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We have developed a new block copolymer gene carrier that comprises of a polyethylene glycol segment and a degradable cationic polyphosphoramidate (PPA) segment. This PEG-b-PPA copolymer carrier formed micelles upon condensation with plasmid DNA in aqueous solution. PEG-b-PPA/DNA micelles exhibited uniform and reduced particle size ranging from 80 to 100 nm and lowered surface charge, compared with complexes of DNA with the corresponding cationic PPA carrier. PEG-b-PPA/DNA micelles maintained similar transfection efficiency as PPA/DNA complexes, which was comparable to that of PEI/DNA complexes in HepG2 cells, but yielded about 16-fold lower transgene expression in primary rat hepatocytes than PPA/DNA complexes. Following bile duct infusion in Wistar rats, PEG-b-PPA/DNA micelles mediated 4-fold higher and more uniform gene expression in the liver than PPA/DNA complexes. Liver function tests and histopathological examination indicated that PEG-b-PPA/DNA micelles showed low toxicity and good biocompatibility in the liver. This study demonstrated the potential of PEG-b-PPA/DNA micelles as an efficient carrier for liver-targeted gene delivery.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.