Understanding Intracellular Biology to Improve mRNA Delivery by Lipid Nanoparticles.

Poor understanding of intracellular delivery and targeting hinders development of nucleic acid-based therapeutics transported by nanoparticles. Utilizing a siRNA-targeting and small molecule profiling approach with advanced imaging and machine learning biological insights is generated into the mechanism of lipid nanoparticle (MC3-LNP) delivery of mRNA. This workflow is termed Advanced Cellular and Endocytic profiling for Intracellular Delivery (ACE-ID).

Heat inactivation of clinical COVID-19 samples on an industrial scale for low risk and efficient high-throughput qRT-PCR diagnostic testing.

We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world.

Secretome screening reveals immunomodulating functions of IFNα-7, PAP and GDF-7 on regulatory T-cells.

Regulatory T cells (Tregs) are the key cells regulating peripheral autoreactive T lymphocytes. Tregs exert their function by suppressing effector T cells. Tregs have been shown to play essential roles in the control of a variety of physiological and pathological immune responses.

Arrayed CRISPR Screening Identifies Novel Targets That Enhance the Productive Delivery of mRNA by MC3-Based Lipid Nanoparticles.

Modified messenger RNAs (mRNAs) hold great potential as therapeutics by using the body's own processes for protein production. However, a key challenge is efficient delivery of therapeutic mRNA to the cell cytosol and productive protein translation. Lipid nanoparticles (LNPs) are the most clinically advanced system for nucleic acid delivery; however, a relatively narrow therapeutic index makes them unsuitable for many therapeutic applications.

Delivering Robust Candidates to the Drug Pipeline through Computational Analysis of Arrayed CRISPR Screens.

Genome-wide arrayed CRISPR screening is a powerful method for drug target identification as it enables exploration of the effect of individual gene perturbations using diverse highly multiplexed functional and phenotypic assays. Using high-content imaging, we can measure changes in biomarker expression, intracellular localization, and cell morphology. Here we present the computational pipeline we have developed to support the analysis and interpretation of arrayed CRISPR screens.

Phenotypic Screen with the Human Secretome Identifies FGF16 as Inducing Proliferation of iPSC-Derived Cardiac Progenitor Cells.

Paracrine factors can induce cardiac regeneration and repair post myocardial infarction by stimulating proliferation of cardiac cells and inducing the anti-fibrotic, antiapoptotic, and immunomodulatory effects of angiogenesis. Here, we screened a human secretome library, consisting of 923 growth factors, cytokines, and proteins with unknown function, in a phenotypic screen with human cardiac progenitor cells. The primary readout in the screen was proliferation measured by nuclear count.

Regulation of hepatitis C virus replication via threonine phosphorylation of the NS5A protein.

The hepatitis C virus non-structural 5A (NS5A) protein is highly phosphorylated and plays roles in both virus genome replication and assembly of infectious virus particles. NS5A comprises three domains separated by low complexity sequences (LCS). Mass spectrometry analysis of NS5A revealed the existence of a singly phosphorylated tryptic peptide corresponding to the end of LCS I and the beginning of domain II that contained a number of potential phosphorylatable residues (serines and threonines).

A novel method for the measurement of hepatitis C virus infectious titres using the IncuCyte ZOOM and its application to antiviral screening.

Hepatitis C virus (HCV) is a significant human pathogen infecting 3% of the world population. An infectious molecular clone capable of replicating and releasing infectious virions in cell culture has only been available since 2005, leaving a significant knowledge gap concerning post-RNA replication events such as particle assembly, trafficking and release. Thus, a fast, efficient and accurate method of measuring infectious viral titres is highly desirable.

Serine phosphorylation of the hepatitis C virus NS5A protein controls the establishment of replication complexes.

Unlabelled: The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is highly phosphorylated and involved in both virus genome replication and virion assembly. We and others have identified serine 225 in NS5A to be a phosphorylation site, but the function of this posttranslational modification in the virus life cycle remains obscure. Here we describe the phenotype of mutants with mutations at serine 225; this residue was mutated to either alanine (S225A; phosphoablatant) or aspartic acid (S225D; phosphomimetic) in the context of both the JFH-1 cell culture infectious virus and a corresponding subgenomic replicon.

Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on!

Since one of us co-authored a review on NS5A a decade ago, the hepatitis C virus (HCV) field has changed dramatically, primarily due to the advent of the JFH-1 cell culture infectious clone, which allowed the study of all aspects of the virus life cycle from entry to exit. This review will describe advances in our understanding of NS5A biology over the past decade, highlighting how the JFH-1 system has allowed us to determine that NS5A is essential not only in genome replication but also in the assembly of infectious virions. We shall review the recent structural insights - NS5A is predicted to comprise three domains; X-ray crystallography has revealed the structure of domain I but there is a lack of detailed structural information about the other two domains, which are predicted to be largely unstructured.

Insights into the complexity and functionality of hepatitis C virus NS5A phosphorylation.

The hepatitis C virus nonstructural NS5A protein has roles in genome replication, virus assembly, and modulation of host pathways. NS5A is a phosphoprotein, and it has been proposed that differential phosphorylation could regulate the various roles of the protein. By SDS-PAGE, two forms of NS5A with distinct apparent molecular weights can be observed, referred to as basally phosphorylated and hyperphosphorylated species.

The C terminus of NS5A domain II is a key determinant of hepatitis C virus genome replication, but is not required for virion assembly and release.

The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and the assembly of infectious virus particles. NS5A comprises three domains, separated by low-complexity sequences. Whilst the function of domain I appears to be predominantly involved with genome replication, the roles of domains II and III are less well defined.