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Recent advances with techniques used for manipulating gene expression have brought us to an era where various gene therapeutic approaches are becoming common therapeutic tools for many previously incurable diseases. The main factor impeding the wider translation of gene therapy is that the active pharmaceutical ingredients used for interfering with gene expression are based on nucleic acids and synthetic oligonucleotides and such molecules do not readily reach their intracellular targets due to their physicochemical properties and therefore they require delivery vectors to cross the cell membrane. Cell-penetrating peptides (CPPs) is one such class of delivery vectors that comprise excellent potential for transporting bioactive cargo molecules across cellular membranes, both in vitro and in vivo conditions. CPPs have shown to be very versatile carriers for various types of bioactive cargo, including different nucleic acids such as plasmids (pDNA), splice-correcting oligonucleotides (SCOs), small interfering RNAs (siRNA) and mRNA, or peptides and proteins or even small molecules. This thesis focuses on characterizing the delivery of various nucleic acids-based molecules with a variety of novel fatty acid modified CPPs. In order to achieve this we utilize the ability of a family of CPPs called PepFects to non-covalently formulate nucleic acids into nanoparticles. More particularly the aim of the thesis is to find and characterize the key parameters of these peptide/nucleic nanoparticles that would improve their potential applicability as a drug formulation and delivery system for future gene therapies. By simultaneously characterizing the role of N-terminal fatty acid modification and the peptide/nucleic acid ratio in the nanoparticles we were able to show in Papers I and II that increasing the hydrophobicity and reducing unbound free fraction of the peptide improves delivery efficiency and decreases toxicity of these nanoparticles both in vitro and in vivo. Based on the findings from Paper I regarding the ability of these amphiphilic peptides to self-associate into supramolecular structures we went deeper in Paper III to study the formation, composition and live cell association of these peptide/nucleic acid complexes at single molecule sensitivity. And finally in Paper IV we enhanced the specificity of these nanoparticles towards in vivo xenograft tumors by incorporating the capacity to be specifically activated in the tumor microenvironment. Conclusively, these findings contribute to the field with identifying and characterizing some of the key factors in developing efficient and safe peptide-based delivery vectors for gene modulating therapeutics. At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.