There is much need in advancing rational design of synthetic cationic polymers as gene carriers. Inspired by the molecular blueprint of the famed branched polyethylenimine (bPEI), here we describe a new series of acid-labile branched poly(ortho ester amino alcohol)s (bPOEAAs) that embodies multiple design principles about polymer degradability, chain branching, and type of cationic amino groups. The bPOEAAs were facilely synthesized via expoxide-amine reaction of monomers with various ratios, allowing a range of branching degrees and amine compositions to be explored. A polymer (bPOEAA2) with an intermediate degree of branching and approximately equal fraction of secondary and tertiary amines was identified to be optimal in forming stable polyplexes with plasmid DNA. Rapid release of DNA from the polymer was achieved by accelerated backbone degradation at endosomal acidic pH. Together, these unique features of bPOEAA2 enabled markedly improved transfection efficiency in cells in physiological medium and much lower cytotoxicity than bPEI. Insights gained from the rational design and screening of bPOEAAs may contribute to deeper understanding of structure-activity relationship of polymeric gene carriers, which may ultimately lead to safer and more effective gene therapies. (C) 2017 Elsevier Ltd. All rights reserved.