?This SBIR project seeks to develop novel technologies for the manipulation and labeling of the interior of living eukaryotic cells for research, diagnostic and eventually therapeutic purposes. Cell-penetrating peptides offer the tantalizing prospect of delivering a broad palette of user-defined protein cargoes through the plasma membranes of eukaryotic cells. Cargo protein delivery via CPPs has many advantages over transfection with respect to variety of deliverable biomolecules, rapidity of protein expression/delivery and control of protein levels over time. Current CPP technologies are either cumbersome and require the production of individually labeled proteins, or suffer from a lack of specificity and low efficiency. Our innovative core technology is based on cell-penetrating peptide-coupled adaptor proteins that allow the spontaneous loading of almost any desired cargo protein for rapid delivery into cells. Our CPP-adaptor technology not only allows delivery of an extended array of cargoes, but has advantages in speed and ease of use as well as safety enhancements compared to current technologies. In addition, cargoes are released from the adaptors in the cytoplasm and the system and can be produced as a modular kit allowing affinity cargo protein purification using the same tag recognized by the CPP-adaptor. Additional features we seek to develop will allow subcellular delivery based on the properties of different CPPs (e.g. nuclear, mitochondrial or lysosomal localization) and enable simultaneous delivery of multiple cargoes to different compartments.
Public Health Relevance Statement: Public Health Relevance: This project seeks to develop an enabling core technology that will allow practical, convenient and safe manipulation of the internal environments of living cells using cell penetrating peptides attached to adaptor molecules that bind payloads with high affinity and release them in the cell interior. The initial products derived from this technology wll be a range of research tools for biomedical sciences that will enable researchers to study a wide array of processes in the cell interior, including signaling cascades related to diseases such as type 2 diabetes, and to reprogram the developmental and metabolic profiles of cells in culture. We also anticipate the development of diagnostic kits built on the ability to deliver payloads such as fluorescent labeled antibodies to the cell interior. Eventually, therapeutic methods may be constructed that use the technology to reset the metabolic profile of cells in vivo.
NIH Spending Category: Bioengineering; Biotechnology
Project Terms: Adaptor Signaling Protein; Address; Affinity; Affinity Chromatography; Antibodies; Back; base; Binding (Molecular Function); Biotechnology; Calmodulin; Cell membrane; Cells; commercial application; commercialization; Confocal Microscopy; Coupled; Cytoplasm; Destinations; Development; Diagnostic; Diagnostics Research; Disease; EF Hand Motifs; Environment; Eukaryotic Cell; Flow Cytometry; Funding; Future; Genetic Transcription; Germany; Goals; Green Fluorescent Proteins; Immunoblotting; in vivo; Incubators; innovation; instrumentation; Intellectual Property; interest; Kinetics; Label; Letters; Life; macromolecule; Mammalian Cell; Metabolic; Methods; Mitochondria; new technology; Nitric Oxide Synthase Type I; Non-Insulin-Dependent Diabetes Mellitus; novel strategies; Nuclear; Peptides; Performance; Phase; Phosphotransferases; Positioning Attribute; Process; Production; Property; protein expression; protein purification; Proteins; prototype; public health relevance; Reagent; Recombinants; Research; research facility; Research Personnel; Safety; Science; Signal Transduction; Small Business Innovation Research Grant; Specificity; Speed (motion); success; Sweden; System; targeted sequencing; Techniques; Technology; Testing; Therapeutic; Time; tool; Training; Trans-Activators; Transfection; Universities; uptake
