Exosome Engineering and Imaging

There are many new pharmaceuticals being developed for the treatment of various diseases. One of the major issues these drugs face is the ability to enter a cell or the area of treatment. The current methods for drug delivery are often arduous to complete and do not make it to the diseased area because they are targeted by the immune system. In order to improve the ability for drugs to reach the desired area we propose the use of exosomes. Exosomes are subcellular vesicles that are responsible for the transport of biomaterials or signals within the cell and to other cells. The size of exosomes is small, around 30 nanometers is the average size, which minimizes the potential for immune targeting and also allows them to cross the blood brain barrier. In this project we look at the ability to tag, or add a protein, to the surface of an exosome. The fluorescent protein illustrates the location of the exosome within the cell and its potential to be tagged with other proteins to target various types of cells. Engineering exosomes has the potential to solve the current issues with drug delivery

Pseudotyping exosomes for enhanced protein delivery in mammalian cells.

Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential, however, is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here, we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG, we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes, and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane, suggesting that the ectodomain is not required for protein loading. Finally, exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain, confirming a role of the ectodomain in cell tropism. In summary, our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells

Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers

Single nucleotide polymorphism (SNP) discovery and genotyping are essential to genetic mapping. There remains a need for a simple, inexpensive platform that allows high-density SNP discovery and genotyping in large populations. Here we describe the sequencing of restriction-site associated DNA (RAD) tags, which identified more than 13,000 SNPs, and mapped three traits in two model organisms, using less than half the capacity of one Illumina sequencing run. We demonstrated that different marker densities can be attained by choice of restriction enzyme. Furthermore, we developed a barcoding system for sample multiplexing and fine mapped the genetic basis of lateral plate armor loss in threespine stickleback by identifying recombinant breakpoints in F2 individuals. Barcoding also facilitated mapping of a second trait, a reduction of pelvic structure, by in silico re-sorting of individuals. To further demonstrate the ease of the RAD sequencing approach we identified polymorphic markers and mapped an induced mutation in Neurospora crassa. Sequencing of RAD markers is an integrated platform for SNP discovery and genotyping. This approach should be widely applicable to genetic mapping in a variety of organisms

AAV-based dual-reporter circuit for monitoring cell signaling in living human cells

Abstract Background High-throughput methods based on molecular reporters have greatly advanced our knowledge of cell signaling in mammalian cells. However, their ability to monitor various types of cells is markedly limited by the inefficiency of reporter gene delivery. Recombinant adeno-associated virus (AAV) vectors are efficient tools widely used for delivering and expressing transgenes in diverse animal cells in vitro and in vivo. Here we present the design, construction and validation of a novel AAV-based dual-reporter circuit that can be used to monitor and quantify cell signaling in living human cells. Results We first design and construct the AAV-based reporter system. We then validate the versatility and specificity of this system in monitoring and quantifying two important cell signaling pathways, inflammation (NFκB) and cell growth and differentiation (AP-1), in cultured HEK293 and MCF-7 cells. Our results demonstrate that the AAV reporter system is both specific and versatile, and it can be used in two common experimental protocols including transfection with plasmid DNA and transduction with packaged viruses. Importantly, this system is efficient, with a high signal-to-background noise ratio, and can be easily adapted to monitor other common signaling pathways. Conclusions The AAV-based system extends the dual-reporter technology to more cell types, allowing for cost-effective and high throughput applications

Pseudotyping exosomes for enhanced protein delivery in mammalian cells

Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential, however, is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here, we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG, we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes, and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane, suggesting that the ectodomain is not required for protein loading. Finally, exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain, confirming a role of the ectodomain in cell tropism. In summary, our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells

Pseudotyping exosomes for enhanced protein delivery in mammalian cells.

Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential, however, is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here, we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG, we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes, and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane, suggesting that the ectodomain is not required for protein loading. Finally, exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain, confirming a role of the ectodomain in cell tropism. In summary, our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells

Sequential deletion of CD63 identifies topologically distinct scaffolds for surface engineering of exosomes in living human cells

Exosomes are cell-derived extracellular vesicles that have great potential in the field of nano-medicine. However, a fundamental challenge in the engineering of exosomes is the design of biocompatible molecular scaffolds on their surface to enable cell targeting and therapeutic functions. CD63 is a hallmark protein of natural exosomes that is highly enriched on the external surface of the membrane. We have previously described engineering of CD63 for use as a molecular scaffold in order to introduce cell-targeting features to the exosome surface. Despite this initial success, the restrictive M-shaped topology of full-length CD63 may hinder specific applications that require N- or C-terminal display of cell-targeting moieties on the outer surface of the exosome. In this study, we describe new and topologically distinct CD63 scaffolds that enable robust and flexible surface engineering of exosomes. In particular, we conducted sequential deletions of the transmembrane helix of CD63 to generate a series of CD63 truncates, each genetically-fused to a fluorescent protein. Molecular and cellular characterization studies showed truncates of CD63 harboring the transmembrane helix 3 (TM3) correctly targeted and anchored to the exosome membrane and exhibited distinct n-, N-, Ω-, or I-shaped membrane topologies in the exosomal membrane. We further established that these truncates retained robust membrane-anchoring and exosome-targeting activities when stably expressed in the HEK293 cells. Moreover, HEK293 cells produced engineered exosomes in similar quantities to cells expressing full-length CD63. Based on the results of our systematic sequential deletion studies, we propose a model to understand molecular mechanisms that underlie membrane-anchoring and exosome targeting features of CD63. In summary, we have established new and topologically distinct scaffolds based on engineering of CD63 that enables flexible engineering of the exosome surface for applications in disease-targeted drug delivery and therapy