The efficient delivery of proteins into cells is needed to fully realize the potential of protein-based therapeutics. Current protein delivery strategies generally suffer from poor endosomal escape and low tolerance for serum. Here, the genetic fusion of a supercharged polypeptide, called SCP, to a protein provides a generic method for intracellular protein delivery. It allows efficient protein endocytosis and endosomal escape and is capable of potently delivering various proteins with a range of charges, sizes, and bioactivities into the nucleus of living cells. SCP is discovered to bind directly to the nuclear import protein importin β1 and gains access to the nucleus. Furthermore, SCP shows minimal hemolytic activity and stability in serum and lacks toxicity and immunogenicity in vivo. Effective gene editing can be achieved by SCP-mediated delivery of Cas9 protein and guide RNA. This study may provide an efficient and useful tool for the design and development of cell-nucleartargeted drug delivery.

Our data have uncovered some of the mechanisms by which PsTag600-FGF21 suppresses hepatic inflammation and further suggest that PsTag600-FGF21 could be an effective approach in NASH treatment.

BACKGROUND AND PURPOSEChemical conjugation of therapeutic proteins with polyethylene glycol (PEG) is an established strategy to extend their biological half-life (t 1/2 ) to a clinically useful range. We developed a novel uncharged and unstructured recombinant polypeptide composed of five amino acids (P, S, T, A and G), named PsTag, as another approach to extend the t 1/2 of human FGF21, with increased hydrodynamic radius. EXPERIMENTAL APPROACHHuman FGF21 was fused with PsTag polymers of differing lengths (200 -600 residues). Three fusion proteins and native FGF21 were produced in Escherichia coli. The biophysical characteristics, metabolic stability, immunogenicity and pharmacokinetics in were assessed in first. In lean and diet-induced obese (DIO) mice, effects on body weight, oral glucose tolerance tests and levels of relevant hormones and metabolites were studied. KEY RESULTSFusion proteins were solubly expressed in E. coli and prolonged the t 1/2 from 0.34h up to 12.9 h in mice. Fusion proteins were also biodegradable, thus avoiding vacuole formation, while lacking immunogenicity in mice. In DIO mice, administration of PsTag fused to FGF21 reduced body weight, blood glucose and lipids levels and reversed hepatic steatosis. CONCLUSIONS AND IMPLICATIONSThe novel recombinant polypeptide, PsTag, should be useful in the development of biological drugs with properties comparable to those achievable by PEGylation, but with potentially less side effects. In mice, fusion of FGF21 to PsTag prolonged and potentiated pharmacological effects of native FGF21, and may offer greater therapeutic effects in treatment of obesity. Abbreviations

Successful and efficient delivery of Cas9 protein and gRNA into cells is critical for genome editing and its therapeutic application. In this study, we developed an improved supercharged polypeptide (SCP) mediated delivery system based on dithiocyclopeptide linker to realize the effective genome editing in tumor cells. The fusion protein Cas9-linker-SCP (Cas9-LS) forms positively charged complexes with gRNA in vitro to provide possibilities for gRNA delivery into cells. Under the microenvironment of tumor cells, the dithiocyclopeptide linker, containing matrix metalloproteinase 2 (MMP-2) sensitive sequence and an intramolecular disulfide bond, can be completely disconnected to promote the release of Cas9 protein with the nuclear localization sequence (NLS) in the cytoplasm and transfer to the cell nucleus for highly efficient genome editing, resulting in an obvious increase of indel efficiency in comparison to fusion protein without dithiocyclopeptide linker (Cas9-SCP). Furthermore, Cas9-LS shows no significant cytotoxicity and minimal hemolytic activity. We envision that the microenvironment-responsive Cas9 protein delivery system can facilitate more efficient genome editing in tumor cells.

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