Strategies to Improve Non-Viral Gene Delivery and the Preclinical Investigation of Nanomedicines

Conventional drug therapy relies on the introduction of therapeutic molecules into the body through various routes of administration (e.g., oral, parenteral, or topical). However, all these applications share a serious drawback: Following administration, the whole organism is exposed to the therapeutic molecule. Consequently, therapeutic molecules can interact with cells or tissues they were not intended to interact with, which is a major driver of side effects. To account for the distribution in the body, much higher doses than necessary are administeredto ensure a sufficiently high concentration of the therapeutic molecule at the target site.
A solution to this problem is to direct the therapeutic molecules to the specific cells where they are needed. On the one hand, this approach, called targeted drug delivery, reduces the number of molecules that end up in the wrong place in the body, thereby reducing side effects. On the other hand, this enables a drastic reduction of the required dose while maintaining the same effect because the therapeutic molecules are directed to the site where they are required instead of being distributed all across the body. There are several strategies to achieve targeted drug delivery but the use of nanoparticles is one of the most common approaches. Due to their small size (<100 nm), nanoparticles are taken up by cells and therefore, enable the intracellular release of therapeutic molecules. Among the different types of nanoparticles of synthetic (non-viral) origin, lipid-based nanoparticles are the best characterized and most used ones. Nanoparticles are usually decorated with molecules that enable prolonged circulation in the bloodstream and with targeting molecules that direct the nanoparticle to a specific cell population.
The benefits offered by encapsulation into targeted nanoparticles are even more pronounced for nucleic acids (e.g., DNA) because naked nucleic acids are rapidly degraded in the blood circulation by serum nucleases which is prevented by encapsulation. Gene therapy offers a number of advantages. Many genetic diseases manifest due to defects in the genetic information that is the blueprint for enzymes responsible for maintenance of normal body functions. Due to the defect in the blueprint, the enzyme cannot be produced or its activity is reduced. The introduction of the correct genetic information (blueprint) in the form of DNA or other nucleic acids can counterbalance the negative effects of the defective native enzyme. This approach is very attractive due to several reasons: First, conventional drug therapy is usually unable to cure genetic diseases but only treats or attenuates the symptoms. Second, with the correct blueprint at hand, the cells own "enzyme factory" can produce the "cure". Third, the restoration of only a small proportion of the native activity of the enzyme is often sufficient for the patients to live without symptoms. Fourth, the long persistence of DNA molecules in the cell nucleus ensures prolonged expression of the enzyme, thereby drastically reducing the number of therapeutic interventions compared to conventional drug therapy (e.g., once every six months compared to daily intake).
Despite these promises, the field of non-viral drug and gene delivery is a very complex topic and the underlying mechanisms and important factors for therapeutic success often remain elusive. Therefore, the first part of this PhD thesis aimed to improve the efficiency of a clinically relevant lipid nanoparticle formulation for gene delivery, as well as our understanding of molecular structures important for successful gene delivery (Chapter 1). Furthermore, the interactions between lipid nanoparticles and DNA molecules are investigated using a fluorescence-based method. The method provides a means to determine the number of DNA molecules per nanoparticle, a question that has only been addressed theoretically so far (Chapter 2). The proposed method enables researchers to draw more precise conclusions from their gene delivery experiments. The third part of the thesis focuses on the improvement of targeting and blood circulation properties of lipid-based drug delivery vehicles. A novel targeting molecule derived from the hepatitis B virus enables highly efficient and selective liposome delivery to hepatocytes whereas a novel nanoparticle shielding molecule demonstrated enhanced blood circulation properties comparable to the gold standard (PEG) while avoiding immune responses associated with PEG (Chapter 3).
Finally, a transparent animal infection model (zebrafish embryo) for the investigation of novel antibiotic compounds is discussed (Chapter 4). The transparency allows the application of fluorescence-based methods to evaluate antibiotic compounds, thereby improving our understanding of antibiotic therapy according to the proverb "seeing is believing". Furthermore, the high reproduction rate and the relatively low regulatory requirements enable the screening of a large number of compounds, thereby possibly accelerating research in the field of antibacterial drug therapy.