Lipid Deposition Profiles Influence Foreign Body Responses.

Fibrosis remains a significant cause of failure in implanted biomedical devices and early absorption of proteins on implant surfaces has been shown to be a key instigating factor. However, lipids can also regulate immune activity and their presence may also contribute to biomaterial-induced foreign body responses (FBR) and fibrosis. Here it is demonstrated that the surface presentation of lipids on implant affects FBR by influencing reactions of immune cells to materials as well as their resultant inflammatory/suppressive polarization. Time-of-flight secondary ion mass spectroscopy (ToF-SIMS) is employed to characterize lipid deposition on implants that are surface-modified chemically with immunomodulatory small molecules. Multiple immunosuppressive phospholipids (phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin) are all found to deposit preferentially on implants with anti-FBR surface modifications in mice. Significantly, a set of 11 fatty acids is enriched on unmodified implanted devices that failed in both mice and humans, highlighting relevance across species. Phospholipid deposition is also found to upregulate the transcription of anti-inflammatory genes in murine macrophages, while fatty acid deposition stimulated the expression of pro-inflammatory genes. These results provide further insights into how to improve the design of biomaterials and medical devices to mitigate biomaterial material-induced FBR and fibrosis.

Rapid, Single-Cell Analysis and Discovery of Vectored mRNA Transfection In Vivo with a loxP-Flanked tdTomato Reporter Mouse.

mRNA therapeutics hold promise for the treatment of diseases requiring intracellular protein expression and for use in genome editing systems, but mRNA must transfect the desired tissue and cell type to be efficacious. Nanoparticle vectors that deliver the mRNA are often evaluated using mRNA encoding for reporter genes such as firefly luciferase (FLuc); however, single-cell resolution of mRNA expression cannot generally be achieved with FLuc, and, thus, the transfected cell populations cannot be determined without additional steps or experiments. To more rapidly identify which types of cells an mRNA formulation transfects in vivo, we describe a Cre recombinase (Cre)-based system that permanently expresses fluorescent tdTomato protein in transfected cells of genetically modified mice. Following in vivo application of vectored Cre mRNA, it is possible to visualize successfully transfected cells via Cre-mediated tdTomato expression in bulk tissues and with single-cell resolution. Using this system, we identify previously unknown transfected cell types of an existing mRNA delivery vehicle in vivo and also develop a new mRNA formulation capable of transfecting lung endothelial cells. Importantly, the same formulations with mRNA encoding for fluorescent protein delivered to wild-type mice did not produce sufficient signal for any visualization in vivo, demonstrating the significantly improved sensitivity of our Cre-based system. We believe that the system described here may facilitate the identification and characterization of mRNA delivery vectors to new tissues and cell types.

Corrigendum: In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.

This corrects the article DOI: 10.1038/nbt.3836.

Synthesis and Biological Evaluation of Ionizable Lipid Materials for the In Vivo Delivery of Messenger RNA to B Lymphocytes.

B lymphocytes regulate several aspects of immunity including antibody production, cytokine secretion, and T-cell activation; moreover, B cell misregulation is implicated in autoimmune disorders and cancers such as multiple sclerosis and non-Hodgkin's lymphomas. The delivery of messenger RNA (mRNA) into B cells can be used to modulate and study these biological functions by means of inducing functional protein expression in a dose-dependent and time-controlled manner. However, current in vivo mRNA delivery systems fail to transfect B lymphocytes and instead primarily target hepatocytes and dendritic cells. Here, the design, synthesis, and biological evaluation of a lipid nanoparticle (LNP) system that can encapsulate mRNA, navigate to the spleen, transfect B lymphocytes, and induce more than 60 pg of protein expression per million B cells within the spleen is described. Importantly, this LNP induces more than 85% of total protein production in the spleen, despite LNPs being observed transiently in the liver and other organs. These results demonstrate that LNP composition alone can be used to modulate the site of protein induction in vivo, highlighting the critical importance of designing and synthesizing new nanomaterials for nucleic acid delivery.

In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.

In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis, which rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;KrasG12D/+;Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5+ stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.

Lipid Nanoparticle Assisted mRNA Delivery for Potent Cancer Immunotherapy.

The induction of a strong cytotoxic T cell response is an important prerequisite for successful immunotherapy against many viral diseases and tumors. Nucleotide vaccines, including mRNA vaccines with their intracellular antigen synthesis, have been shown to be potent activators of a cytotoxic immune response. The intracellular delivery of mRNA vaccines to the cytosol of antigen presenting immune cells is still not sufficiently well understood. Here, we report on the development of a lipid nanoparticle formulation for the delivery of mRNA vaccines to induce a cytotoxic CD 8 T cell response. We show transfection of dendritic cells, macrophages, and neutrophils. The efficacy of the vaccine was tested in an aggressive B16F10 melanoma model. We found a strong CD 8 T cell activation after a single immunization. Treatment of B16F10 melanoma tumors with lipid nanoparticles containing mRNA coding for the tumor-associated antigens gp100 and TRP2 resulted in tumor shrinkage and extended the overall survival of the treated mice. The immune response can be further increased by the incorporation of the adjuvant LPS. In conclusion, the lipid nanoparticle formulation presented here is a promising vector for mRNA vaccine delivery, one that is capable of inducing a strong cytotoxic T cell response. Further optimization, including the incorporation of different adjuvants, will likely enhance the potency of the vaccine.

Ultrasound-Mediated Delivery of RNA to Colonic Mucosa of Live Mice.

BACKGROUND & AIMS: It is a challenge to deliver nucleic acids to gastrointestinal (GI) tissues due to their size and need for intracellular delivery. They are also extremely susceptible to degradation by nucleases, which are ubiquitous in the GI tract. We investigated whether ultrasound, which can permeabilize tissue through a phenomenon known as transient cavitation, can be used to deliver RNA to the colonic mucosa of living mice. METHODS: We investigated delivery of fluorescently labeled permeants to colon tissues of Yorkshire pigs ex vivo and mice in vivo. Colon tissues were collected and fluorescence was measured by confocal microscopy. We then evaluated whether ultrasound is effective in delivering small interfering (si)RNA to C57BL/6 mice with dextran sodium sulfate-induced colitis. Some mice were given siRNA against tumor necrosis factor (Tnf) mRNA for 6 days; colon tissues were collected and analyzed histologically and TNF protein levels measured by enzyme-linked immunosorbent assay. Feces were collected and assessed for consistency and occult bleeding. We delivered mRNA encoding firefly luciferase to colons of healthy C57BL/6 mice. RESULTS: Exposure of ex vivo pig colon tissues to 20 kHz ultrasound for 1 minute increased the level of delivery of 3 kDa dextran 7-fold compared with passive diffusion (P = .037); 40 kHz ultrasound application for 0.5 seconds increased the delivery 3.3-fold in living mice (P = .041). Confocal microscopy analyses of colon tissues from pigs revealed regions of punctuated fluorescent dextran signal, indicating intracellular delivery of macromolecules. In mice with colitis, ultrasound delivery of unencapsulated siRNA against Tnf mRNA reduced protein levels of TNF in colon tissues, compared with mice with colitis given siRNA against Tnf mRNA without ultrasound (P ≤ .014), and reduced features of inflammation (P ≤ 4.1 × 10-5). Separately, colons of mice administered an mRNA encoding firefly luciferase with ultrasound and the D-luciferin substrate had levels of bioluminescence 11-fold greater than colons of mice given the mRNA alone (P = .0025). Ultrasound exposures of 40 kHz ultrasound for 0.5 seconds were well tolerated, even in mice with acute colitis. CONCLUSIONS: Ultrasound can be used to deliver mRNAs and siRNAs to the colonic mucosa of mice and knock down expression of target mRNAs.

mRNA vaccine delivery using lipid nanoparticles.

mRNA vaccines elicit a potent immune response including antibodies and cytotoxic T cells. mRNA vaccines are currently evaluated in clinical trials for cancer immunotherapy applications, but also have great potential as prophylactic vaccines. Efficient delivery of mRNA vaccines will be key for their success and translation to the clinic. Among potential nonviral vectors, lipid nanoparticles are particularly promising. Indeed, lipid nanoparticles can be synthesized with relative ease in a scalable manner, protect the mRNA against degradation, facilitate endosomal escape, can be targeted to the desired cell type by surface decoration with ligands, and as needed, can be codelivered with adjuvants.

Ultrasound-enhanced transdermal delivery: recent advances and future challenges.

The skin is a formidable diffusion barrier that restricts passive diffusion to small (<500 Da) lipophilic molecules. Methods used to permeabilize this barrier for the purpose of drug delivery are maturing as an alternative to oral drug delivery and hypodermic injections. Ultrasound can reversibly and non-invasively permeabilize the diffusion barrier posed by the skin. This review discusses the mechanisms of ultrasound-permeability enhancement, and presents technological innovations in equipment miniaturization and recent advances in permeabilization capabilities. Additionally, potentially exciting applications, including protein delivery, vaccination, gene therapy and sensing of blood analytes, are discussed. Finally, the future challenges and opportunities associated with the use of ultrasound are discussed. It is stressed that developing ultrasound for suitable applications is key to ensure commercial success.

Glycan arrays containing synthetic Clostridium difficile lipoteichoic acid oligomers as tools toward a carbohydrate vaccine.

Clostridium difficile is a leading cause of severe nosocomial infections. Cell-surface carbohydrate antigens are promising vaccine candidates. Here we report the first total synthesis of oligomers of the lipoteichoic acid antigen repeating unit. Synthetic glycan microarrays revealed anti-glycan antibodies in the blood of patients that help to define epitopes for vaccine development.

Immunological evaluation of a synthetic Clostridium difficile oligosaccharide conjugate vaccine candidate and identification of a minimal epitope.

Clostridium difficile is the cause of emerging nosocomial infections that result in abundant morbidity and mortality worldwide. Thus, the development of a vaccine to kill the bacteria to prevent this disease is highly desirable. Several recently identified bacterial surface glycans, such as PS-I and PS-II, are promising vaccine candidates to preclude C. difficile infection. To circumvent difficulties with the generation of natural PS-I due to its low expression levels in bacterial cultures, improved chemical synthesis protocols for the pentasaccharide repeating unit of PS-I and oligosaccharide substructures were utilized to produce large quantities of well-defined PS-I related glycans. The analysis of stool and serum samples obtained from C. difficile patients using glycan microarrays of synthetic oligosaccharide epitopes revealed humoral immune responses to the PS-I related glycan epitopes. Two different vaccine candidates were evaluated in the mouse model. A synthetic PS-I repeating unit CRM197 conjugate was immunogenic in mice and induced immunoglobulin class switching as well as affinity maturation. Microarray screening employing PS-I repeating unit substructures revealed the disaccharide Rha-(1→3)-Glc as a minimal epitope. A CRM197-Rha-(1→3)-Glc disaccharide conjugate was able to elicit antibodies recognizing the C. difficile PS-I pentasaccharide. We herein demonstrate that glycan microarrays exposing defined oligosaccharide epitopes help to determine the minimal immunogenic epitopes of complex oligosaccharide antigens. The synthetic PS-I pentasaccharide repeating unit as well as the Rha-(1→3)-Glc disaccharide are promising novel vaccine candidates against C. difficile that are currently in preclinical evaluation.

A general method for Suzuki-Miyaura coupling reactions using lithium triisopropyl borates.

Conditions for the Suzuki-Miyaura coupling of lithium triisopropyl borates are reported, as well as a procedure for a one-pot lithiation, borylation, and subsequent Suzuki-Miyaura coupling of various heterocycles with aryl halides. These borate species are much more stable toward protodeboronation than the corresponding boronic acids and can conveniently be stored on benchtop at room temperature.

A possible oligosaccharide-conjugate vaccine candidate for Clostridium difficile is antigenic and immunogenic.

Nosocomial infections with the Gram-positive pathogen Clostridium difficile pose a major risk for hospitalized patients and result in significant costs to health care systems. Here, we present the chemical synthesis of a PS-II hapten of a cell wall polysaccharide of hypervirulent ribotype 027 of C. difficile. Mice were immunized with a conjugate consisting of the synthetic hexasaccharide and the diphtheria toxoid variant CRM(197). The immunogenicity of the glycan repeating unit was demonstrated by the presence of specific IgG antibodies in the serum of immunized mice. Murine monoclonal antibodies interact with the synthetic hexasaccharide, as determined by microarray analysis. Finally, we found that specific IgA antibodies in the stool of hospital patients infected with C. difficile recognize the synthetic PS-II hexasaccharide hapten.

Identification of an African Bacillus anthracis lineage that lacks expression of the spore surface-associated anthrose-containing oligosaccharide.

The surfaces of Bacillus anthracis endospores expose a pentasaccharide containing the monosaccharide anthrose, which has been considered for use as a vaccine or target for specific detection of the spores. In this study B. anthracis strains isolated from cattle carcasses in African countries where anthrax is endemic were tested for their cross-reactivity with monoclonal antibodies (MAbs) specific for anthrose-containing oligosaccharides. Unexpectedly, none of the isolates collected in Chad, Cameroon, and Mali were recognized by the MAbs. Sequencing of the four-gene operon encoding anthrose biosynthetic enzymes revealed the presence of premature stop codons in the aminotransferase and glycosyltransferase genes in all isolates from Chad, Cameroon, and Mali. Both immunological and genetic findings suggest that the West African isolates are unable to produce anthrose. The anthrose-deficient strains from West Africa belong to a particular genetic lineage. Immunization of cattle in Chad with a locally produced vaccine based on anthrose-positive spores of the B. anthracis strain Sterne elicited an anti-carbohydrate IgG response specific for a synthetic anthrose-containing tetrasaccharide as demonstrated by glycan microarray analysis. Competition immunoblots with synthetic pentasaccharide derivatives suggested an immunodominant role of the anthrose-containing carbohydrate in cattle. In West Africa anthrax is highly endemic. Massive vaccination of livestock in this area has taken place over long periods of time using spores of the anthrose-positive vaccine strain Sterne. The spread of anthrose-deficient strains in this region may represent an escape strategy of B. anthracis.

Surface characterization of carbohydrate microarrays.

Carbohydrate microarrays are essential tools to determine the biological function of glycans. Here, we analyze a glycan array by time-of-flight secondary ion mass spectrometry (ToF-SIMS) to gain a better understanding of the physicochemical properties of the individual spots and to improve carbohydrate microarray quality. The carbohydrate microarray is prepared by piezo printing of thiol-terminated sugars onto a maleimide functionalized glass slide. The hyperspectral ToF-SIMS imaging data are analyzed by multivariate curve resolution (MCR) to discern secondary ions from regions of the array containing saccharide, linker, salts from the printing buffer, and the background linker chemistry. Analysis of secondary ions from the linker common to all of the sugar molecules employed reveals a relatively uniform distribution of the sugars within the spots formed from solutions with saccharide concentration of 0.4 mM and less, whereas a doughnut shape is often formed at higher-concentration solutions. A detailed analysis of individual spots reveals that in the larger spots the phosphate buffered saline (PBS) salts are heterogeneously distributed, apparently resulting in saccharide concentrated at the rim of the spots. A model of spot formation from the evaporating sessile drop is proposed to explain these observations. Saccharide spot diameters increase with saccharide concentration due to a reduction in surface tension of the saccharide solution compared to PBS. The multivariate analytical partial least squares (PLS) technique identifies ions from the sugars that in the complex ToF-SIMS spectra correlate with the binding of galectin proteins.

Molecular analysis of carbohydrate-antibody interactions: case study using a Bacillus anthracis tetrasaccharide.

The process for selecting potent and effective carbohydrate antigens is not well-established. A combination of synthetic glycan microarray screening, surface plasmon resonance analysis, and saturation transfer difference NMR spectroscopy was used to dissect the antibody-binding surface of a carbohydrate antigen, revealing crucial binding elements with atomic-level detail. This analysis takes the first step toward uncovering the rules for structure-based design of carbohydrate antigens.

Determination of carbohydrate-binding preferences of human galectins with carbohydrate microarrays.

Galectins are a class of carbohydrate-binding proteins named for their galactose-binding preference and are involved in a host of processes ranging from homeostasis of organisms to immune responses. As a first step towards correlating the carbohydrate-binding preferences of the different galectins with their biological functions, we determined carbohydrate recognition fine-specificities of galectins with the aid of carbohydrate microarrays. A focused set of oligosaccharides considered relevant to galectins was prepared by chemical synthesis. Structure-activity relationships for galectin-sugar interactions were determined, and these helped in the establishment of redundant and specific galectin actions by comparison of binding preferences. Distinct glycosylations on the basic lactosyl motifs proved to be key to galectin binding regulation--and therefore galectin action--as either high-affinity ligands are produced or binding is blocked. High-affinity ligands such as the blood group antigens that presumably mediate particular functions were identified.

Synthesis of a hexasaccharide repeating unit from Bacillus anthracis vegetative cell walls.

The first synthesis of hexasaccharide 1 representing a repeat unit of a polysaccharide specific to the vegetative cell wall of Bacillus anthracis is reported. The synthetic hexasaccharide is equipped with an n-pentenyl handle at the reducing terminus to allow for further functionalization. Key transformations during the synthesis are the conversion of a glucose into a mannosazide residue, a (2+2) coupling, followed by double alpha-galactosylation to furnish the hexasaccharide, and global deprotection under Birch conditions.

Diradical-promoted (N + 2 - 1) ring expansion: an efficient reaction for the synthesis of macrocyclic ketones.

[reaction: see text] A diradical-promoted (n + 2 - 1) ring expansion reaction based on vinyl side chain insertion (+2C) and decarbonylation (-1C) has been developed. Flash vacuum pyrolysis (FVP) of medium- and large-ring 2-trimethylsilyloxy-2-vinyl-cycloalkanones at 500-600 degrees C affords the one-carbon ring-expanded cycloalkanones in good yields. Methyl groups on the vinyl moiety are transformed regiospecifically as corresponding alpha- and beta-substituents, respectively. 2-Ethynyl precursor analogues react in a manner similar to give alpha,beta-unsaturated cyclic ketones.