Recent developments in intracellular protein delivery

Abstract

Protein therapeutics based on transcription factors, gene editing enzymes, signaling proteins and protein antigens, have the potential to provide cures for a wide number of untreatable diseases, but cannot be developed into therapeutics due to challenges in delivering them into the cytoplasm. There is therefore great interest in developing strategies that can enable proteins to enter the cytoplasm of cells. In this review article we will discuss recent progress in intracellular protein therapeutics, which are focused on the following four classes of therapeutics, Firstly, vaccine development, secondly, transcription factor therapies, thirdly, gene editing and finally, cancer therapeutics. These exciting new advances raise the prospect of developing cures for several un-treatable diseases.

Figure 1. Protein therapeutics that function inside of cells: An emerging class of therapeutics

Overview of the four major applications of intracellular protein delivery: Vaccine development, transcription factor therapy, gene editing and cancer therapy. Schematic diagram describing intracellular protein delivery. Green and blue items represent a cell and a protein therapeutic, respectively.

Figure 2. CRISPR–Gold can deliver Cas9 RNP and donor DNA in vivo and induce HDR

A) CRISPR–Gold is composed of 15 nm GNPs conjugated to thiol modified oligonucleotides (DNA-Thiol), which are hybridized with single-stranded donor oligonucleotides and subsequently complexed with Cas9 RNP and the endosomal disruptive polymer PAsp(DET), where ‘DET’ is diethylenetriamine. B) CRISPR–Gold is internalized by cells in vitro and in vivo via endocytosis. This triggers endosomal disruption and releases Cas9 RNP and donor DNA into the cytoplasm. Nuclear delivery results in HDR.

Figure 3. Strategies for delivering protein based cancer therapeutics intracellularly

(a) Cell-penetrating peptides (CPPs) co-administered with macromolecules can disrupt the endosomal membrane and enable the cytosolic delivery of macromolecular therapeutics. (b) Endosomolytic micelles conjugated with antibodies target cancer cells and deliver BIND1 intracellularly (an inhibitor of BHFR1), and treat lymphatic cancers caused from EBV infections. (c) Encapsulation of caspase-3 in a positively charged disulfide cross-linked polymer micelle increases its serum stability, cellular uptake and intracellular delivery.