UC Berkeley

Despite the growing interest in protein-based therapeutics, one of the major limitations towards their development is the fact that proteins cannot simply diffuse into the cell interior. Instead, most proteinaceous cargo must be taken up via the biological process of endocytosis. Proteins become engulfed by the endocytic vesicle that travels into the cell and many of these protein cargoes remained trapped within endosomes. While some protein therapeutics, such as Cerezyme and Fabrazyme, take advantage of this entrapment to exhibit their functions within the endocytic pathway, the inability to access the cytosol and other organelles within the cell prevents development of intracellular-targeting proteins. A number of delivery vehicles have been proposed, including liposomes, nanoparticles, polymers, and cell-penetrating peptides (CPPs). Unfortunately, each of these techniques must be optimized for their respective cargo and target and may still suffer from overall low cytosolic delivery. Cell-permeant miniature proteins (CPMPs) provide a promising solution as they reach the cytosol intact at high concentrations, have a defined mechanism, and can deliver protein cargo.

Here, I describe progress from the evaluation of CPMPs alone to delivery of therapeutically relevant proteins and enzymes in both mammalian cells and mice. First, I present a summary of CPMP development, from designing miniature proteins that installed structural elements of traditionally flexible CPPs, to describing mechanistic details of endosomal uptake and escape, to delivering enzymatic cargo to the liver of mice. Next, I describe fluorescence correlation spectroscopy (FCS) in conjunction with flow cytometry experiments for monitoring proteins in live cells and evaluating endosomal escape. This methodology is followed by FCS studies that establish diffusion properties of free dye and CPMPs to assess whether intact CPMPs reach the cytosol of Saos-2 cells. The following chapter illustrates that among a panel of CPPs and CPMPs, the CPMP ZF5.3 is a superior delivery vehicle for a model cargo in multiple cell lines. Next, I describe the development and use of ZF5.3 conjugated to argininosuccinate synthetase (ZF-AS), an enzyme involved in an inborn error of metabolism. ZF-AS was the first ZF5.3-containing molecule to participate in animal studies to evaluate clinical utility of CPMPs. To continue improving cellular delivery of therapeutically relevant proteins, I describe functional and cellular uptake studies in which ZF5.3 is appended to the well-studied endonuclease Cas9 and initial development towards ZF5.3 conjugates of NS1, a monobody that binds Ras proteins with high affinity. Finally, I conclude with remarks on CPMP-mediated delivery. Together, these projects demonstrate that the CPMP ZF5.3 is a superior delivery vehicle compared to canonical and cyclic CPPs, illustrate ZF5.3-mediated delivery of three protein cargos, and support further ZF5.3 clinical development as a tool for protein-based therapeutics.