Hastened Fusion-Dependent Endosomal Escape Improves Activity of Delivered Enzyme Cargo

There is enormous interest in strategies to traffic biologics into the mammalian cell cytosol. Not only must these materials reach the appropriate cellular locale intact and in therapeutically relevant concentrations, they must also retain activity upon arrival. The question of residual activity is especially critical when delivery involves the late endocytic pathway, whose acidic environment can denature and/or degrade internalized material. ZF5.3 is a compact mini-protein that escapes efficiently from late endocytic vesicles, with or without covalently linked protein cargo. Here, we redesign the sequence of ZF5.3 to hasten the timing of endosomal escape. The new mini-protein we describe, AV5.3, escapes earlier than ZF5.3 along the endocytic pathway with no loss in efficiency, with or without enzyme cargo. More importantly, earlier endosomal escape translates into higher enzymatic activity of a pH-sensitive enzyme upon arrival in the cytosol. Delivery of the pH-sensitive enzyme DHFR with AV5.3 results in substantial catalytic activity in the cytosol, whereas delivery with ZF5.3 does not. The activity of delivered AV5.3-DHFR successfully rescues a DHFR deletion in CHO cells. AV5.3 represents an improved strategy for the efficient and direct delivery of active therapeutic proteins and enzymes.

Rational redesign of a zinc finger mini-protein delivery agent hastens the timing of endosomal escape and avoids irreversible denaturation of enzyme cargo in late endocytic vesicles.