Potent Protein Delivery into Mammalian Cells via a Supercharged Polypeptide

Abstract

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.

Non-viral delivery of the CRISPR/Cas system: DNAversusRNAversusRNP

“…Gao and colleagues genetically fused a supercharged polypeptide (SCP) to proteins to facilitate direct intracellular transduction without requirement of additional delivery agents. 231 They found that K4tagged proteins achieved highest cellular uptake and distribution into the nucleus. For genome editing, Cas9-K4/sgRNA induced 15.2% indels in the CCR5 gene in HeLa cells.…”

Strategies for High-Efficiency Mutation Using the CRISPR/Cas System

“…However, few studies have been reported on CPP-mediated CRISPR component delivery at present. Moreover, both delivery efficiency and subsequent editing efficiency were usually at a low level of just 10-20% (Ramakrishna et al, 2014b;Yin et al, 2018b;Del'Guidice et al, 2018;Yin et al, 2019a). This likely stems from the indefinite mechanism of CPP internalization and requirement for extensive optimization for targeting each type of cargo and cell.…”

CRISPR/Cas9 Delivery System Engineering for Genome Editing in Therapeutic Applications

“…The cell-penetrating peptides (CPPs) are short cationic polypeptides with 10-30 amino acids in length, which could covalently conjugate to the Cas9 protein to enable the delivery of cargoes into the cytosol through passive or active endocytic pathways. For example, the Cas9 RNP fused with a supercharged peptide called SCP could be efficiently internalized into the target cells, followed by an escape from the endosomes, and translocation into the nucleus [ 61 ]. However, the covalent strategies have underlying risks of structure alteration and bioactivity reduction.…”