Nanodelivery of a functional membrane receptor to manipulate cellular phenotype.

Modification of membrane receptor makeup is one of the most efficient ways to control input-output signals but is usually achieved by expressing DNA or RNA-encoded proteins or by using other genome-editing methods, which can be technically challenging and produce unwanted side effects. Here we develop and validate a nanodelivery approach to transfer in vitro synthesized, functional membrane receptors into the plasma membrane of living cells. Using β2-adrenergic receptor (β2AR), a prototypical G-protein coupled receptor, as an example, we demonstrated efficient incorporation of a full-length β2AR into a variety of mammalian cells, which imparts pharmacologic control over cellular signaling and affects cellular phenotype in an ex-vivo wound-healing model. Our approach for nanodelivery of functional membrane receptors expands the current toolkit for DNA and RNA-free manipulation of cellular function. We expect this approach to be readily applicable to the synthesis and nanodelivery of other types of GPCRs and membrane receptors, opening new doors for therapeutic development at the intersection between synthetic biology and nanomedicine

A Noninvasive, Orally Stable, Mucosa-Penetrating Polyvalent Vaccine Platform Based on Hepatitis E Virus Nanoparticle

Hepatitis E virus nanoparticle (HEVNP) is an orally stable, mucosa-penetrating delivery platform for noninvasive, targeted delivery of therapeutic and diagnostic agents. HEVNP does not carry HEV genomic RNA and is incapable of replication. The key characteristics that make HEVNP an ideal and unique vehicle for diagnostic and therapeutic delivery include surface plasticity, resistance to the harsh environment of the gastrointestinal (GI) tract, significant payload capacity, platform sustainability, and safety. Furthermore, HEVNP is easily produced using currently available expression/purification technologies; can be easily formulated as a liquid, powder, or solid; and can be distributed (and stored) without the need for a temperature-controlled supply chain

Structural Insight into CVB3-VLP Non-Adjuvanted Vaccine

Coxsackievirus B (CVB) enteroviruses are common pathogens that can cause acute and chronic myocarditis, dilated cardiomyopathy, aseptic meningitis, and they are hypothesized to be a causal factor in type 1 diabetes. The licensed enterovirus vaccines and those currently in clinical development are traditional inactivated or live attenuated vaccines. Even though these vaccines work well in the prevention of enterovirus diseases, new vaccine technologies, like virus-like particles (VLPs), can offer important advantages in the manufacturing and epitope engineering. We have previously produced VLPs for CVB3 and CVB1 in insect cells. Here, we describe the production of CVB3-VLPs with enhanced production yield and purity using an improved purification method consisting of tangential flow filtration and ion exchange chromatography, which is compatible with industrial scale production. We also resolved the CVB3-VLP structure by Cryo-Electron Microscopy imaging and single particle reconstruction. The VLP diameter is 30.9 nm on average, and it is similar to Coxsackievirus A VLPs and the expanded enterovirus cell-entry intermediate (the 135s particle), which is similar to 2 nm larger than the mature virion. High neutralizing and total IgG antibody levels, the latter being a predominantly Th2 type (IgG1) phenotype, were detected in C57BL/6J mice immunized with non-adjuvanted CVB3-VLP vaccine. The structural and immunogenic data presented here indicate the potential of this improved methodology to produce highly immunogenic enterovirus VLP-vaccines in the future.Peer reviewe

The F1 loop of the talin head domain acts as a gatekeeper in integrin activation and clustering

Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the beta integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn2+. Here we show that kindlin-1 can replace Mn2+ to mediate beta 3 integrin clustering induced by the talin head, but not that induced by the F2-F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for beta 3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of alpha- and beta 3-integrins, in order to activate the beta 3 integrin heterodimer, further detailing the mechanism by which the talin-kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the beta 3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer. This article has an associated First Person interview with the first author of the paper.Peer reviewe

Protein-based nanoplatform for detection of tumorigenic polyps in the colon via noninvasive mucosal routes

The use of nanoparticulate systems to diagnose and treat tumors has gained momentum with the rapid development of nanomedicine. Many nanotheranostics fail due to insufficient bioavailability and low accumulation at the tumor site, resulting in undesirable side effects. We describe the use of an engineered hepatitis E viral nanoparticle (HEVNP) with enhanced bioavailability, tissue retention and mucosal penetration capacities. HEVNP is a modular nanocapsule that can encapsulate heterologous nucleotides, proteins and inorganic metals, such as ferrite oxide nanoparticles. Additionally, the exterior protruding arms of HEVNP is composed of loops that are used for chemical coupling of targeting and therapeutic peptides. We propose the use of HEVNP to target colorectal cancer (i.e., polyps) with imaging-guided delivery using colonoscopy

Structural characterization of site-modified nanocapsid with monodispersed gold clusters

Hepatitis E Virus-like particles self-assemble in to noninfectious nanocapsids that are resistant to proteolytic/acidic mucosal delivery conditions. Previously, the nanocapsid was engineered to specifcally bind and enter breast cancer cells, where successful tumor targeting was demonstrated in animal models. In the present study, the nanocapsid surface was modifed with a solvent-exposed cysteine to conjugate monolayer protected gold nanoclusters (AuNC). Unlike commercially available gold nanoparticles, AuNCs monodisperse in water and are composed of a discrete number of gold atoms, forming a crystalline gold core. Au102pMBA44 (Au102) was an ideal conjugate given its small 2.5nm size and detectability in cryoEM. Au102 was bound directly to nanocapsid surface cysteines via direct ligand exchange. In addition, Au102 was functionalized with a maleimide linker (Au102_C6MI) for maleimide-thiol conjugation to nanocapsid cysteines. The AuNC-bound nanocapsid constructs were conjugated in various conditions. We found Au102_C6MI to bind nanocapsid more efciently, while Au102 remained more soluble over time. Nanocapsids conjugated to Au102_C6MI were imaged in cryoEM for single particle reconstruction to localize AuNC position on the nanocapsid surface. We resolved fve unique high intensity volumes that formed a ring-shaped density at the 5-fold symmetry center. This fnding was further supported by independent rigid modeling.peerReviewe

Structural characterization of site-modified nanocapsid with monodispersed gold clusters.

Hepatitis E Virus-like particles self-assemble in to noninfectious nanocapsids that are resistant to proteolytic/acidic mucosal delivery conditions. Previously, the nanocapsid was engineered to specifically bind and enter breast cancer cells, where successful tumor targeting was demonstrated in animal models. In the present study, the nanocapsid surface was modified with a solvent-exposed cysteine to conjugate monolayer protected gold nanoclusters (AuNC). Unlike commercially available gold nanoparticles, AuNCs monodisperse in water and are composed of a discrete number of gold atoms, forming a crystalline gold core. Au102 pMBA44 (Au102) was an ideal conjugate given its small 2.5 nm size and detectability in cryoEM. Au102 was bound directly to nanocapsid surface cysteines via direct ligand exchange. In addition, Au102 was functionalized with a maleimide linker (Au102_C6MI) for maleimide-thiol conjugation to nanocapsid cysteines. The AuNC-bound nanocapsid constructs were conjugated in various conditions. We found Au102_C6MI to bind nanocapsid more efficiently, while Au102 remained more soluble over time. Nanocapsids conjugated to Au102_C6MI were imaged in cryoEM for single particle reconstruction to localize AuNC position on the nanocapsid surface. We resolved five unique high intensity volumes that formed a ring-shaped density at the 5-fold symmetry center. This finding was further supported by independent rigid modeling

Structural Insight into CVB3-VLP Non-Adjuvanted Vaccine

Coxsackievirus B (CVB) enteroviruses are common pathogens that can cause acute and chronic myocarditis, dilated cardiomyopathy, aseptic meningitis, and they are hypothesized to be a causal factor in type 1 diabetes. The licensed enterovirus vaccines and those currently in clinical development are traditional inactivated or live attenuated vaccines. Even though these vaccines work well in the prevention of enterovirus diseases, new vaccine technologies, like virus-like particles (VLPs), can offer important advantages in the manufacturing and epitope engineering. We have previously produced VLPs for CVB3 and CVB1 in insect cells. Here, we describe the production of CVB3-VLPs with enhanced production yield and purity using an improved purification method consisting of tangential flow filtration and ion exchange chromatography, which is compatible with industrial scale production. We also resolved the CVB3-VLP structure by Cryo-Electron Microscopy imaging and single particle reconstruction. The VLP diameter is 30.9 nm on average, and it is similar to Coxsackievirus A VLPs and the expanded enterovirus cell-entry intermediate (the 135s particle), which is ~2 nm larger than the mature virion. High neutralizing and total IgG antibody levels, the latter being a predominantly Th2 type (IgG1) phenotype, were detected in C57BL/6J mice immunized with non-adjuvanted CVB3-VLP vaccine. The structural and immunogenic data presented here indicate the potential of this improved methodology to produce highly immunogenic enterovirus VLP-vaccines in the future

Positron emission tomography imaging of novel AAV capsids maps rapid brain accumulation.

Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid (ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-specific AAV capsids (e.g. PHP.eB) have been shown to enhance brain delivery in rodents via the LY6A receptor on brain endothelial cells. Here, we create a non-invasive positron emission tomography (PET) methodology to track viruses. To provide the sensitivity required to track AAVs injected at picomolar levels, a unique multichelator construct labeled with a positron emitter (Cu-64, t1/2 = 12.7 h) is coupled to the viral capsid. We find that brain accumulation of the PHP.eB capsid 1) exceeds that reported in any previous PET study of brain uptake of targeted therapies and 2) is correlated with optical reporter gene transduction of the brain. The PHP.eB capsid brain endothelial receptor affinity is nearly 20-fold greater than that of AAV9. The results suggest that novel PET imaging techniques can be applied to inform and optimize capsid design