  8. + + + * + * * * + * * + * * * + + * * * * * * * * + + + * * * + * * * + * * * + + * * * * * * * + In vivo Luciferase Imaging PEI /DNAcomplexes(unshielded, positive charge) gene expression in lungs and tail (application site) I. Hildebrandt et al. Gene Ther. 2003

  9. PEG EGF EGF Polyplexes for Systemic Targeting HUH-7 s.c. SCID mice 0.8-1 µm <+4 mV 2 days mouse 1 – pCMVLuc mouse 2 – control Wolschek 2002; Prasmickaite, 2003

  10. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * DNA NUCLEUS * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Endosomal Release Cellular Uptake of Synthetic Viruses siRNA pIC

  11. Influenza virus HA-2 acidic Endosomolytic peptides: (as alternative or in combination) D-melittin, N-terminal Source peptide optimal pH PEI INF6: GLFGAIEG FIEN GWEG WEGn IDGW WYGG CG Melittin-SH:CIGA VLKV LTTG LPAL ISWI KRKR QQ neutral Bee venom Melittin Plank 1992 ; Ogris 2001; Boeckle 2005 Endosomal Escape: Membrane-active Polymers or/and Peptides Endosomolytic polymers: e.g. polyethylenimine (PEI) proton sponge effect,discovered by J.P. Behr (Boussif et al. 1995)

  12. EGFR-targeted Poly IC: Intracellular Distribution +Mel +Mel Fluorescein -labeled polyIC -Mel -Mel A. Shir, M. Ogris, E. Wagner, A. Levitzki PLOS Medicine 2006

  13. Bioreversible hydrazone linker N N N C H H DNA PEG polyplex deshieldingpH 5: less than 1 hr / 37°CpH 7: no deshielding in 6 hrs /37°C ++++ ++++ PC-PEG + + ++++ + + pH responsive linker - stable at pH 7, labile at pH 5.5 pH-Specific Endosomal PEG Deshielding extra - intra- cellular Endosomal acidification G. Walker et al, Mol. Ther. 2005

  14. +7 * * * +6 * * * * log RLU * DNA * +5 * * * * +4 or EGF +8 +7 EGFR-targeting Renca-EGFR log RLU +6 * +5 Reversibly shielded Non shielded Stable shielded Ligand plus pH-reversible PEG Shield TfR-targeting K562 Tf G. Walker et al, Mol. Ther. 2005

  15. * * * * * * * * DNA * * * * * * stable shielded reversibly shielded non shielded pH-reversible PEG Shield: in vivo EGFR-targeted reversibly shielded small polyplexes mediate highest gene expression in sc HUH7 tumors 1E+7 1E+6 EGF log RLU/ organ 1E+5 1E+4 1E+3 spleen kidney liver heart lung tumor G. Walker et al, Mol. Ther. 2005

  16. Therapeutic Applications ....

  17. Cancer gene therapy: GDEPT CPA: Chemotherapy vs. GDEPT

  18. P450/CPA Gene Therapy • Human Hepatoma in SCID mice 1600 control 1400 CPA 1200 CYP/CPA 1000 tumorvolume bGal/CPA 800 600 400 200 0 0 5 10 15 20 25 days after tumor setting

  19. Synthetic RNA: a therapeutic approach EGF Receptor-Targeted Synthetic Double- Stranded RNA Eliminates Glioblastoma, Breast Cancer, and Adenocarcinoma Tumors in Mice A. Shir, M. Ogris, E. Wagner, A. Levitzki PLoS Medicine, 2006

  20. dsRNA/ PEI ++ ++ ++ dsRNA dsRNA Complex Formation Targeting domain: EGF (select desired interaction) Compacting domain: PEI Shielding domain: PEG (block undesired interactions) Endosomal domain: Melittin (intracellular release)

  21. 100 0 0 20 40 60 80 100 120 140 160 180 200 Survival of mice (%) Targeted Poly (I• C) EliminatesGlioblastoma Tumors From Mouse Brains 20 days tumors 10 days tumors conjugate control No treatment pIC/PEI-PEG-EGF+Mel No treatment

  22. Key Issues for Cancer Gene Therapy Improve targeting • Overcome systemic barriers • Target tumor cells/tumor endothelium Enhance efficiency • intracellular release by membrane-active peptides • optimize into bioresponsivesystems (e.g. PEG deshielding) Choice of therapeutic concept • Combined effects with chemotherapeutics • Induction of immune responses

  23. Ernst Wagner Sabine Boeckle Julia Fahrmeir Carolin Fella Katharina v. Gersdorff Julia Klöckner Veronika Knorr Nicole Tietze Collaborators: LMU Christoph Bräuchle Karla de Bruin Ralf Bausinger Jerusalem U Alexei Shir Alexander Levitzki Arkadi Zintchenko, postdoc Greg Walker, postdoc EC FP6 ‘GIANT‘ DFG SFB486 ‘NanoMan‘ Sanders Stiftung Dr. Mildred Scheel-Stiftung

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