Project Summary We propose to develop hybrid exosomes combining synthetic lipid-based nanoparticles with defined exosome cargo to recreate the properties and biological functions of the native exosome but with the added benefit of increased delivery efficiency and controlled therapeutic encapsulate. The rationale is that only some components in the native exosomes are required for their proper functioning in receiving cells. We hypothesize that generating hybrid exosomes loaded with only the active components would increase their biological functioning, e.g. promoting axonal growth. Further, the synthetic lipid nanoparticle, with higher intracellular delivery efficiency than the native exosomes, would enhance the therapeutic delivery and promote the tissue regeneration. To accomplish the objective of this application, we propose the following two Specific Aims: Specific Aim 1. Engineering hybrid exosomes derived from SCs to promote axonal growth. The goal is to study the capability of the hybrid lipid-based nanoparticles for simultaneously delivery of multiple different cargos, including proteins and RNAs. We will isolate the interior components from SC-derived exosomes, which can promote axonal growth of DRG, and load them into the synthetic nanoparticle formulation. Cargo delivery of such engineered hybrid exosomes will be evaluated on efficacy to induce DRG axonal growth in vitro. The success of this aim will provide a nanoparticle formulation that enables the efficient delivery of multiple cargos simultaneously. From this Aim, we expect to i) successfully identify the synthetic nanoparticle formulation for multiple cargo delivery and ii) an effective formulation to promote axonal growth that is useful for peripheral nerve regeneration. Specific Aim 2. Profiling the components of exosomes and identifying the factors that promote neurite outgrowth. The goal is to profile the components in the exosomes, including proteins and RNAs. The protein and RNA components will be isolated and profiled using LC-MS and gene sequencing respectively. The identified individual component or combinations will be loaded into the synthetic lipid nanoparticles to evaluate their biological function, e.g. in promoting axonal growth in DRG. From this Aim, we expect to i) successfully profile the protein and RNA content in the SC-derived exosomes and ii) identify the functional miRNA, mRNA, proteins or their combinations that promote the neuronal growth. These results will provide useful guidance for generation of synthetic exosomes with defined cargos.