The delivery of genes of therapeutic relevance to their correct molecular target requires overcoming a complex series of barriers. Since naked nucleic acids are no match to our bodies’ defense mechanisms, delivery vectors are being developed to guide these therapeutic genes across a myriad of hindrances. In the last decades, nonviral vectors have emerged as promising carrier systems as they possess several
desirable traits such as ease of production, ability to carry large payloads, increased safety profile compared to viral alternatives, etc. Despite their many advantages, nonviral vectors (a.k.a. nanoparticles) – unfortunately – seem to lack therapeutic efficiency. In order to reach sufficient efficiency, scientists worldwide believe that nanoparticle design needs to take into account the numerous biological barriers a
nanomedicine encounter after administration. However, the rational design of nanomedicines depends on our fundamental knowledge about these barriers and especially how nanomedicines interact with them. The design of a new generation of non-viral gene therapeutics, based on fundamental knowledge regarding the biological barriers, could bring about the long-awaited success of ‘nano’ in gene therapy.
In Chapter 1 we start by providing an overview of the different biological barriers a nanomedicine encounters upon administration. After shortly discussing the extracellular barriers, an extensive overview of the intracellular barriers is provided.
The plasma membrane, exocytosis, endosomal release, autophagy, vector unpacking, cytoplasmic degradation and nuclear uptake are all prominent intracellular barriers that
prevent effective delivery via non-viral gene therapeutics. After discussing the physiology of the different barriers and why they pose a hindrance to gene delivery, we focus on methodologies (both well-known as state-of-the-art) that allow visualization and quantification of nanoparticle-barrier interactions. These assays and techniques should enable the scientific community to gain a better fundamental understanding of the delivery barriers to non-viral gene therapy.
Of these intracellular delivery barriers, endosomal escape is considered to be one of the major hurdles in gene therapy. After endocytic uptake, it is well-known that the majority of nanomedicines remain entrapped inside the endosomal compartment.
Upon endosomal maturation, the nanomedicines become prone to degradation by lysosomal enzymes, apart from the fact that the endosomal membrane prevents translocation of the nucleic acid cargo into the cytosol. A commonly used strategy to overcome the endosomal barrier is the use of cationic polymeric vectors that are thought to induce osmotic endosomal bursting through the proton sponge effect. In
Chapter 2 we review the conflicting reports that have been published on this subject.
The debate about the validity of the proton sponge hypothesis has divided the scientific community for more than 2 decades. By systematically analyzing the individual reports and including recent findings on additional factors that contribute to the proton sponge hypothesis, we come to the conclusion that the various reports are not that conflicting after all. With this we hope to arrive at a consensus that the endosomal escape capacity of proton sponge-based polymers depends on a delicate balance between osmotic forces, polymer swelling and membrane destabilization.
In Chapter 3 we set out to gain a deeper understanding of the mechanistic factors that govern effective proton sponge-based endosomal escape. Therefore, we perform a detailed comparative study of the endosomal escape capacity of
JetPEI/pDNA polyplexes in HeLa cells vs ARPE-19 cells. We observed that JetPEI/pDNA polyplexes were able to induce higher levels of transfection in HeLa cells than in ARPE19 cells, which we could attribute to an increased endosomal escape frequency in HeLa cells. After evaluation of several endosomal properties, we found that both endosomal
size and endosomal membrane leakiness can have a considerable impact on proton sponge-mediated endosomal escape. Larger endosomes require a higher number of
polyplexes to create an osmotic pressure that is sufficiently high to induce endosomal bursting. Membrane leakiness – the loss of its semi-permeable property for small molecules – prevents the build-up of osmotic pressure inside the endosome, thereby abolishing proton sponge-based endosomal rupture. The importance of these intercellular variations was confirmed with additional experiments on A549 and H1299 cells. We conclude that the effectivity of proton sponge-based endosomal escape is very much cell-dependent, with the endosomal size and endosomal membrane
leakiness being two important factors that have been largely overlooked until now.
When cells have comparable endosomal membrane leakiness, endosomal size will play a determining role. However, at high levels of leakiness, build-up of the osmotic pressure is no longer possible, regardless of endosomal size.
In Chapter 4 we investigate the application of plasmonic nanoparticles coupled with laser irradiation to induce photothermally-triggered endosomal escape of pDNA.
Besides the opportunity to overcome one of the most prominent intracellular barriers in gene therapy, this strategy would allow to obtain spatio-temporal control over the
cytosolic delivery of pDNA, rendering it a suitable tool to conduct fundamental studies regarding the endosomal barrier. In particular, we examined the potential of JetPEI/pDNA/Au complexes to induce endosomal escape of functional pDNA in HeLa cells after irradiation in a heating regime (low laser energy) or after the formation of explosive vapour nanobubbles (high laser energy). Unfortunately, we observed that although both regimes could induce endosomal rupture, they nevertheless failed to generate efficient transfection in HeLa cells. We believe this is primarily due to dysfunctionality of the pDNA after being subject to the aforementioned photothermal
effects. Other nanoparticle designs should be considered that provide better protection to the pDNA upon laser irradiation.
Finally, Chapter 5 discusses the broader international context of the work in this thesis and its relevance to the field. We started by giving a general overview of the key challenges and successes of gene therapy. As the development of efficient
non-viral vectors poses a significant challenge, we discuss some factors that hinder the progression of nanomedicines in the field of gene therapy. The main reason for the
inefficiency of non-viral gene therapeutics is a their inability to overcome one or more biological barriers. We propose that fundamental studies, focused on the properties of these biological barriers and the interaction of nanomedicines with these barriers, could lead to renewed insights that could enable the development of more effective rationally-designed nanomedicines for gene therapy.