Chemical evolution of 2D sequences and 3D topologies within lipo-xenopeptides comprising succinyl tetraethylene pentamine (Stp) and lipoamino fatty acid (LAF) as the artificial amino acids revealed highly potent double pH-responsive carriers for pDNA, mRNA, siRNA or CRISPR Cas9 mRNA/sgRNA cargos when formulated as polyplexes or lipid nanoparticles (LNPs). Herein, in a next chemical evolution step the effects of spacer amino acids were investigated. In contrast to published molecules, the novel carriers were synthesized by connecting Stp and LAFs with L-ornithine instead of L-lysine. The introduction of ornithine resulted in shorter interspaces between the polar (Stp) and the apolar (LAF) cationizable domains. Original and new analogue structures were evaluated for both luciferase pDNA and mRNA delivery on Huh7 hepatocellular carcinoma and HeLa carcinoma cells. In direct comparison and for both investigated (U1 and B2) carrier topologies, the novel ornithine-spaced xenopeptides (ID 1827 and ID 1813) mediated high transfection efficacies at low dose (50 ng/well), exceeding the previous lysine-spaced top-performing carriers (ID 1611 and 1621).