Genetic and Covalent Protein Modification Strategies to Facilitate Intracellular Delivery

Abstract

Protein-based therapeutics represent a rapidly growing segment of approved disease treatments. Successful intracellular delivery of proteins is an important precondition for expanded in vivo and in vitro applications of protein therapeutics. Direct modification of proteins and peptides for improved cytosolic translocation are a promising method of increasing delivery efficiency and expanding the viability of intracellular protein therapeutics. In this Review, we present recent advances in both synthetic and genetic protein modifications for intracellular delivery. Active endocytosis-based and passive internalization pathways are discussed, followed by a review of modification methods for improved cytosolic delivery. After establishing how proteins can be modified, general strategies for facilitating intracellular delivery, such as chemical supercharging or inclusion of cell-penetrating motifs, are covered. We then outline protein modifications that promote endosomal escape. We finally examine the delivery of two potential classes of therapeutic proteins, antibodies and associated antibody fragments, and gene editing proteins, such as cas9.

Figure 1.

Schematic depicting routes for internalization of extracellular proteins including (a) direct translocation, (b) membrane disruption, and (c) endocytosis.

Figure 2.

(a) Genetic methods of protein modification include addition of individual point mutations, addition of entire peptide domains and tags, and fusion to other globular protein domains via flexible linkers. (b) Chemical modification can occur via residue-specific modification of abundant residues such as lysines, aspartates and glutamates, or site-specific modification of cysteines or the N-terminus. Examples of bioconjugation reactions include modification of the N-terminus with 2-pyridinecarboxyaldehyde functionalized substrates, Michael addition of maleimide-functionalized substrates with cysteine thiolates, esterification of carboxylic acids with 2-diazo-2(p-methylphenyl)-N,N-dimethylacetamide, or reaction of activated esters with the primary amines of lysine.

Figure 3.

(a) List of prominent CPP’s used for intracellular delivery with cationic residues in blue. (b, top) Confocal microscopy images of the delivery of 0.4 μM CGC-aPP and CGC-aPP-K to HeLa cells in 10% FBS. Proteins were incubated with the cells for 20 h and at 16 h 150 μM chloroquine was added to increase the permeability of lysosomes. Hoechst-3342 (blue) and LysoTracker Red DND-99 (red) were used to track the cell nucleus and lysosomes, while protein localization was monitored via fluorescein-labeling of the microproteins (green). Scale bar, 10 μm. (b, bottom) Flow cytometry shows CGC motifs allowed for equivalent uptake of microproteins after replacing arginine residues with lysine. Flow cytometry data showing the fluorescence of HeLa cells after incubation with 0.4 μM of each miniprotein in 10% FBS medium. 150 μM chloroquine was added to increase the permeability of lysosomes after incubation with the miniproteins for 24 h. Mean fluorescence intensity (MFI) for each sample was normalized to a control sample treated with 0.4 μM S-aPP. Data are presented as mean ± SD. Adapted with permission from ACS Chem. Biol. 2018, 13, 11, 3078–3086. Copyright 2018 American Chemical Society. (c) Proteins can be supercharged either through removal of anionic residues or addition of cationic residues through many chemical and genetic modification routes. (d, top) Protein carboxyl side chains were targeted for modification via esterification with a hydrophobic reagent. Decreasing negative charge and increasing hydrophobicity increased direct translocation of the model protein into the cell. (d, bottom) The viability HeLa cells after addition of WT human RNase 1 and human RNase 1 cloaked with the hydrophobic reagent was monitored. Cell viability decreased at lower protein concentrations for the cloaked RNase 1, implying improved cellular uptake. Adapted with permission from ACS Chem. Biol. 2019, 14, 4, 599–602. Copyright 2019 American Chemical Society. Figure adapted with permission from. (e, top) Supercharging of GFP occurred via addition of histidine residues by site-directed mutagenesis. The resulting GFPs were supercharged only at acidic pH, allowing for selective delivery to cells in an acidic environment. (e, bottom). Confocal microscopy images of supercharged and histidine-supercharged GFP, delivered to HeLa cells at different pH. The histidine-supercharged GFP showed pH-responsive delivery, only reaching the cytosol at pH 6.5. Reproduced from Ref. 98 with permission from the Royal Society of Chemistry.

Figure 4.

(a) A range of nanoscale particles can be used to encapsulate and deliver protein cargo. (b, top) Uptake of proteins into polycationic carriers via anionic supercharging was accomplished using poly-glutamate sequences (E5–20). These charged peptide tags were fused to the N-terminus of cas9, allowing for complexation with arginine-decorated gold nanoparticles. (b, bottom) Microscopy images of HeLa cells after addition of gold nanoparticle-E-tagged cas9 complexes. Cas9 was labeled with FITC and showed successful cytosolic delivery above a critical tag length of 15 glutamates. Adapted with permission from ACS Nano 2017, 11, 3, 2452–2458. Copyright 2017 American Chemical Society. (c, top) Proteins were functionalized with an aryl boronic acid, followed by conjugation to a salicylhydroxamate-functionalized polymer. Conjugation resulted in the formation of nanoscale protein-polymer assemblies that were redox-responsive and successfully delivered native proteins to the cytosol. (c, bottom) RNase A nanoassemblies were successfully delivered to HeLa cells and decreased cell viability up to 60%. WT RNase A saw no decrease in cell viability upon addition as it was not readily internalized. Adapted with permission from ACS Chem. Biol. 2018, 13, 11, 3078–3086. Copyright 2018 American Chemical Society. (d) Proteins that are internalized are frequently trapped inside endosomal vesicles, several strategies to assist with endosomal escape have been developed. (e, top) Endosomal escape efficiency was increased by the addition of a cell-penetrating disulfide (CPD). (e, bottom) CPD-conjugated antibodies (green) were successfully delivered to HeLa cells stained with Hoechst nuclear stain (blue), with CPD’s showing higher internalization in the cytosol than non-CPD-conjugated antibodies. Adapted with permission from J. Am. Chem. Soc. 2015, 137, 37, 12153–12160. Copyright 2015 American Chemical Society. (f, top) TMab4 was identified as a cell-penetrating protein domain, capable of endosomal disruption upon acidification of the endosome. (f, bottom) pH-responsive membrane disruption was induced through modification of a YYH motif within CDR3, with tryptophan-containing motifs selectively permitting uptake into Ramos cells only at pH 5.5. Reprinted from Journal of Controlled Release, Volume 235, Ji-Sun Kim, Dong-Ki Choi, Ju-Yeon Shin, Seung-Min Shin, Seong-Wook Park, Hyun-Soo Cho, Yong-Sung Kim, “Endosomal acidic pH-induced conformational changes of a cytosol-penetrating antibody mediate endosomal escape”, Pages 165–175 Copyright 2016, with permission from Elsevier.