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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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Gene therapy

Gene therapy is one of the most promising treatment options for future advanced therapies in a broad range of inherited and life threatening diseases caused by genetic deficiencies and abnormalities, including monogenetic disorders such as many blood diseases, acquired immune deficiency syndrome, cardiovascular diseases, cancer among others.[1-4]

Gene therapy generally encompasses all applications that employ therapeutic DNA to replace, inactivate or introduce a gene to treat a disorder. Here, the therapeutic nucleic acids can act at different stages of the gene expression process. Controlled gene delivery describes the specific process to bring the genes to the target tissues or cells to regulate or replace abnormal genes.

In the process of gene delivery, a multitude of biological obstacles have to be overcome to provide an efficient 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 inside of the target cell.

In systemic delivery, DNA therapeutics can be rapidly degraded by endonucleases in the blood stream or the extracellular space. Therefore, a complexation or encapsulation is desirable to protect the DNA and to prolong circulation. Classically, viral vectors have been employed as gene carries. They usually show efficient delivery capabilities but have considerable limitations such as low DNA/RNA loading capacity. Furthermore, immunogenicity and toxicity issues of viral vectors have stimulated new approaches towards the development of non-viral carriers. Often, self-assembled complexes with cationic polymers (polyplexes) or zwitterionic/neutral lipids (lipoplexes) are used to condense and protect the negatively charged DNA.[6-7]

Further biological hurdles are the recognition of target cells as well as cytosolic and nucleosolic uptake of the gene which needs to be considered to realize a successful gene delivery. In currently available technologies, more than 98% of the therapeutic gene is transported to the cell but then it accumulates inside cellular compartments called endosomes and is finally degraded. 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. In particular, we aim for an efficient, safe, and cost-effective transfer of gene therapeutics into the cytosol.

Saponins as endosomal escape enhancers

As mentioned above, a major bottleneck of current gene delivery approaches is the efficient cytosolic release of the genetic material from intracellular vesicles in the endocytic pathway such as endosomes and endolysosomes. When the DNA formulation is not released from these vesicles, it is prone to degradation in endolysosomes or is recycled back to the cell surface and is not able to reach the site of action in the cytosol or nucleus. To enable an efficient transfer of gene therapeutics to the cytosol, particular substances from plants are employed as endosomal escape enhancers (EEEs). In our group, we demonstrated that this class of secondary metabolites belonging to the group of saponins modulates the intracellular trafficking of plant toxins and other macromolecules.[8]

We and our partner at the Free University of Berlin have discovered and studied the EEEs focusing on the question if and how they mediate the specific uptake of potential pharmaceutical drugs. This includes targeted toxins, toxins without targeting moieties, small molecular weight drugs, and gene therapeutics. For more information how EEEs enhance the endosomal escape, see our projects on Targeted Tumor Therapies and our Glossary entry on EEEs. In the framework of the European project ENDOSCAPE the DNA-based therapeutics are of particular interest.

In recent years, first studies have been conducted to evaluate the potential of EEEs to improve the delivery outcome of targeted, non-viral gene therapies. Weng et al. showed that specific saponins can augment 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 endosomal membranes and destabilize them, preventing gene degradation in lysosomes by endosomal escape and providing sufficient amount of the therapeutic gene to localize in the cell nucleus. In another study, different DNA- and RNA-nanoplexes were formulated with liposomes, polyethylenimine (PEI) or targeted and non-targeted oligo-lysine peptides. Although the effect of EEEs varied within the cell lines, each transfection method was significantly improved by co-administration of EEEs.[10]

ENDOSCAPE project

So far, EEEs were always added separately to the DNA formulations as supporting agent in independent doses and distribution. The ENDOSCAPE technology aims to create a polymeric scaffold carrying all required components, the EEEs, the targeting ligand, and the effector gene in one single construct. We aim to develop novel non-viral gene delivery vectors based on cationic (bio)polymers to protect and transport the therapeutic DNA. In addition, EEEs will be included in the design of the carrier system and therefore covalently attached to the polymer. This enables the concerted delivery of the DNA therapeutic and EEEs to the site of action to efficiently augment the endosomal release.

To ensure recognition of target cells and include specificity of the gene delivery system, the scaffold will be endowed with targeting ligands specific for the intended disease.  The aim of the ENDOSCAPE technology is to provide targeting for any addressable cell type with all known genetic agents; thereby, ensuring better patient therapy not limited to inherited disorders, but also for cancer therapy. Thus, the technology will be of importance for large patient groups. We focus on certain model ligands specifically addressing either liver cells to address hemophilia or tumor cells to introduce suicide genes. The ligands comprise designed mutants or chemically modified versions of epidermal growth factor, transferrin, apolipoprotein A1 and the pre-S1 domain from hepatitis B virus. Using click chemistry, the targeting ligand can be easily added to the ENDOSCAPE module, allowing for customized drug applications and a wide range of tissue and cell targeting techniques. Therefore, the technology is designed as a toolbox allowing that building blocks can be combined for customized drug applications and for future developments in the field of cell targeting techniques.

Furthermore, the combination of the building blocks in one construct will facilitate the application in vivo as the gene therapeutic and all supporting compounds have the same pharmacokinetics. The designed constructs will be investigated in vivo regarding the toxicity and immunogenicity profiles as well as pharmacokinetic and pharmacodynamic studies for the developed gene therapeutics.

In the present project, we will focus on commonly used targeting concepts to demonstrate proof of concept of the technology. Here, we aim to achieve a higher efficacy of non-viral targeted gene therapy, using EEEs and to allow for efficient intracellular delivery to a targeted cell type in the body, in particular to treat monogenetic diseases and cancer. ENDOSCAPE will aim to provide a good alternative to viral gene delivery technology while incorporating already existing medical drug candidates. The long-term vision of ENDOSCAPE is market uptake of a novel technology that can be used for intracellular delivery of any applicable drug for medical treatment, as well as its application in personalized medicine.

See https://endoscape-2020.eu for further information and follow us at LinkedIn: https://www.linkedin.com/company/endoscapeproject/

The ENDOSCAPE project is funded by the European Union under Horizon 2020 contract no. 825730, https://cordis.europa.eu/project/id/825730/de.

 

References:

[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.