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Extracellular vesicles (EVs) are promising tools to transfer macromolecular therapeutic molecules to recipient cells, however, efficient functional intracellular protein delivery by EVs remains challenging. Here, we have developed novel and versatile systems that leverage selected molecular tools to engineer EVs for robust cytosolic protein delivery bothin vitroandin vivo. These systems, termed VSV-G plus EV-sorting Domain-Intein-Cargo (VEDIC) and VSV-G-Foldon-Intein-Cargo (VFIC), exploit an engineered mini-intein (intein) protein with self-cleavage activity to link cargo to an EV-sorting domain and release it from the EV membrane inside the EV lumen. In addition, we utilize the fusogenic protein VSV-G to facilitate endosomal escape and cargo release from the endosomal system to the cytosol of recipient cells. Importantly, we demonstrate that the combination of the self-cleavage intein, fusogenic protein and EV-sorting domain are indispensable for efficient functional intracellular delivery of cargo proteins by engineered EVs. As such, nearly 100% recombination and close to 80% genome editing efficiency in reporter cells were observed by EV-transferred Cre recombinase and Cas9/sgRNA RNPs, respectively. Moreover, EV-mediated Cre delivery by VEDIC or VFIC engineered EVs resulted in significantin vivorecombination in Cre-LoxP R26-LSL-tdTomato reporter mice following both local and systemic injections. Finally, we applied these systems for improved treatment of LPS-induced systemic inflammation by delivering a super-repressor of NF-ĸB activity. Altogether, this study describes a platform by which EVs can be utilized as a vehicle for the efficient intracellular delivery of macromolecular therapeutics for treatments of disease.Abstract FigureGraphic summary: Development of VEDIC and VFIC systems for high-efficiency intracellular protein delivery in vitro and in vivo.Intein in tripartite fusion protein (EV-sorting Domain-Intein-Cargo) performs C-terminal cleavage during the process of EV-biogenesis, resulting in enriched free cargo proteins inside of vesicles. Together with fusogenic protein, VSV-G, these engineered EVs achieve high-efficiency intracellular delivery of cargo protein (Cre and super repressor of NF-ĸB) or protein complex (Cas9/sgRNA RNPs) both in reporter cells and in mice models.

Messenger RNA (mRNA) has emerged as an attractive therapeutic molecule for a range of clinical applications. Forin vivofunctionality, mRNA therapeutics require encapsulation into effective, stable, and safe delivery systems to protect the cargo from degradation and reduce immunogenicity. Here, we developed a bioengineering platform for efficient mRNA loading and functional delivery using naturally-derived nanoparticles, Extracellular Vesicles (EVs). By expressing the highly specific PUFe RNA-binding domain fused to CD63 in EV producer cells stably expressing the target mRNA, our system exceeds mRNA loading efficiencies by up to 4.5-fold over previously reported EV-based approaches. By combining with an mRNA-stabilizing protein, PABPc, and a fusogenic endosomal escape moiety, VSVg, functional mRNA deliveryin vivois achieved at doses substantially lower than clinically used lipid nanoparticles. Our technology solves substantial drawbacks currently associated with EV-based nucleic acid delivery systems and could enable new applications for mRNA therapeutics.

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.

Extracellular vesicles (EVs) have recently emerged as a highly promising cell-free bio-therapeutics. While a range of engineering strategies have been developed to functionalize the EV surface, current approaches fail to address the limitations associated with endogenous surface display, pertaining to the heterogeneous display of commonly used EV-loading moieties among different EV subpopulations. Here we present a novel engineering platform to display multiple protein therapeutics simultaneously on the EV surface. As proof-of-concept, we screened multiple endogenous display strategies for decorating the EV surface with cytokine binding domains derived from tumor necrosis factor receptor 1 (TNFR1) and interleukin 6 signal transducer (IL6ST), which can act as decoys for the pro-inflammatory cytokines TNFα and IL6, respectively. Combining synthetic biology and systematic screening of loading moieties, resulted in a three-component system which increased the display and decoy activity of TNFR1 and IL6ST, respectively. Further, this system allowed for combinatorial functionalization of two different receptors on the same EV surface. These cytokine decoy EVs significantly ameliorated disease phenotypes in three different inflammatory mouse models for systemic inflammation, neuroinflammation, and intestinal inflammation. Importantly, significantly improvedin vitroandin vivoefficacy of these engineered EVs was observed when compared directly to clinically approved biologics targeting the IL6 and TNFα pathways.

Extracellular vesicles (EV) convey biological information by transmitting macromolecules between cells and tissues and are of great promise as pharmaceutical nanocarriers, and as therapeutic per se. Strategies for customizing the EV surface and cargo are being developed to enable their tracking, visualization, loading with pharmaceutical agents and decoration of the surface with tissue targeting ligands. While much progress has been made in the engineering of EVs, an exhaustive comparative analysis of the most commonly exploited EV-associated proteins, as well as a quantification at the molecular level are lacking. Here, we selected 12 EV-related proteins based on MS-proteomics data for comparative quantification of their EV engineering potential. All proteins were expressed with fluorescent protein (FP) tags in EV-producing cells; both parent cells as well as the recovered vesicles were characterized biochemically and biophysically. Using Fluorescence Correlation Spectroscopy (FCS) we quantified the number of FP-tagged molecules per vesicle. We observed different loading efficiencies and specificities for the different proteins into EVs. For the candidates showing the highest loading efficiency in terms of engineering, the molecular levels in the vesicles did not exceed ca 40–60 fluorescent proteins per vesicle upon transient overexpression in the cells. Some of the GFP-tagged EV reporters showed quenched fluorescence and were either non-vesicular, despite co-purification with EVs, or comprised a significant fraction of truncated GFP. The co-expression of each target protein with CD63 was further quantified by widefield and confocal imaging of single vesicles after double transfection of parent cells. In summary, we provide a quantitative comparison for the most commonly used sorting proteins for bioengineering of EVs and introduce a set of biophysical techniques for straightforward quantitative and qualitative characterization of fluorescent EVs to link single vesicle analysis with single molecule quantification. CA extern

Heusermann et al. use a single-vesicle dye-tracing analysis in live cells showing that exosomes enter cells as intact vesicles, primarily at filopodia-active regions, and sort into endocytic vesicle circuits that are targeted to scan the ER before being directed to lysosomes., Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.

Synthetic RNAi activators have shown considerable potential for therapeutic application to silencing of pathology-causing genes. Typically these exogenous RNAi activators comprise duplex RNA of approximately 21 bp with 2 nt overhangs at the 3' ends. To improve efficacy of siRNAs, chemical modification at the 2'-OH group of ribose has been employed. Enhanced stability, gene silencing and attenuated immunostimulation have been demonstrated using this approach. Although promising, efficient and controlled delivery of highly negatively charged nucleic acid gene silencers remains problematic. To assess the potential utility of introducing positively charged groups at the 2' position, our investigations aimed at assessing efficacy of novel siRNAs containing 2'-O-guanidinopropyl (GP) moieties. We describe the formation of all four GP-modified nucleosides using the synthesis sequence of Michael addition with acrylonitrile followed by Raney-Ni reduction and guanidinylation. These precursors were used successfully to generate antihepatitis B virus (HBV) siRNAs. Testing in a cell culture model of viral replication demonstrated that the GP modifications improved silencing. Moreover, thermodynamic stability was not affected by the GP moieties and their introduction into each position of the seed region of the siRNA guide strand did not alter the silencing efficacy of the intended HBV target. These results demonstrate that modification of siRNAs with GP groups confers properties that may be useful for advancing therapeutic application of synthetic RNAi activators.

Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the world's population and carriers of the virus are at risk for complicating hepatocellular carcinoma. Current treatment options have limited efficacy and chronic HBV infection is likely to remain a significant global medical problem for many years to come. Silencing HBV gene expression by harnessing RNA interference (RNAi) presents an attractive option for development of novel and effective anti HBV agents. However, despite significant and rapid progress, further refinement of existing technologies is necessary before clinical application of RNAi-based HBV therapies is realized. Limiting off target effects, improvement of delivery efficiency, dose regulation and preventing reactivation of viral replication are some of the hurdles that need to be overcome. To address this, we assessed the usefulness of the recently described class of altritol-containing synthetic siRNAs (ANA siRNAs), which were administered as lipoplexes and tested in vivo in a stringent HBV transgenic mouse model. Our observations show that ANA siRNAs are capable of silencing of HBV replication in vivo. Importantly, non specific immunostimulation was observed with unmodified siRNAs and this undesirable effect was significantly attenuated by ANA modification. Inhibition of HBV replication of approximately 50% was achieved without evidence for induction of toxicity. These results augur well for future application of ANA siRNA therapeutic lipoplexes.

To advance the use of cationic lipids for non-viral nucleic acid vector formulation, a panel of novel nitrogen heterocycle cholesteryl derivatives containing a biodegradable carbamate linker was synthesised. Optimally acting piperazine and cyclen compounds had nucleic acid-binding and lipoplex nanoparticle formation properties that were suitable for their use as non-viral vectors. It was found that the lipoplexes formed were capable of efficient non-toxic nucleic acid delivery to cells in culture. The chemical structure of individual cationic lipids, which is likely to influence lipoplex formation, affected efficiency of DNA or RNA transfection. The results indicated that the cyclen containing compound possessing two cholesteryl moieties resulted in efficient siRNA-mediated target gene silencing but was a poor reagent for DNA transfection.

Chronic HBV infection remains an important public health problem and currently licensed therapies rarely prevent complications of viral persistence. Silencing HBV gene expression using gene therapy, particularly with exogenous activators of RNAi, holds promise for developing an HBV gene therapy. However, immune stimulation, off-targeting effects and inefficient delivery of RNAi activators remain problematic. Several new approaches have recently been employed to address these issues. Chemical modifications to anti-HBV synthetic siRNAs have been investigated and a variety of vectors are being developed for delivery of RNAi effectors. In this article, we review the potential utility of gene therapy for treating HBV infection.

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT). Fractionation and membrane co-immune precipitations further confirm that siRNA-loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC-associated double-stranded siRNA, diagnostic of RISC loading, and RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA-independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA-mediated RNA silencing., The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing This detailed characterization of membrane association in siRNA-mediated gene silencing establishes the rough ER as major site for canonical RISC loading and target RNA cleavage.