Gene delivery enables the treatment of genetic disorders by introducing nucleic acids into target cells. While gene delivery vectors based on viruses are equipped with elegant molecular mechanisms enabling delivery of nucleic acids to the intended target location inside cells, they face notable safety concerns. Consequently, there is growing interest in developing safer non-viral gene delivery systems. However, current non-viral vectors continue to face substantial challenges, particularly in achieving efficient cytoplasmic delivery of genetic cargo following cellular uptake, a process known as endosomal escape. Despite extensive research aimed at overcoming this barrier, endosomal escape remains a major bottleneck in non-viral gene delivery due to its inherently low efficiency. Therefore, the development of novel strategies to facilitate endosomal escape is essential for advancing the field of non-viral gene delivery towards safe and effective therapeutic applications. Chapter 1 provides an introduction to gene therapy and outlines current gene delivery strategies, highlighting the major challenges that hinder the efficient delivery of nucleic acids. Particular emphasis is placed on endosomal escape, which plays a critical role in the success of non-viral gene delivery systems. The chapter explores natural strategies employed by viruses and bacteria to gain cytoplasmic access and discusses the endosomal escape mechanisms of non-viral delivery vectors. Chapter 2 presents a comprehensive and critical overview of peptide- and protein-based endosomal escape enhancers, discussing both their potential and limitations. The chapter also provides an overview on the evolution of peptide- and protein-based delivery approaches and identifies key aspects for the development of more effective non-viral gene delivery agents. In Chapter 3, the objectives of this thesis are discussed and summarized. This chapter outlines strategies to enhance the endosomal escape efficiency of the non-viral TFAMoplex gene delivery system and approaches to make it more biocompatible through the use of human-derived proteins only. The TFAMoplex deoxyribonucleic acid (DNA) delivery agent, which is based on human mitochondrial transcription factor A (TFAM), enables efficient transfection of cells. TFAM is naturally present in mitochondria and involved in regulation of mitochondrial DNA. For its application as a delivery agent, TFAM was repurposed to bind and condense plasmid DNA (pDNA), which can harbor any gene of interest, into nanoparticles exhibiting a diameter of ~100 nm. To overcome the endosomal barrier, TFAM was fused to a bacterial phosphatidylcholine-specific phospholipase C (pc-PLC) enabling endosomal escape via hydrolysis of endosomal limiting membranes following cellular uptake of the nanoparticles. The subsequent chapters investigate strategies to enhance this escape mechanism and explore the replacement of the bacterial pcPLC with a human analogue to reduce potential immunogenicity. Chapter 4 describes the development of a modular calcium-responsive system designed to improve phospholipase-mediated endosomal escape in the TFAMoplex gene delivery platform. This system utilizes human split calbindin-D9k (Cal9k) domains in order to capture pc-PLC to TFAMoplexes and subsequently release it in a calcium-dependent manner inside the endosomal compartment. By additional Summary 6 targeting of pc-PLC to endosomal membranes via integration of a nanobody directed against the membrane protein CD9, this novel system significantly improved endosomal disruption and transfection efficiency. The optimized TFAMoplex system achieved high transfection at remarkably low pc-PLC concentrations, outperforming existing non-viral delivery systems including the original TFAMoplexes. Owing to its modular design, this system holds promise for broad application across diverse gene delivery systems currently limited by poor endosomal escape, thereby overcoming a major hurdle in the cytosolic delivery of macromolecular therapeutics. Chapter 5 investigates human von Willebrand factor A domain-containing protein 7 (VWA7) as a potential pc-PLC candidate, with the aim of identifying a less immunogenic alternative to the bacterial phospholipase used in the TFAMoplex system. Structure-based analyses using AlphaFold predictions identified VWA7 as a structural homolog of bacterial pc-PLCs, harboring a conserved active site. Functional assays demonstrated modest cytotoxicity and basal pc-PLC activity for recombinant VWA7 variants produced in bacteria, although activity levels were substantially lower than bacterial counterparts. The detected low activity suggests possible dependence on unknown activation mechanisms, post-translational modifications, or specific cellular environments. While the obtained VWA7 variants are not yet suitable for integration into TFAMoplexes, this chapter lays the basis for future optimization and highlights VWA7 as a promising candidate for human-derived endosomal escape agents. Chapter 6 provides a comprehensive summary and critical discussion of the key findings and limitations presented throughout this thesis. Moreover, potential directions for future research on the development of potent and safe endosomal escape enhancers are discussed. Collectively, the work presented in this thesis advances the field by contributing novel insights and strategies towards the design of safer and more effective non-viral gene delivery platforms.