Clustered regularly interspaced short palindromic repeat (CRISPR) has arisen as a frontrunner for efficient genome engineering. However, the potentially broad therapeutic implications are largely unexplored. Here, to investigate the therapeutic potential of CRISPR/Cas9 in a diverse set of genetic disorders, we establish a pipeline that uses readily obtainable cells from affected individuals. We show that an adapted version of CRISPR/Cas9 increases the amount of utrophin, a known disease modifier in Duchenne muscular dystrophy (DMD). Furthermore, we demonstrate preferential elimination of the dominant-negative FGFR3 c.1138G>A allele in fibroblasts of an individual affected by achondroplasia. Using a previously undescribed approach involving single guide RNA, we successfully removed large genome rearrangement in primary cells of an individual with an X chromosome duplication including MECP2. Moreover, removal of a duplication of DMD exons 18-30 in myotubes of an individual affected by DMD produced full-length dystrophin. Our findings establish the far-reaching therapeutic utility of CRISPR/Cas9, which can be tailored to target numerous inherited disorders.

Endogenous pancreatic multipotent progenitors (PMPs) are ideal candidates for regenerative approaches to compensate for b-cell loss since their b-cell-producing capacities as well as strategic location would eliminate unnecessary invasive manipulations. However, little is known about the status and potentials of PMPs under diabetic conditions. Here we show that b-cell metabolic stress and hyperglycemia enhance the proliferation capacities of adult PMP cells and bias their production of progeny toward b-cells in mouse and human. These effects are dynamic and correlate with functional b-cell regeneration when conditions allow.

BACKGROUND The endothelial glycocalyx (EG) on the luminal surface of endothelial cells contributes to the permeability barrier of vessels and prevents activation of the coagulation cascade. Endothelial glycocalyx damage, which occurs in the shock state, results in endotheliopathy. Interleukin (IL)-22 is a cytokine with both proinflammatory and anti-inflammatory properties, and how IL-22 affects the EG has not been studied. We hypothesized that IL-22:Fc, a recombinant fusion protein with human IL-22 and the Fc portion of human immunoglobulin G1 (which extends the protein half-life), would not affect EG shedding in endothelium after injury. METHODS Human umbilical vein endothelial cells (HUVECs) were exposed to 1 μg/mL lipopolysaccharide (LPS). Lipopolysaccharide-injured cells (n = 284) were compared with HUVECs with LPS injury plus 0.375 μg/mL of IL-22:Fc treatment (n = 293) for 12 hours. These two cohorts were compared with control HUVECs (n = 286) and HUVECs exposed to IL-22:Fc alone (n = 269). Cells were fixed and stained with fluorescein isothiocyanate-labeled wheat germ agglutinin to quantify EG. Total RNA was collected, and select messenger RNAs were quantified by real time - quantitative polymerase chain reaction (RT-qPCR) using SYBR green fluorescence. RESULTS Exposure of HUVECs to LPS resulted in degradation of the EG compared with control (5.86 vs. 6.09 arbitrary unit [AU], p = 0.01). Interleukin-22:Fc alone also resulted in degradation of EG (5.08 vs. 6.09 AU, p = 0.01). Treatment with IL-22:Fc after LPS injury resulted in less degradation of EG compared with LPS injury alone (5.86 vs. 5.08 AU, p = 0.002). Expression of the IL-22Ra1 receptor was not different for IL-22:Fc treated compared with LPS injury only (0.69 vs. 0.86 relative expression, p = 0.10). Treatment with IL-22:Fc after LPS injury resulted in less matrix metalloproteinase 2 (0.79 vs. 1.70 relative expression, p = 0.005) and matrix metalloproteinase 14 (0.94 vs. 2.04 relative expression, p = 0.02). CONCLUSIONS Interleukin-22:Fc alone induces EG degradation. However, IL-22:Fc treatment after LPS injury appears to mitigate EG degradation. This protective effect appears to be mediated via reduced expression of metalloproteinases.