Resolvin D1 (RvD1) was shown to be a potent anti-inflammatory and proresolution lipid mediator in several animal models of inflammation, but its mechanism of action in humans is not clear. We show that the RvD1 receptor GPR32 is present on resting, proinflammatory M(LPS) and alternatively activated primary human M(IL-4) macrophages, whereas TGF-β and IL-6 reduce its membrane expression. Accordingly, stimulation of resting primary human macrophages with 10 nM RvD1 for 48 h maximally reduced the secretion of the proinflammatory cytokines IL-1β and IL-8; abolished chemotaxis to several chemoattractants like chemerin, fMLF, and MCP-1; and doubled the phagocytic activity of these macrophages toward microbial particles. In contrast, these functional changes were not accompanied by surface expression of markers specific for alternatively activated M(IL-4) macrophages. Similar proresolution effects of RvD1 were observed when proinflammatory M(LPS) macrophages were treated with RvD1. In addition, we show that these RvD1-mediated effects are GPR32 dependent because reduction of GPR32 expression by small interfering RNA, TGF-β, and IL-6 treatment ablated these proresolution effects in primary human macrophages. Taken together, our results indicate that in humans RvD1 triggers GPR32 to polarize and repolarize macrophages toward a proresolution phenotype, supporting the role of this mediator in the resolution of inflammation in humans.

Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced Sp Cas9 prime editor (PE) lacking the RNaseH domain (PE2 Δ RnH ) and an intein-split construct (PE2 p.1153) for adeno-associated virus–mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2 Δ RnH via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pah enu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 10 14 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.

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.

Phenylalanine hydroxylase (PAH) deficiency, colloquially known as phenylketonuria (PKU), is among the most common inborn errors of metabolism and in the past decade has become a target for the development of novel therapeutics such as gene therapy. PAH deficient mouse models have been key to new treatment development, but all prior existing models natively express liver PAH polypeptide as inactive or partially active PAH monomers, which complicates the experimental assessment of protein expression following therapeutic gene, mRNA, protein, or cell transfer. The mutant PAH monomers are able to form hetero-tetramers with and inhibit the overall holoenzyme activity of wild type PAH monomers produced from a therapeutic vector. Preclinical therapeutic studies would benefit from a PKU model that completely lacks both PAH activity and protein expression in liver. In this study, we employed CRISPR/Cas9-mediated gene editing in fertilized mouse embryos to generate a novel mouse model that lacks exon 1 of the Pah gene. Mice that are homozygous for the Pah exon 1 deletion are viable, severely hyperphenylalaninemic, accurately replicate phenotypic features of untreated human classical PKU and lack any detectable liver PAH activity or protein. This model of classical PKU is ideal for further development of gene and cell biologics to treat PKU.

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The most common ureagenesis defect is X-linked ornithine transcarbamylase (OTC) deficiency which is a main target for novel therapeutic interventions. The spf ash mouse model carries a variant (c.386G>A, p.Arg129His) that is also found in patients. Male spf ash mice have a mild biochemical phenotype with low OTC activity (5%-10% of wild-type), resulting in elevated urinary orotic acid but no hyperammonemia. We recently established a dried blood spot method for in vivo quantification of ureagenesis by Gas chromatography-mass spectrometry (GC-MS) using stable isotopes. Here, we applied this assay to wild-type and spf ash mice to assess ureagenesis at different ages. Unexpectedly, we found an age-dependency with a higher capacity for ammonia detoxification in young mice after weaning. A parallel pattern was observed for carbamoylphosphate synthetase 1 and OTC enzyme expression and activities, which may act as pacemaker of this ammonia detoxification pathway. Moreover, high ureagenesis in younger mice was accompanied by elevated periportal expression of hepatic glutamine synthetase, another main enzyme required for ammonia detoxification. These observations led us to perform a more extensive analysis of the spf ash mouse in comparison to the wildtype, including characterization of the corresponding metabolites, enzyme activities in the liver and plasma and the gut microbiota. In conclusion, the comprehensive enzymatic and metabolic analysis of ureagenesis performed in the presented depth was only possible in animals. Our findings suggest such analyses being essential when using the mouse as a model and revealed age-dependent activity of ammonia detoxification. K E Y W O R D Sage-dependency, gut microbiome, hyperammonemia, ornithine transcarbamylase (OTC) deficiency, spf ash mouse model, urea cycle disorders, ureagenesis Gabriella Allegri and Sereina Deplazes contributed equally to this study.

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