The success of gene therapies fundamentally depends on the efficient delivery of nucleic acid cargo into the target cells of patients—a process known as gene delivery or transfection. Numerous delivery vehicles, commonly referred to as vectors, have been developed to achieve this goal. These vectors can be broadly categorized into two types: engineered viruses and nonviral systems. Engineered viruses are viruses which are modified to replace their natural genetic material with a therapeutic cargo, allowing them to deliver it into host cells. Nonviral vectors, on the other hand, are manufactured with diverse materials such as lipids, polymers, peptides, or proteins. While each vector type offers unique advantages, both have significant limitations that must be addressed to fully realize the transformative potential of gene therapies. In Chapter 1, we introduce the concept of gene delivery by presenting the biological barriers that vectors must overcome during the transfection process. Strategies employed by both viral and nonviral vectors to address these challenges are examined, highlighting their respective mechanisms and limitations. The chapter provides a comparative overview of six vector systems - three of each vector group - culminating in the description of the TFAMoplex, a novel nonviral vector for DNA delivery that forms the central focus of this thesis. The TFAMoplex is based on the human transcription factor A (TFAM) protein. Physiologically, TFAM is located in mitochondria, where it performs functions analogous to histones in the nucleus by condensing and organizing mitochondrial DNA. Interestingly, this property is retained when TFAM is isolated and combined with foreign DNA, such as plasmids, forming compact DNA-TFAM particles approximately 100 nm in diameter—referred to as TFAMoplexes. Through protein engineering, TFAM can be modified to include functional proteins, effectively transforming the TFAMoplex into a transfection agent. The subsequent chapters explore additional modifications, functional enhancements, and the further optimization and characterization of the TFAMoplex system. The first optimization step, detailed in Chapter 2, addresses the intracellular transportation of the genetic material delivered by the TFAMoplex. Large macromolecular complexes, including the TFAMoplex and its DNA cargo, generally require active transport within the cell, a process mediated by motor proteins. To enable this, we fused dynein light chains to the TFAMoplex, creating a bridge between the vector and motor proteins. This modification significantly enhanced gene delivery efficiency compared to unmodified TFAMoplexes. 6 Furthermore, we exploited a pull-down proteomics approach to identify cytosolic proteins interacting with the TFAMoplex and that a high performing version had more nucleolar interactors. Leveraging this knowledge, we introduced a domain of one of these nucleolar interacting proteins directly to the complex, resulting in improved expression levels of the delivered gene. In Chapter 3, we further functionalized the TFAM protein by incorporating additional protein moieties. Specifically, we included DNA-binding sequences from transcription factors, known as bZIP domains, into the system. This approach aimed to improve particle stability by enhancing DNA binding. While the modified TFAMoplexes exhibited increased transfection efficiency, our findings revealed that the improvement was likely due to enhanced cell association rather than increased particle stability. We demonstrated that the bZIP domains bind directly to the cell membrane, effectively tethering the TFAMoplex to the cell surface. Finally, we compared the transfection of the best performing bZIP-TFAMoplex to one of the gold standards in gene delivery: Adeno-associated viruses (AAVs). Chapter 4 provides a general discussion of the results, highlighting the strengths and limitations of the TFAMoplex system. This chapter also outlines potential strategies for future improvements, building on the insights gained throughout this thesis. This research underscores the versatility and potential of the TFAMoplex as a nonviral gene delivery vector, laying the groundwork for further advancements in gene therapy