Gene editing-based targeted integration for correction of Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a severe X-linked primary immunodeficiency resulting from a diversity of mutations distributed across all 12 exons of the WAS gene. WAS encodes a hematopoietic-specific and developmentally regulated cytoplasmic protein (WASp). The objective of this study was to develop a gene correction strategy potentially applicable to most WAS patients by employing nuclease-mediated, site-specific integration of a corrective WAS gene sequence into the endogenous WAS chromosomal locus. In this study, we demonstrate the ability to target the integration of WAS2-12-containing constructs into intron 1 of the endogenous WAS gene of primary CD34+ hematopoietic stem and progenitor cells (HSPCs), as well as WASp-deficient B cell lines and WASp-deficient primary T cells. This intron 1 targeted integration (TI) approach proved to be quite efficient and restored WASp expression in treated cells. Furthermore, TI restored WASp-dependent function to WAS patient T cells. Edited CD34+ HSPCs exhibited the capacity for multipotent differentiation to various hematopoietic lineages in vitro and in transplanted immunodeficient mice. This methodology offers a potential editing approach for treatment of WAS using patient's CD34+ cells.

Convenient synthesis of deazaflavin cofactor FO and its activity in F(420)-dependent NADP reductase.

F420 and FO are phenolic 5-deazaflavin cofactors that complement nicotinamide and flavin redox coenzymes in biochemical oxidoreductases and photocatalytic systems. Specifically, these 5-deazaflavins lack the single electron reactivity with O2 of riboflavin-derived coenzymes (FMN and FAD), and, in general, have a more negative redox potential than NAD(P)(+). For example, F420-dependent NADP(+) oxidoreductase (Fno) is critical to the conversion of CO2 to CH4 by methanogenic archaea, while FO functions as a light-harvesting agent in DNA repair. The preparation of these cofactors is an obstacle to their use in biochemical studies and biotechnology. Here, a convenient synthesis of FO was achieved by improving the redox stability of synthetic intermediates containing a polar, electron-rich aminophenol fragment. Improved yields and simplified purification techniques for FO are described. Additionally, Fno activity was restored with FO in the absence of F420. Investigating the FO-dependent NADP(+)/NADPH redox process by stopped-flow spectrophotometry, steady state kinetics were defined as having a Km of 4.00 ± 0.39 µM and a kcat of 5.27 ± 0.14 s(-1). The preparation of FO should enable future biochemical studies and novel uses of F420 mimics.

Cost effectiveness of tobacco control policies in Vietnam: the case of population-level interventions.

BACKGROUND: Tobacco smoking is one of the leading public health problems in the world. It is also possible to prevent and/or reduce the harm from tobacco use through the use of cost-effective tobacco control measures. However, most of this evidence comes from developed countries and little research has been conducted on this issue in developing countries. OBJECTIVE: The objective of this study was to analyse the cost effectiveness of four population-level tobacco control interventions in Vietnam. METHODS: Four tobacco control interventions were evaluated: excise tax increase; graphic warning labels on cigarette packs; mass media campaigns; and smoking bans (in public or in work places). A multi-state life table model was constructed in Microsoft® Excel to examine the cost effectiveness of the tobacco control intervention options. A government perspective was adopted, with costing conducted using a bottom-up approach. Health improvement was considered in terms of disability-adjusted life-years (DALYs) averted. All assumptions were subject to sensitivity and uncertainty analysis. RESULTS: All the interventions fell within the definition of being very cost effective according to the threshold level suggested by the WHO (i.e.