This work describes the development and investigation of a family of novel "smart" copolymers as non-viral gene delivery vectors. The copolymers have five blocks, and thus named pentablock, with a central block of a hydrophobic polymer, surrounded by two blocks of a hydrophilic polymer, and capped at each terminal end with cationic polymer blocks, arranged in an architecture to provide temperature and pH sensitivity to the copolymers. They are derived from commercially available triblock Pluronic copolymers. The cationic copolymers can efficiently condense negatively charged plasmid DNA in nanostructures with efficient cellular uptake. The amphiphilic nature of copolymers causes them to exist as micelles in aqueous solutions that help them traverse cellular membranes with minimal cell membrane damage. Intra-cellular trafficking of copolymer/DNA complexes revealed that they are up-taken by the cells predominately via endocytosis and are able to deliver the ferried gene into the nuclei. The copolymers efficiently protect the condensed DNA against degradation by nucleases while their protonation capability at low pH assists them in escape from endosomal vesicles into the cytoplasm. The efficiency of the copolymers to deliver condensed DNA into the cells in vitro was comparable to the commercially available polymeric transfection vectors, and they were also found to be significantly less cytotoxic. Adding non-ionic Pluronic copolymers to the formulation of pentablock copolymer/DNA complexes sterically shielded their surface charge and protected them against aggregation with serum proteins. These stabilized formulations were able to retain their ability to transfect cells even in complete growth media supplemented with serum proteins, warranting efficient transfection efficiency in an in vivo application. The amphiphilic nature of copolymers further permits copolymer/DNA complexes to form thermo-reversible hydrogels at physiological temperatures. At concentrations above 15 wt%, copolymer/DNA complexes existed as solutions at room temperature and formed elastic hydrogels at 37°C that dissolved over seven days in excess buffers to release colloidally stable polyplexes. The system thus permits an injectable aqueous pharmaceutical preparation at room temperature that can be injected subcutaneously in tissues/cavities to form a localized depot in situ, which provides a long-term sustained release of therapeutic genes well protected inside the copolymer/DNA complexes.