Topoisomerase Inhibitors Increase Episomal DNA Expression by Inducing the Integration of Episomal DNA in Hepatic Cells.

Gene therapy is a promising strategy to treat and cure most inherited metabolic liver disorders. Viral vectors such as those based on adeno-associated viruses (AAVs) and lentiviruses (LVs) are used as vehicles to deliver functional genes to affected hepatocytes. Adverse events associated with the use of high vector doses have motivated the use of small molecules as adjuvants to reduce the dose. In this study, we showed that a one-hour treatment with topoisomerase inhibitors (camptothecin and etoposide) prior to viral transduction is enough to increase AAV and LV reporter expression in non-dividing hepatic cells in culture. Topoisomerase inhibitors increased both integration-competent (ICLV) and integration-deficient (IDLV) LV-derived expression, with a much stronger increase in the IDLV transduction system. In agreement with that, topoisomerase inhibitors increased viral genome integration in both strains, with a greater impact on the IDLV strain, supporting the idea that topoisomerase inhibitors increased episomal DNA integration, especially when viral integrase activity is abolished. These effects correlated with an increase in the DNA damage response produced by the treatments. Our study highlights the need to monitor DNA damage and undesired integration of viral episomal DNAs into the host genome when studying chemical compounds that increase viral transduction.

Magnetogenetics: remote activation of cellular functions triggered by magnetic switches.

During the last decade, the possibility to remotely control intracellular pathways using physical tools has opened the way to novel and exciting applications, both in basic research and clinical applications. Indeed, the use of physical and non-invasive stimuli such as light, electricity or magnetic fields offers the possibility of manipulating biological processes with spatial and temporal resolution in a remote fashion. The use of magnetic fields is especially appealing for in vivo applications because they can penetrate deep into tissues, as opposed to light. In combination with magnetic actuators they are emerging as a new instrument to precisely manipulate biological functions. This approach, coined as magnetogenetics, provides an exclusive tool to study how cells transform mechanical stimuli into biochemical signalling and offers the possibility of activating intracellular pathways connected to temperature-sensitive proteins. In this review we provide a critical overview of the recent developments in the field of magnetogenetics. We discuss general topics regarding the three main components for magnetic field-based actuation: the magnetic fields, the magnetic actuators and the cellular targets. We first introduce the main approaches in which the magnetic field can be used to manipulate the magnetic actuators, together with the most commonly used magnetic field configurations and the physicochemical parameters that can critically influence the magnetic properties of the actuators. Thereafter, we discuss relevant examples of magneto-mechanical and magneto-thermal stimulation, used to control stem cell fate, to activate neuronal functions, or to stimulate apoptotic pathways, among others. Finally, although magnetogenetics has raised high expectations from the research community, to date there are still many obstacles to be overcome in order for it to become a real alternative to optogenetics for instance. We discuss some controversial aspects related to the insufficient elucidation of the mechanisms of action of some magnetogenetics constructs and approaches, providing our opinion on important challenges in the field and possible directions for the upcoming years.

The Host Factor Erlin-1 is Required for Efficient Hepatitis C Virus Infection.

Development of hepatitis C virus (HCV) infection cell culture systems has permitted the identification of cellular factors that regulate the HCV life cycle. Some of these cellular factors affect steps in the viral life cycle that are tightly associated with intracellular membranes derived from the endoplasmic reticulum (ER). Here, we describe the discovery of erlin-1 protein as a cellular factor that regulates HCV infection. Erlin-1 is a cholesterol-binding protein located in detergent-resistant membranes within the ER. It is implicated in cholesterol homeostasis and the ER-associated degradation pathway. Silencing of erlin-1 protein expression by siRNA led to decreased infection efficiency characterized by reduction in intracellular RNA accumulation, HCV protein expression and virus production. Mechanistic studies revealed that erlin-1 protein is required early in the infection, downstream of cell entry and primary translation, specifically to initiate RNA replication, and later in the infection to support infectious virus production. This study identifies erlin-1 protein as an important cellular factor regulating HCV infection.