Publikationsdatenbank

Ex vivo and in vivo testing of non-viral gene delivery systems mediated by endosomal escape Enhancers for advanced cancer therapy‎ (2025)

Art

Hochschulschrift

Autor

Kuhlmann, Philipp Jochen (WE 14)

Quelle

Berlin: Mensch und Buch Verlag, 2025 — X, 100 Seiten

ISBN: 978-3-96729-291-6

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/48516

Kontakt

Institut für Pharmakologie und Toxikologie

Koserstr. 20
14195 Berlin
+49 30 838 53221
pharmakologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

The delivery of therapeutic nucleic acids into cells is the goal of gene therapy, which is still a challenge. Therefore, the focus of this work was to identify carriers capable of transfecting target cells with therapeutic plasmids in vivo in order to develop a platform technology for the treatment of cancer. The dendrimer poly(amidoamine) (PAMAM) and the peptide K16C and, based on these, various prototypes with different surface modifications were tested as carrier systems for the delivery of the so-called suicide gene encoding for the protein toxin saporin. To be successful as a carrier for transferring deoxyribonucleic acid (DNA) to target cells, a carrier must have a number of properties. For example, the carrier must form polyplexes that are stable in the blood, protect the DNA from degradation and, above all, be well tolerated by the body at the dose required for the therapeutic purpose. The DNA can only develop its potential to kill the cancer cell if the polyplex can circulate in the bloodstream long enough to reach its target. The experiments of this study clearly showed that the applied carrier molecules were able to protect the plasmid deoxyribonucleic acid (pDNA) from enzymatic digestion and were stable even after one hour of incubation in blood serum. In addition, the tested prototypes did not cause any measurable hemolysis at the doses planned for the in vivo experiments. However, the carrier PAMAM in particular showed a strong tendency to clump the erythrocytes. The influence of the carrier molecules and their polyplexes on blood coagulation cannot be denied. The retarding effect of the carrier PAMAM and its polyplex on the coagulation parameter prothrombin time (PT) was much stronger than that of the peptide K16C and its polyplex. Nevertheless, the values of all tested substances at a concentration of 1 μM were above the reference range. The effect on the partial thromboplastin time (PTT) parameter was striking. Almost all tested substances completely prevented coagulation starting with a concentration of 0.5 μM. The prototypes therefore carry the risk of an increased tendency to bleed after intravenous administration. Nevertheless, the starting point for further evaluation of the prototypes in in vivo toxicity studies was obtained. The no observed adverse effect level (NOAEL) is a threshold above which adverse effects or signs of toxicity occur. The aim of these studies was to determine an appropriate dose for the in vivo efficacy studies, in other words, to find the NOAEL for the prototypes. For the dendrimer-based prototypes and the peptidebased prototypes, the NOAEL was greater than the maximum dose administered. Consequently, all treatments were well tolerated with the exception of local inflammation at the injection site. The maximum intravenous dose administered was 45 μg pDNA for the dendrimer-based prototype and 15 μg pDNA for the peptide-based prototype. There was no evidence of elevated liver enzyme parameters and urea in the blood samples tested, which would be an indication of organ damage. The pathohistological examinations showed a clear activation of the spleen parenchyma. The suspicion that the treatments had activated the immune system was confirmed by testing the blood for specific antibodies against the carrier molecules using an enzyme-linked immunosorbent assay (ELISA). It was found that the amount of specific antibodies formed against the carrier molecules increased with increasing dose and number of injections. Once the maximum dose for the treatments had been determined and a comprehensive picture of the behavior of the prototypes in the blood had been obtained, the groundwork was laid for efficacy studies. A total of four peptide-based prototypes and four dendrimer-based prototypes were tested at two different doses for their tumor inhibitory properties in a colon cancer xenograft model. At a dose of 5 μg pDNA, the dendrimer-based carriers significantly reduced tumor growth. In particular, the prototype D5(SO1861)0.5 reduced the tumor volume by 75% and the prototype D5 by as much as 50%, resulting in prolonged survival in these groups compared to the vehicle control group. Follow-up experiments with a 3-fold higher dose of 15 μg pDNA were designed to confirm the results but did not yield the desired results. Nevertheless, further studies with other tumor mouse models will be useful to further investigate the tumor inhibitory effect of the dendrimer vectors. In contrast to the dendrimer-based prototypes, the overall performance of the peptide prototypes was less convincing. Although the peptide-based prototypes PP(SO1861)0.25 and PP + 30 μg SO1861 were able to significantly reduce tumor growth at a dose of 5 μg pDNA compared to the vehicle control group, but this did not lead to prolonged survival of the animals. Given that promising approaches with similar constructs have already been pursued in other projects, the developed prototypes should still be further investigated in other routes of administration and in other tumor models. It would also be worth investigating whether conjugation of SO1861 to the vector or co-administration with free SO1861 would be an improvement. Gene therapy offers the hope of taking a significant step closer to the ultimate goal of successfully treating currently incurable cancers, making further efforts in this project worthwhile.