Genetic engineering of T-cells with chimeric antigen receptors (CARs) has revolutionised the treatment of cancer, but the requirement for viral vectors to deliver nucleic acids encoding the CAR construct into T-cells is costly, requires complex manufacturing procedures, and raises safety issues due to the risk of insertional mutagenesis. Non-viral delivery methods are therefore being actively explored, but need to overcome the challenging nature of T-cells as being hard-to-transfect. RALA is a cell penetrating peptide that forms nanoparticles upon complexation with nucleic acids and has been shown to deliver plasmid DNA (pDNA), mRNA, and siRNA into mammalian cells. This thesis explored whether RALA could deliver nucleic acids encoding green fluorescent protein (GFP), in the form of pDNA or mRNA, or CAR constructs, into human T-cells, and characterised its mechanism of uptake. RALA successfully delivered pDNA and mRNA encoding GFP into the Jurkat leukaemic T-cell line with similar transfection efficiencies (TEs) of 10-20%. RALA outperformed the non-viral delivery platforms Trans-IT-Jurkat and Lipofectamine LTX but was not as efficient as nucleofection. RALA/pDNA complexes were internalised by Jurkat T-cells via endocytosis, and primarily co-localised with the lysosome thereafter. Further studies revealed that endosomal/lysosomal trafficking and intracellular nucleic acid-sensing pathways limited RALA-mediated GFP expression. Modulating these pathways led to a two-fold increase in GFP expression and enabled robust RALA-mediated CAR expression, which was otherwise undetectable. Proteomic analysis of three independent Jurkat T-cell stocks with significantly different TEs identified endophilin-B2 as a potential regulator of transgene delivery. RALA yielded negligible levels of GFP expression in primary human T-cells with either pDNA or mRNA. These studies demonstrated that RALA can deliver nucleic acids into Jurkat T-cells, identified novel barriers that limit non-viral transgene delivery and expression, and provide a pathway for further exploration of RALA or its derivatives for nucleic acid delivery into human T-cells.