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

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

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

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. Keywords: Antisense Therapy; Ulcerative Colitis; Inflammatory Bowel Disease; Ultrasound-Mediated Gastrointestinal Drug DeliveryNational Institutes of Health (U.S.) (Grant EB-000244

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.Novartis (Firm) (Postdoctoral Fellowship

Reproducibility of Thermal History Reconstruction From Apatite Fission-Track and (U-Th)/He Data

International audienceWe report a new interlaboratory exercise to evaluate the reproducibility of apatite fission-track (AFT) and (U-Th)/He (AHe) data and thermal history analysis. Twelve laboratory groups participated, analyzing apatite separates from two previously studied localities. Ten groups returned AFT data from 13 analysts, five groups returned AHe data, one contributed apatite U/Pb data, and nine contributed thermal history models. Submitted AFT age data were generally consistent with the original studies and each other to within uncertainties, although there were departures, particularly among results obtained using laser ablation inductively coupled plasma mass spectrometry for uranium determination. AFT-confined track-length data showed more variation, which correlated between samples, suggesting that they may reflect analyst-specific factors. Accounting for anisotropy using c axis projection reduced the variation and correlation. AHe ages showed more dispersion than observed in the previous studies, with one sample showing many ages consistent with prior work but also many significantly older ages, and the other roughly matching prior work but showing symmetrical scatter suggesting a 1SE uncertainty of 17-21%. Thermal history models generally agreed in their major features but showed considerable variation in detail, due to differences in data and data entry, model setup, and modeling software approach. Addressing pitfalls in data entry and model setup improved congruence, as would greater emphasis on AFT length and etch figure calibration. Because of scatter, AHe data had to be entered selectively into the models to achieve reasonable results. Although cautionary in several respects, study results point to promising avenues for improving the reproducibility of thermal history modeling. Abstract Copyright (2018). American Geophysical Union. All Rights Reserved