Quantifying mutant huntingtin protein in human cerebrospinal fluid to support the development of huntingtin-lowering therapies.

Huntington's disease (HD) is caused by a cytosine adenine guanine-repeat expansion in the huntingtin gene. This results in the production of toxic mutant huntingtin protein (mHTT), which has an elongated polyglutamine (polyQ) stretch near the protein's N-terminal end. The pharmacological lowering of mHTT expression in the brain targets the underlying driver of HD and is one of the principal therapeutic strategies being pursued to slow or stop disease progression. This report describes the characterisation and validation of an assay designed to quantify mHTT in the cerebrospinal fluid of individuals with HD, for use in registrational clinical trials. The assay was optimised, and its performance was characterised with recombinant huntingtin protein (HTT) varying in overall and polyQ-repeat length. The assay was successfully validated by two independent laboratories in regulated bioanalytical environments and showed a steep signal increase as the polyQ stretch of recombinant HTTs pivoted from wild-type to mutant protein forms. Linear mixed effects modelling confirmed highly parallel concentration-response curves for HTTs, with only a minor impact of individual slopes of the concentration-response for different HTTs (typically < 5% of the overall slope). This implies an equivalent quantitative signal behaviour for HTTs with differing polyQ-repeat lengths. The reported method may be a reliable biomarker tool with relevance across the spectrum of HD mutations, which can facilitate the clinical development of HTT-lowering therapies in HD.

Single-Point Mutations in Qß Virus-like Particles Change Binding to Cells.

Virus-like particles (VLPs) constitute large, polyvalent platforms onto which a wide variety of functional units can be grafted. Their use in biological settings often depends on their specific binding to cells or receptors of interest; this can be compromised by excessive nonspecific association with other cells. We found that lysine residues mediate such nonspecific interactions, presumably by virtue of protonation and interaction with anionic membrane lipid headgroups and/or complementary residues of cell surface proteins and polysaccharides. Chemical acylation of surface-exposed amines of the Qß VLP led to a significant reduction in the association of particles with mammalian cells. Single-point mutations of particular lysine residues to either glutamine, glutamic acid, tryptophan, or phenylalanine were mostly well-tolerated and formed intact capsids, but the introduction of double and triple mutants was far less forgiving. Introduction of glutamic acid at position 13 (K13E) led to a dramatic increase in cellular binding, whereas removal of the lysine at position 46 (K46Q) led to an equally striking reduction. Several plasma membrane components were found to specifically interact with the Qß capsid irrespective of surface charge. These results suggest that specific cellular interactions are engaged or obviated by such mutations and provide us with more "benign" particles to which can be added binding functionality for targeted delivery applications.

Pharmacodynamic Monitoring of RO5459072, a Small Molecule Inhibitor of Cathepsin S.

Major histocompatibility complex class II (MHCII)-restricted antigen priming of CD4+ T cells is both involved in adaptive immune responses and the pathogenesis of autoimmune diseases. Degradation of invariant chain Ii, a protein that prevents premature peptide loading, is a prerequisite for nascent MHCII-peptide complex formation. A key proteolytic step in this process is mediated by cathepsin S. Inhibition of this cysteine protease is known to result in the intracellular accumulation of Lip10 in B cells. Here, we describe the development and application of a neoepitope-based flow cytometry assay measuring accumulation of Lip10. This novel method enabled the investigation of cathepsin S-dependent MHCII maturation in professional antigen-presenting cell (APC) subsets. Inhibition of cathepsin S by a specific inhibitor, RO5459072, in human PBMC ex vivo resulted in accumulation of Lip10 in B cells and myeloid dendritic cells, but not in plasmacytoid dendritic cells and only to a minor degree in monocytes. We qualified Lip10 as a pharmacodynamic biomarker by showing the cathepsin S inhibitor-dependent accumulation of Lip10 in vivo in cynomolgus monkeys treated with RO5459072. Finally, dosing of RO5459072 in a first-in-human clinical study (www.ClinicalTrials.gov, identifier NCT02295332) exhibited a dose-dependent increase in Lip10, confirming target engagement and demonstrating desired pharmacologic inhibition in vivo. The degree of cathepsin S antagonist-induced maximum Lip10 accumulation in APCs varied significantly between individuals both in vitro and in vivo. This finding has not been reported previously using alternative, less sensitive methods and demands further investigation as to the potential of this biomarker to predict response to treatment. These results will help guide subsequent clinical studies investigating the pharmacokinetic and pharmacodynamic relationship of cathepsin S inhibitor RO5459072 after multiple dosing.

Metabolomics: Strategies to Define the Role of Metabolism in Virus Infection and Pathogenesis.

Metabolomics is an analytical profiling technique for measuring and comparing large numbers of metabolites present in biological samples. Combining high-throughput analytical chemistry and multivariate data analysis, metabolomics offers a window on metabolic mechanisms. Because they intimately utilize and often rewire host metabolism, viruses are an excellent choice to study by metabolomics techniques. Studies of the effects of viruses on metabolism during replication in vitro and infection in animal models or human subjects have provided novel insights into these networks and provided new targets for therapy and biomarker development. Identifying the common metabolic pathways utilized by viruses has the potential to reveal those that can be targeted by broad-spectrum antiviral and vaccine approaches.

Alterations in Spinal Cord Metabolism during Treatment of Neuropathic Pain.

Therapeutic options for neuropathic pain have improved over the last 20 years yet still only provide partial relief with numerous side effects. Recently, metabolomics revealed that the concentration of the endogenous metabolite N,N-dimethylsphingosine (DMS) is increased in the spinal cord in a model of neuropathic pain. Additionally, it was shown that introduction of DMS to the central nervous system (CNS) resulted in mechanical allodynia. Here, we have examined two compounds; pregabalin (Lyrica®), a drug used to treat neuropathic pain, and N-oleoylethanolamine (NOE), an endogenous endocannabinoid-like compound that is known to affect multiple lipid pathways. We found that the concentration of DMS in the spinal cord was not significantly altered upon pregabalin treatment of rats suffering from neuropathic pain. We further explored whether modulating lipid metabolism may impact neuropathic pain by testing NOE as a potential novel therapeutic.

Virus-Based Nanoparticles as Versatile Nanomachines.

Nanoscale engineering is revolutionizing the way we prevent, detect, and treat diseases. Viruses have played a special role in these developments because they can function as prefabricated nanoscaffolds that have unique properties and are easily modified. The interiors of virus particles can encapsulate and protect sensitive compounds, while the exteriors can be altered to display large and small molecules in precisely defined arrays. These properties of viruses, along with their innate biocompatibility, have led to their development as actively targeted drug delivery systems that expand on and improve current pharmaceutical options. Viruses are naturally immunogenic, and antigens displayed on their surface have been used to create vaccines against pathogens and to break self-tolerance to initiate an immune response to dysfunctional proteins. Densely and specifically aligned imaging agents on viruses have allowed for high-resolution and noninvasive visualization tools to detect and treat diseases earlier than previously possible. These and future applications of viruses have created an exciting new field within the disciplines of both nanotechnology and medicine.

Encapsidated atom-transfer radical polymerization in Qß virus-like nanoparticles.

Virus-like particles (VLPs) are unique macromolecular structures that hold great promise in biomedical and biomaterial applications. The interior of the 30 nm-diameter Qß VLP was functionalized by a three-step process: (1) hydrolytic removal of endogenously packaged RNA, (2) covalent attachment of initiator molecules to unnatural amino acid residues located on the interior capsid surface, and (3) atom-transfer radical polymerization of tertiary amine-bearing methacrylate monomers. The resulting polymer-containing particles were moderately expanded in size; however, biotin-derivatized polymer strands were only very weakly accessible to avidin, suggesting that most of the polymer was confined within the protein shell. The polymer-containing particles were also found to exhibit physical and chemical properties characteristic of positively charged nanostructures, including the ability to easily enter mammalian cells and deliver functional small interfering RNA.

Lysine addressability and mammalian cell interactions of bacteriophage λ procapsids.

Chemically or genetically modified virus particles, termed viral nanoparticles (VNPs), are being explored in applications such as drug delivery, vaccine development, and materials science. Each virus platform has inherent properties and advantages based on its structure, molecular composition, and biomolecular interactions. Bacteriophage λ was studied for its lysine addressability, stability, cellular uptake, and the ability to modify its cellular uptake. λ procapsids could be labeled primarily at a single residue on the gpE capsid protein as determined by tandem mass spectrometry, providing a unique attachment site for further capsid modification. Bioconjugation of transferrin to the procapsids mediated specific interaction with transferrin receptor-expressing cells. These studies demonstrate the utility of bacteriophage λ procapsids and their potential use as targeted drug delivery vehicles.

Readily accessible fluorescent probes for sensitive biological imaging of hydrogen peroxide.

Hydrogen peroxide is a major component of oxygen metabolism in biological systems that, when present in high concentrations, can lead to oxidative stress in cells. Noninvasive molecular imaging of H(2)O(2) using fluorogenic systems represents an effective way to detect and measure the accumulation of this metabolite. Herein, we detail the development of robust H(2)O(2)-sensitive fluorescent probes using a boronic ester trigger appended to the fluorophore through a benzyl ether linkage. A major advantage of the probes presented here is their synthetic accessibility, with only one step needed to generate the probes on the gram scale. The sensitivity of the probes was evaluated in simulated physiological conditions, showing micromolar sensitivity to H(2)O(2). The probes were tested in biological model systems, demonstrating effective imaging of unstimulated, endogenous H(2)O(2) levels in RAW 264.7 cells and murine brain tissue.

Endocytic uptake pathways utilized by CPMV nanoparticles.

Cowpea mosaic virus (CPMV) has been used as a nanoparticle platform for biomedical applications including vaccine development, in vivo vascular imaging, and tissue-targeted delivery. A better understanding of the mechanisms of CPMV targeting and cell internalization would enable enhanced targeting and more effective delivery. Previous studies showed that, following binding and internalization by mammalian cells, CPMV localizes in a perinuclear late-endosome compartment where it remains for as long as several days. To further investigate endocytic trafficking of CPMV within the cell, we used multiple approaches including pharmacologic inhibition of pathways and colocalization with endocytic vesicle compartments. CPMV internalization was clathrin-independent and utilized a combination of caveolar endocytosis and macropinocytosis pathways for entry. CPMV particles colocalized with Rab5(+) early endosomes to traffic ultimately to a lysosomal compartment. These studies facilitate the further development of effective intracellular drug-delivery strategies using CPMV.

A view from above: cloud plots to visualize global metabolomic data.

Global metabolomics describes the comprehensive analysis of small molecules in a biological system without bias. With mass spectrometry-based methods, global metabolomic data sets typically comprise thousands of peaks, each of which is associated with a mass-to-charge ratio, retention time, fold change, p-value, and relative intensity. Although several visualization schemes have been used for metabolomic data, most commonly used representations exclude important data dimensions and therefore limit interpretation of global data sets. Given that metabolite identification through tandem mass spectrometry data acquisition is a time-limiting step of the untargeted metabolomic workflow, simultaneous visualization of these parameters from large sets of data could facilitate compound identification and data interpretation. Here, we present such a visualization scheme of global metabolomic data using a so-called "cloud plot" to represent multidimensional data from septic mice. While much attention has been dedicated to lipid compounds as potential biomarkers for sepsis, the cloud plot shows that alterations in hydrophilic metabolites may provide an early signature of the disease prior to the onset of clinical symptoms. The cloud plot is an effective representation of global mass spectrometry-based metabolomic data, and we describe how to extract it as standard output from our XCMS metabolomic software.

Localization of gadolinium-loaded CPMV to sites of inflammation during central nervous system autoimmunity.

Contrast-enhanced magnetic resonance imaging (MRI) allows rapid non-invasive diagnosis of central nervous system (CNS) pathologies such as multiple sclerosis (MS). Current gadolinium-based contrast agents must be administered at high doses, are excreted by the kidney, and some formulations are associated with toxicity in patients with renal insufficiency. The development of nanoparticle carriers for targeted delivery of gadolinium to sites of disease would increase specificity, as well as decrease the dose of gadolinium required to obtain sufficient contrast for disease diagnosis. The plant virus, cowpea mosaic virus (CPMV), is a biocompatible nanoparticle platform for imaging applications. Gadolinium is rapidly incorporated into the interior of the CPMV capsid without disrupting particle integrity, and CPMV-Gd particles have relaxivity comparable to gadolinium chelates used clinically. Here we examine the ability of gadolinium-loaded CPMV particles (CPMV-Gd) to localize to lesions in the CNS in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). The in vivo distribution of gadolinium-loaded CPMV (CPMV-Gd) was examined within the periphery and central nervous system (CNS). CPMV accumulated in inflammatory lesions within the brain and spinal cord, and specifically associated with CD11b+ and CD11c+ cells. These results demonstrate that CPMV is an attractive nanoparticle chelate for gadolinium for in vivo applications and may have clinical utility as a contrast agent for the detection of autoimmune demyelinating diseases of the CNS.

Differential uptake of chemically modified cowpea mosaic virus nanoparticles in macrophage subpopulations present in inflammatory and tumor microenvironments.

There remains a tremendous need to develop targeted therapeutics that can both image and localize the toxic effects of chemotherapeutics and antagonists on diseased tissue while reducing adverse systemic effects. These needs have fostered the development of a nanotechnology-based approach that can combine targeting and toxicity potential. In this study, CPMV nanoparticles were chemically modified with the dye Alexa Flour 488 and were also tandemly modified with PEG1000 followed by AF488; and the derivatized nanoparticles were subsequently added to macrophages stimulated with either LPS (M1) or IL-4 (M2). Previously published studies have shown that M1/M2 macrophages are both present in an inflammatory microenvironment (such as a tumor microenvironment and atherosclerosis) and play opposing yet balancing roles; M2 macrophages have a delayed and progressive onset in the tumor microenvironment (concomitant with an immunosuppression of M1 macrophages). In this study, we show higher uptake of CPMV-AF488 and CPMV-PEG-AF488 by M2 macrophages compared to M1 macrophages. M1 macrophages showed no uptake of CPMV-PEG-AF488. More specifically, M2 macrophages are known to be up-regulated in early atherosclerosis plaque. Indeed, previous work showed that M2 macrophages in plaque also correlate with CPMV internalization. These studies emphasize the potential effectiveness of CPMV as a tailored vehicle for targeting tumor macrophages involved in cancer metastasis or vascular inflammation and further highlight the potential of CPMV in targeted therapeutics against other diseases.

Guiding plant virus particles to integrin-displaying cells.

Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors.

Delayed toxicity associated with soluble anthrax toxin receptor decoy-Ig fusion protein treatment.

Soluble receptor decoy inhibitors, including receptor-immunogloubulin (Ig) fusion proteins, have shown promise as candidate anthrax toxin therapeutics. These agents act by binding to the receptor-interaction site on the protective antigen (PA) toxin subunit, thereby blocking toxin binding to cell surface receptors. Here we have made the surprising observation that co-administration of receptor decoy-Ig fusion proteins significantly delayed, but did not protect, rats challenged with anthrax lethal toxin. The delayed toxicity was associated with the in vivo assembly of a long-lived complex comprised of anthrax lethal toxin and the receptor decoy-Ig inhibitor. Intoxication in this system presumably results from the slow dissociation of the toxin complex from the inhibitor following their prolonged circulation. We conclude that while receptor decoy-Ig proteins represent promising candidates for the early treatment of B. anthracis infection, they may not be suitable for therapeutic use at later stages when fatal levels of toxin have already accumulated in the bloodstream.

Interaction of cowpea mosaic virus nanoparticles with surface vimentin and inflammatory cells in atherosclerotic lesions.

AIMS: Detection of atherosclerosis has generally been limited to the late stages of development, after cardiovascular symptoms present or a clinical event occurs. One possibility for early detection is the use of functionalized nanoparticles. The aim of this study was the early imaging of atherosclerosis using nanoparticles with a natural affinity for inflammatory cells in the lesion. MATERIALS & METHODS: We investigated uptake of cowpea mosaic virus by macrophages and foam cells in vitro and correlated this with vimentin expression. We also examined the ability of cowpea mosaic virus to interact with atherosclerotic lesions in a murine model of atherosclerosis. RESULTS & CONCLUSION: We found that uptake of cowpea mosaic virus is increased in areas of atherosclerotic lesion. This correlated with increased surface vimentin in the lesion compared with nonlesion vasculature. In conclusion, cowpea mosaic virus and its vimentin-binding region holds potential for use as a targeting ligand for early atherosclerotic lesions, and as a probe for detecting upregulation of surface vimentin during inflammation.

Metabolomics implicates altered sphingolipids in chronic pain of neuropathic origin.

Neuropathic pain is a debilitating condition for which the development of effective treatments has been limited by an incomplete understanding of its chemical basis. We show by using untargeted metabolomics that sphingomyelin-ceramide metabolism is altered in the dorsal horn of rats with neuropathic pain and that the upregulated, endogenous metabolite N,N-dimethylsphingosine induces mechanical hypersensitivity in vivo. These results demonstrate the utility of metabolomics to implicate unexplored biochemical pathways in disease.

Multivalent display of proteins on viral nanoparticles using molecular recognition and chemical ligation strategies.

Multivalent display of heterologous proteins on viral nanoparticles forms a basis for numerous applications in nanotechnology, including vaccine development, targeted therapeutic delivery, and tissue-specific bioimaging. In many instances, precise placement of proteins is required for optimal functioning of the supramolecular assemblies, but orientation- and site-specific coupling of proteins to viral scaffolds remains a significant technical challenge. We have developed two strategies that allow for controlled attachment of a variety of proteins on viral particles using covalent and noncovalent principles. In one strategy, an interaction between domain 4 of anthrax protective antigen and its receptor was used to display multiple copies of a target protein on virus-like particles. In the other, expressed protein ligation and aniline-catalyzed oximation was used to display covalently a model protein. The latter strategy, in particular, yielded nanoparticles that induced potent immune responses to the coupled protein, suggesting potential applications in vaccine development.

Cowpea mosaic virus nanoparticles target surface vimentin on cancer cells.

AIMS: Vimentin, a type III intermediate filament, is upregulated during epithelial-mesenchymal transition and tumor progression. Vimentin is surface-expressed on cells involved in inflammation; the function remains unknown. We investigated the expression of surface vimentin on cancer cells and evaluated targeting nanoparticles to tumors exploiting vimentin. MATERIALS & METHODS: Cowpea mosaic virus nanoparticles that interact with surface vimentin were used as probes. Tumor homing was tested using the chick chorioallantoic membrane model with human tumor xenografts. RESULTS & DISCUSSION: Surface vimentin levels varied during cell cycle and among the cell lines tested. Surface vimentin expression correlated with cowpea mosaic virus uptake, underscoring the utility of cowpea mosaic virus to detect invasive cancer cells. Targeting to tumor xenografts was observed; homing was based on the enhanced permeability and retention effect. Our data provide novel insights into the role of surface vimentin in cancer and targeting nanoparticles in vivo.

Viral nanoparticles and virus-like particles: platforms for contemporary vaccine design.

Current vaccines that provide protection against infectious diseases have primarily relied on attenuated or inactivated pathogens. Virus-like particles (VLPs), comprised of capsid proteins that can initiate an immune response but do not include the genetic material required for replication, promote immunogenicity and have been developed and approved as vaccines in some cases. In addition, many of these VLPs can be used as molecular platforms for genetic fusion or chemical attachment of heterologous antigenic epitopes. This approach has been shown to provide protective immunity against the foreign epitopes in many cases. A variety of VLPs and virus-based nanoparticles are being developed for use as vaccines and epitope platforms. These particles have the potential to increase efficacy of current vaccines as well as treat diseases for which no effective vaccines are available.