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Nahaufnahme mehrerer Probenröhrchen, die auf einem Tisch liegen. Im Hintergrund ist unscharf der Schriftzug "Charité" auf einem Formular erkennbar.

Controlled Gene Delivery

In the process of gene delivery, a multitude of biological obstacles have to be overcome to provide an efficient therapy. One of the major problems in the field of non-viral gene therapy is the inefficient and safe delivery of genetic material to the site of action inside of the target cell.

Within the framework of a collaborative European project under the Horizon 2020 initiative, we aspire to overcome these hurdles in the field of gene delivery.


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Controlled Gene Delivery

Gene therapy

Gene therapy is one of the most promising treatment options for future therapies for a wide range of inherited and life-threatening diseases caused by genetic defects and abnormalities, including monogenetic diseases such as many blood disorders, acquired immunodeficiency syndrome, cardiovascular disease, cancer, and others [1-4]. Gene therapies generally include all applications in which therapeutic DNA is used to replace, inactivate, or introduce a gene to treat a disease. In this context, the therapeutic nucleic acids can act at different stages of the gene expression process. Controlled gene transfer describes the specific process by which genes are delivered to target tissues or cells to regulate or replace abnormal genes. Gene delivery must overcome numerous biological obstacles to enable effective therapy [5].

One of the major problems in the field of non-viral gene therapy is the inefficient and safe delivery of genetic material to the site of action in the target cell. When administered systemically, DNA therapeutics can be rapidly degraded by endonucleases in the bloodstream or extracellular space. Therefore, complexation or encapsulation is required to protect the DNA and allow circulation in the body. Classically, viral vectors have been used as gene carriers. They are usually efficient but have significant limitations, such as low loading capacity. In addition, problems with immunogenicity and toxicity of viral vectors have led to new approaches in the development of non-viral carriers. Self-assembled complexes with cationic polymers (polyplexes) or zwitterionic/neutral lipids (lipoplexes) are often used to condense and protect the negatively charged DNA [6-7]. Other biological hurdles include target cell recognition and cytosolic and nucleosolic uptake of the gene, which must be considered to realize successful gene transfer. With currently available technologies, more than 98% of the therapeutic gene is transported into the cell, but there it accumulates in cellular compartments, the endosomes, and is eventually degraded. Within the framework of the European joint project ENDOSCAPE, we aim to overcome these hurdles.

Glycosylated Triterpenoids as Endosomal Escape Enhancer

A major bottleneck in current gene delivery approaches is the efficient cytosolic release of genetic material from intracellular vesicles of the endocytic pathway such as endosomes and endolysosomes. If DNA is not released from these vesicles in time, it is degraded or transported back to the cell surface. To enable efficient transfer of gene therapeutics into the cytosol, we use endosomal escape enhancers (EEEs), which are secondary plant compounds from the group of glycosylated triterpenoids that can mediate intracellular transport of plant toxins and other macromolecules [8]. Together with our partners at Freie Universität Berlin, we discovered and studied EEEs, focusing on whether and how they mediate the specific uptake of potential drugs. These include targeted toxins, toxins without targeted components, small molecule drugs, and gene therapeutics.

In the context of the European ENDOSCAPE project, DNA-based therapeutics are of particular interest. In recent years, initial studies have been conducted to evaluate the potential of EEEs to improve targeted non-viral gene therapies. Weng et al. showed that certain glycosylated triterpenoids can enhance the delivery of peptide- and lipid-based DNA complexes with plasmid DNA, minicircle DNA, as well as siRNA (small interfering RNA) [9]. The EEEs specifically accumulate in and destabilize endosomal membranes, preventing DNA degradation in lysosomes by timely endosomal escape. In another study, various DNA and RNA nanoplexes were formulated with liposomes, polyethylenimine, or targeted oligo-lysine peptides. Although the effect of EEEs varied within cell lines, each transfection method was significantly improved by the co-administration of EEEs [10].


Until now, the endosomal escape enhancers (EEEs) have always been added separately to DNA formulations as supporting agents in independent doses and distributions. The ENDOSCAPE technology aims to create a polymeric scaffold containing all the required components – the EEEs, the ligands for target cell recognition, and the effector gene – in a single construct. We aim to develop novel non-viral gene delivery vectors based on cationic (bio)polymers to protect and transport therapeutic DNA. In addition, the EEEs will be incorporated into the design of the delivery system and thus covalently bound to the polymer. This enables concerted delivery of the DNA and EEEs to the site of action to efficiently enhance endosomal release.

The goal of the ENDOSCAPE technology is to be able to potentially produce genetic drugs for any addressable cell type, thus making progress not only in hereditary diseases but also in cancer therapy. Thus, the technology will be important for large patient populations. We are focusing on specific model ligands that target either liver cells for the treatment of hemophilia or tumor cells for the introduction of suicide genes. The ligands are engineered mutants or chemically modified versions of epidermal growth factor, transferrin, apolipoprotein A1, and the pre-S1 domain of hepatitis B virus. Using click chemistry, the ligand can be added to the ENDOSCAPE module, enabling tailored drug applications and a wide range of techniques for targeting tissues and cells. Therefore, the technology is designed as a modular toolbox that also allows building blocks to be combined for future developments in the field of target cell-directed methods.

In addition, combining the building blocks into a single construct will facilitate application in vivo, as the gene therapy drug and all supporting compounds will share the same pharmacokinetics. The designed constructs will be tested in vivo for toxicity and immunogenicity profiles, as well as in pharmacokinetic and pharmacodynamic studies.

In the present project, we aim to achieve higher efficacy of non-viral targeted gene therapy using EEEs and to enable efficient intracellular delivery to a specific cell type in the body, especially for the treatment of monogenetic diseases and cancer. ENDOSCAPE is intended to provide an alternative to viral gene therapy while incorporating existing medical drug candidates. The long-term vision of ENDOSCAPE is market acceptance of a novel technology that can be used for intracellular delivery of drugs for medical treatment as well as its application in personalized medicine.

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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 825730,

Cited Literature

[1] M. Cavazzana, F. D. Bushman, A. Miccio, I. André-Schmutz, E. Six, Nature Reviews Drug Discovery 2019, 18, 447-462.
[2] J.-J. Nie, B. Qiao, S. Duan, C. Xu, B. Chen, W. Hao, B. Yu, Y. Li, J. Du, F.-J. Xu, Advanced Materials 2018, 30, 1801570.
[3] H. Yin, R. L. Kanasty, A. A. Eltoukhy, A. J. Vegas, J. R. Dorkin, D. G. Anderson, Nature Reviews Genetics 2014, 15, 541-555.
[4] D. B. Kohn, Current Opinion in Biotechnology 2019, 60, 39-45.
[5] I. M. S. Degors, C. Wang, Z. U. Rehman, I. S. Zuhorn, Accounts of Chemical Research 2019, 52, 1750-1760.
[6] J. Buck, P. Grossen, P. R. Cullis, J. Huwyler, D. Witzigmann, ACS Nano 2019, 13, 3754-3782.
[7] U. Lächelt, E. Wagner, Chemical Reviews 2015, 115, 11043-11078.
[8] A. Weng, M. Thakur, B. von Mallinckrodt, F. Beceren-Braun, R. Gilabert-Oriol, B. Wiesner, J. Eichhorst, S. Böttger, M. F. Melzig, H. Fuchs, Journal of Controlled Release 2012, 164, 74-86.
[9] A. Weng, M. D. I. Manunta, M. Thakur, R. Gilabert-Oriol, A. D. Tagalakis, A. Eddaoudi, M. M. Munye, C. A. Vink, B. Wiesner, J. Eichhorst, M. F. Melzig, S. L. Hart, Journal of Controlled Release 2015, 206, 75-90.
[10 S. Sama, G. Jerz, P. Schmieder, E. Woith, M. F. Melzig, A. Weng, International Journal of Pharmaceutics 2017, 534, 195-205.

Further information on the research of the AG Fuchs