Discovery and characterization of a peptide that enhances endosomal escape of delivered proteins in vitro and in vivo.

The inefficient delivery of proteins into mammalian cells remains a major barrier to realizing the therapeutic potential of many proteins. We and others have previously shown that superpositively charged proteins are efficiently endocytosed and can bring associated proteins and nucleic acids into cells. The vast majority of cargo delivered in this manner, however, remains in endosomes and does not reach the cytosol. In this study we designed and implemented a screen to discover peptides that enhance the endosomal escape of proteins fused to superpositively charged GFP (+36 GFP). From a screen of peptides previously reported to disrupt microbial membranes without known mammalian cell toxicity, we discovered a 13-residue peptide, aurein 1.2, that substantially increases cytosolic protein delivery by up to ∼5-fold in a cytosolic fractionation assay in cultured cells. Four additional independent assays for nonendosomal protein delivery collectively suggest that aurein 1.2 enhances endosomal escape of associated endocytosed protein cargo. Structure-function studies clarified peptide sequence and protein conjugation requirements for endosomal escape activity. When applied to the in vivo delivery of +36 GFP-Cre recombinase fusions into the inner ear of live mice, fusion with aurein 1.2 dramatically increased nonendosomal Cre recombinase delivery potency, resulting in up to 100% recombined inner hair cells and 96% recombined outer hair cells, compared to 0-4% recombined hair cells from +36-GFP-Cre without aurein 1.2. Collectively, these findings describe a genetically encodable, endosome escape-enhancing peptide that can substantially increase the cytoplasmic delivery of cationic proteins in vitro and in vivo.

Targeted disruption of ß-arrestin 2-mediated signaling pathways by aptamer chimeras leads to inhibition of leukemic cell growth.

UNLABELLED: ß-arrestins, ubiquitous cellular scaffolding proteins that act as signaling mediators of numerous critical cellular pathways, are attractive therapeutic targets because they promote tumorigenesis in several tumor models. However, targeting scaffolding proteins with traditional small molecule drugs has been challenging. Inhibition of ß-arrestin 2 with a novel aptamer impedes multiple oncogenic signaling pathways simultaneously. Additionally, delivery of the ß-arrestin 2-targeting aptamer into leukemia cells through coupling to a recently described cancer cell-specific delivery aptamer, inhibits multiple ß-arrestin-mediated signaling pathways known to be required for chronic myelogenous leukemia (CML) disease progression, and impairs tumorigenic growth in CML patient samples. The ability to target scaffolding proteins such as ß-arrestin 2 with RNA aptamers may prove beneficial as a therapeutic strategy. HIGHLIGHTS: An RNA aptamer inhibits ß-arrestin 2 activity.Inhibiting ß-arrestin 2 impedes multiple tumorigenic pathways simultaneously.The therapeutic aptamer is delivered to cancer cells using a cell-specific DNA aptamer.Targeting ß-arrestin 2 inhibits tumor progression in CML models and patient samples.

Rapid, robotic, small-scale protein production for NMR screening and structure determination.

Three-dimensional protein structure determination is a costly process due in part to the low success rate within groups of potential targets. Conventional validation methods eliminate the vast majority of proteins from further consideration through a time-consuming succession of screens for expression, solubility, purification, and folding. False negatives at each stage incur unwarranted reductions in the overall success rate. We developed a semi-automated protocol for isotopically-labeled protein production using the Maxwell-16, a commercially available bench top robot, that allows for single-step target screening by 2D NMR. In the span of a week, one person can express, purify, and screen 48 different (15)N-labeled proteins, accelerating the validation process by more than 10-fold. The yield from a single channel of the Maxwell-16 is sufficient for acquisition of a high-quality 2D (1)H-(15)N-HSQC spectrum using a 3-mm sample cell and 5-mm cryogenic NMR probe. Maxwell-16 screening of a control group of proteins reproduced previous validation results from conventional small-scale expression screening and large-scale production approaches currently employed by our structural genomics pipeline. Analysis of 18 new protein constructs identified two potential structure targets that included the second PDZ domain of human Par-3. To further demonstrate the broad utility of this production strategy, we solved the PDZ2 NMR structure using [U-(15)N,(13)C] protein prepared using the Maxwell-16. This novel semi-automated protein production protocol reduces the time and cost associated with NMR structure determination by eliminating unnecessary screening and scale-up steps.