Transposon-Nanocarrier auf Basis von Poly(Propylenimin) für die gezielte Einschleusung von therapeutischer DNA in Tumorzellen
Molekulare und zelluläre Neurologie und Neuropathologie
Präparative und Physikalische Chemie von Polymeren
Zellbiologie
Zusammenfassung der Projektergebnisse
The delivery of nucleic acids, encoding for example for suicide genes, is a promising therapeutic option to treat cancer. Due to the utilization of polycationic carrier molecules, delivery of therapeutic nucleic acids became applicable for in vivo approaches. So far, systemic delivery of such carrier-complexed therapeutic nucleic acids, designated dendriplexes suffers from unspecific side effects upon systemic administration as the preferential mode of application in a therapeutic setting. One approach to avoid unwanted off-target effects is the use of minicircle DNAs (MC), which are devoid of bacterial backbone DNA and therefore minimizes the risk of unspecific activation of pattern recognition receptors (PRR). In addition, the concept of targeted delivery of nanoparticles based on the introduction of targeting devices (ligands, antibodies), that specifically targets tumor cells and induces a receptor-mediated endocytosis, further minimizes side effects. More specifically, biocompatible maltose-modified poly(propylenimine) (mPPI) dendrimers, coupled to single chain fragment variables (scFv) or peptide ligands targeting tumor-associated antigens on the surface of tumor cells were employed to selectively deliver MCs to tumor cells. To enable stable expression in targeted tumor cells, MCs encoding for a Sleeping Beauty (SB) transposase and a MC transposon encoding for a suicide gene or for a small hairpin RNA (shRNA) for abrogating cell proliferation were delivered simultaneously. This study thus explored the combination of differently chemically modified PPI-DNA nanoparticles equipped with targeting devices (scFv and ligands) for targeting tumor cells displaying tumor-associated antigens such as prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), and epidermal growth factor receptor variant III (EGFRvIII). The extra modifications of the PPIs included direct chemical coupling such as a peptide-based PSMA-ligand and biotinylation of PPIs to enable a biotinneutravidin driven assembly of PPI-DNA dendriplexes with biotinylated scFv molecules or biotinylated ligands. Yet, copper-free click chemistry of all tested peptide ligands resulted in water-insoluble PPI-products not suited for subsequent experiments. However, additional work with the so-called polyplex system, in which biotinylated dendriplexes were bioconjugated via neutravidin to mono-biotinylated PSMA-ligand and additionally to EGFR and folatreceptor-ligands, enabled a selective targeting to tumor cells with expression of the cognate receptor. Experiments with polyplexes equipped with humanized anti-PSCA-scFv and loaded with MC encoding for reporter genes and a shRNA against the inhibitor of apoptosis protein (IAP) Survivin revealed insufficient DNA transfer efficiencies. Yet, when loaded with siRNAs targeting Survivin, a selective delivery to PSCA-positive cells without activation of the PRR Toll-like receptor 3 (TLR3) and significant anti-proliferative effects in a xenograft tumor model was achieved. To improve the transfer of DNA, polyplexes were further modified by adding cyclodextrin-functionalized PPIs. This modification resulted in nanoparticles competent for improved endosomal escape of MCs, which enabled the transposition of the tumor suppressor p53 in PSCA-positive tumor cells, and furthermore achieved suppression of tumor cell growth without activation of TLR9. Yet, significant differences in gene transfer efficiencies between tumor cell types indicates effects of intrinsic restriction factors for exogenous DNA. Our work provides the basis for subsequent studies aiming at further improving the efficiency of targeted delivery of MCs, in particular for transfection-resistant cells, towards a clinical translation.
Projektbezogene Publikationen (Auswahl)
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Biohybrid structures and their targeting properties: Considering the potential use of mono- and polyassociation steps. IDS11 - 11th International Dendrimer Symposium. Funchal, PT, 14.07.19-18.07.19
Appelhans D., Fingernagel J., Daeg J., Boye S., Lederer, A. & Voit B.
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Bivalent Peptide- and Chelator-Containing Bioconjugates as Toolbox Components for Personalized Nanomedicine. Biomacromolecules, 21(1), 199–213.
Daeg, Jennifer; Xu, Xiaoying; Zhao, Lingzhou; Boye, Susanne; Janke, Andreas; Temme, Achim; Zhao, Jinhua; Lederer, Albena; Voit, Brigitte; Shi, Xiangyang & Appelhans, Dietmar
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Design and synthesis of bioconjugates for personalized medicine. IDS11 - 11th International Dendrimer Symposium. Funchal, PT, 14.07.19-18.07.19
Daeg J., Xu X., Voit B., Shi X., Temme A. & Appelhans D.
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Targeted delivery of oligonucleotides into eukaryotic cells using hybrid maltose/cyclodextrin polyplexes. TU Dresden (TUD138EP), application number: EP22160227.9
Temme A., Appelhans D., Jugel W. & Tietze S.
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Targeted RNAi of BIRC5/Survivin Using Antibody-Conjugated Poly(Propylene Imine)-Based Polyplexes Inhibits Growth of PSCA-Positive Tumors. Pharmaceutics, 13(5), 676.
Jugel, Willi; Aigner, Achim; Michen, Susanne; Hagstotz, Alexander; Ewe, Alexander; Appelhans, Dietmar; Schackert, Gabriele; Temme, Achim & Tietze, Stefanie
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Development of selective nanoparticles for p53-replacement therapy for gliomas. 73rd Annual Meeting of the German Society for Neurosurgery (DGNC) 2024, Cologne, 29.05.2022. - 01.06.2022
Jugel W., Broghammer F., Schackert G. & Temme A.
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Targeted Transposition of Minicircle DNA Using Single-Chain Antibody Conjugated Cyclodextrin-Modified Poly (Propylene Imine) Nanocarriers. Cancers, 14(8), 1925.
Jugel, Willi; Tietze, Stefanie; Daeg, Jennifer; Appelhans, Dietmar; Broghammer, Felix; Aigner, Achim; Karimov, Michael; Schackert, Gabriele & Temme, Achim
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Considering the potential of dendritic (glyco)architectures in the application field of neurodegenerative diseases. IDS12 - 12th International Dendrimer Symposium, Singapore, 26.06.-31.06.2023, Singapore.
Appelhans D., Kösterke T., Firdaus S., Xu X., Moreno S., Lederer A. & Voit B.
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Synthesis and characterisation of pseudo-glycodendrimers for biomedical applications. EPF Summer School. Bertinoro, ITA, 02.05.-05.05.2023
Kösterke T., Appelhans D. & Voit, B.
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Antibody-TLR3 agonist-conjugates for immunotherapy of tumors. Annual Meeting of the American Association for Immunologists (AAI), Chicago, 03.05.2024 – 07.05.2024
Temme A., Hagstotz A., Schau I., Seifert M., Sürün D., Zimmermann S., Buchholz F., Rupp L., Wehner R., Schmitz M., Schackert G. & Eyüpoglu I.Y.
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Antibody-TLR3 agonist-conjugates induce innate immune responses and immune memory eradicating experimental EGFRvIII tumors. Annual Meeting of the American Association Cancer Research (AACR), San Diego, 05.04.2024 – 10.04.2024
Temme A., Hagstotz A., Schau I., Seifert M., Sürün D., Zimmermann S., Buchholz F., Rupp L., Wehner R., Schmitz M., Schackert G. & Eyüpoglu I.Y.
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Pseudomonas Exotoxin PE38 suicide gene therapy for the treatment of glioblastoma. 75th Annual Meeting of the German Society for Neurosurgery (DGNC) 2024, Göttingen, 09.06.2024 — 09.06.2024
Jilek L., Jugel W., Lange-Osborn M., Schackert G., Eyüpoglu I.Y. & Temme A.
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Synthesis and characterisation of pseud-glycodendrimers for biomedical applications. IUPAC MACRO 2024 - The 50th World Polymer Congress. Warwick University, GBR, 01.07.2024-04.07.2024
Kösterke T., Appelhans D. & Voit, B.
