Novel polyaspartamide non-viral carriers for gene therapy were synthesized by introducing, on the same polymer backbone, positively charged groups, for electrostatic interactions with DNA, and thiol groups for the formation of disulfide bridges between polymer chains. The introduction of thiols was aimed to have a vector with low redox potential sensitivity: disulfide crosslinking in fact, being stable in extracellular environment, allowed either to have stable complexes in plasma, that can protect DNA from metabolism, or to be reduced inside the cell, where the excess of glutathion in reduced form maintains a low redox potential. The consequent destabilization of the complex after disulfide cleavage can release DNA selectively inside the cells. Alpha,beta-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) was used as starting polymer being a highly water-soluble synthetic polymer, already proposed with success as therapeutic carrier by our group. In this study, PHEA was firstly functionalised with ethylendiamine, obtaining a well defined copolymer with pendant primary amine groups (PHEA-EDA), to which N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and 3-(carboxypropyl)trimethyl-ammonium chloride (CPTA) were linked in two subsequent steps, allowing the introduction of thiol and cationic groups respectively. Finally DTT treatment lead to the final PHEA-EDA-SH-CPTA thiopolycation, named PESC. The present work describes the synthesis and characterization of the thiopolycation PESC. 1H NMR spectroscopy detected the derivatization molar degrees in SPDP and CPTA; the formation of DNA complexes (thiopolyplexes), their stability in the presence of polyanions and the ability to release DNA under reductive conditions were studied by agarose gel electrophoresis. DNase II degradation study was carried out to detect the ability of thiopolyplex to stabilize DNA towards enzymatic metabolism. Thiopolyplexes were then characterized by Dynamic Light Scattering (DLS) and Zeta Potential analysis. Finally, in vitro toxicity profile (MTT) and gene transfer efficiency (Luciferase assay) were carried out to evaluate thiopolyplex biocompatibility, safety and efficacy to be used as gene delivery system.