Development of carbamate modified PEI for delivery of a granzyme B inhibitor gene to protect against cytotoxic lymphocyte killing

Abstract

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells protect vertebrates by killing infected or transformed cells using granzyme B (GrB) to induce apoptosis. However, GrB-induced apoptosis causes inflammatory disease and allograft rejection and is an important disease target. The aim of this project is to prevent apoptosis of the target cells by delivering a plasmid encoding GrB inhibitor proteinase inhibitor-9 (PI-9) using cationic polymers. Polyethylenimine (PEI, branched, Mw 25 kDa) gives high gene transfection efficiency in many cell lines, but it is highly cytotoxic. To reduce its cytotoxicity, we modified PEI by blocking primary amines through nucleophilic addition between primary amine and a protected functionalized cyclic carbonate, generating a carbamate linkage through the ring-opening of the cyclic carbonate. Different hydrophobic-groups or sugar functionalized carbonates were substituted onto PEI and the optimum substitution ratios to achieve high gene transfection efficiency and minimal cytotoxicity in various cell lines were determined. Among these polymers, PEI with 7 or 20 of 56 primary amine groups substituted by mannose had similar gene binding ability as unmodified PEI, leading to almost 100% transfection efficiency of a GFP plasmid in HEK293T cells as well as a large decrease in cytotoxicity. However, PEI with all primary amine groups blocked was unable to form complexes with DNA, and gene transfection was negligible. The PI-9 encoding plasmid was transfected into HEK293T cells effectively using the optimally modified PEIs, protecting up to 80% HEK293T cells from killing by human natural killer-like YT cells. Furthermore, PEI with 25 primary amine groups substituted by mannose successfully delivered PI-9 plasmid into Balb/3T3 fibroblasts and protected them from killing by GrB. Therefore, the carbamate-mannose modified PEI/PI-9 encoding plasmid complexes have potential clinical utility in the prevention of allograft rejection and inflammatory disease caused by GrB.