Protein metabolism in leg muscle following an endotoxin injection in healthy volunteers

A B S T R A C T The human endotoxin model has been used to study the early phase of sepsis. The aim of the present study was to assess leg muscle protein kinetics after an endotoxin challenge given to healthy human volunteers. Six healthy male subjects were studied in the post-absorptive state before and during 4 h following an intravenous endotoxin bolus (4 ng/kg of body weight). Primed continuous infusion of [ 2 H 5 ]phenylalanine and [ 2 H 3 ]3-methylhistidine in combination with sampling from the radial artery, femoral vein and muscle tissue were used to assess leg muscle protein kinetics. Both two-and three-compartment models were used to calculate protein kinetics. In addition 26S proteasome activity and protein ubiquitination were assessed. An increase in the net release of phenylalanine from the leg following the endotoxin challenge was observed; however, this phenylalanine originates from the free intracellular pool and not from protein. Net protein balance was unchanged, whereas both protein synthesis and breakdown were decreased. Degradation rates of contractile proteins were not affected by endotoxin, as indicated by an unchanged rate of appearance of 3-methylhistidine from leg muscle. In addition, proteasome activity and protein ubiquitination were unaffected by endotoxaemia. In conclusion, intravenous endotoxin administration to healthy volunteers resulted in an increased release of free phenylalanine from skeletal muscle, whereas protein balance was unaffected. Both protein synthesis and breakdown were decreased to a similar extent

Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective. Keywords: CRISPR; FRGN; ex vivo; genome editing; hepatocyte transplantation; liver-humanized mouse; primary hepatocytes; urea cycle disorder