The favourable outcome of gene therapy depends on the developing efficient and safe gene delivery systems. Recently, nonviral vectors have gained considerable attention due to their improved safety profiles and their potential application in viral vector manufacturing. In this study, we report the design, synthesis, and evaluation of novel short-chain (C6) ionizable lipids with varying hydrophobic amino acid headgroups phenylalanine (C6F-IL), tryptophan (C6W-IL), and glycine (C6G-IL) along with a cationic lipid containing twin C12 alkyl chains (C12-CL). Liposomes composed solely of either C6 ionizable amino lipids (C6-IL) or C12-CL were unable to efficiently deliver plasmid DNA. However, an optimized combination of C12-CL and C6 ionizable lipids, particularly C6W-IL (Lipo-3), demonstrated superior transfection efficiency across HEK-293T, SKHEP, and HEPA cell lines, comparable to that of Lipofectamine 3000. Furthermore, when Lipo-3 was applied in lentivirus production, it yielded viral titres equivalent to those obtained using commercial Lipofectamine 3000. These findings demonstrate that a combination of two individually transfection-incompetent lipids can be engineered into a highly efficient liposomal system for gene delivery. This approach provides new opportunities for harnessing existing transfection-incompetent lipids in gene therapy applications.