Arginase deficiency is caused by biallelic mutations in arginase 1 (ARG1), the final step of the urea cycle, and results biochemically in hyperargininemia and the presence of guanidino compounds, while it is clinically notable for developmental delays, spastic diplegia, psychomotor function loss, and (uncommonly) death. There is currently no completely effective medical treatment available. While preclinical strategies have been demonstrated, disadvantages with viral-based episomal-expressing gene therapy vectors include the risk of insertional mutagenesis and limited efficacy due to hepatocellular division. Recent advances in messenger RNA (mRNA) codon optimization, synthesis, and encapsulation within biodegradable liver-targeted lipid nanoparticles (LNPs) have potentially enabled a new generation of safer, albeit temporary, treatments to restore liver metabolic function in patients with urea cycle disorders, including ARG1 deficiency. In this study, we applied such technologies to successfully treat an ARG1-deficient murine model. Mice were administered LNPs encapsulating human codon-optimized ARG1 mRNA every 3 d. Mice demonstrated 100% survival with no signs of hyperammonemia or weight loss to beyond 11 wk, compared with controls that perished by day 22. Plasma ammonia, arginine, and glutamine demonstrated good control without elevation of guanidinoacetic acid, a guanidino compound. Evidence of urea cycle activity restoration was demonstrated by the ability to fully metabolize an ammonium challenge and by achieving near-normal ureagenesis; liver arginase activity achieved 54% of wild type. Biochemical and microscopic data showed no evidence of hepatotoxicity. These results suggest that delivery of ARG1 mRNA by liver-targeted nanoparticles may be a viable gene-based therapeutic for the treatment of arginase deficiency.

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Carbamoyl phosphate synthetase 1 (CPS1) is a urea cycle enzyme that forms carbamoyl phosphate from bicarbonate, ammonia and ATP. Bi-allelic mutations of the CPS1 gene result in a urea cycle disorder presenting with hyperammonemia, often with reduced citrulline, and without orotic aciduria. CPS1 deficiency is particularly challenging to treat and lack of early recognition typically results in early neonatal death. Therapeutic interventions have limited efficacy and most patients develop long-term neurologic sequelae. Using transgenic techniques, we generated a conditional Cps1 knockout mouse. By loxP/Cre recombinase technology, deletion of the Cps1 locus was achieved in adult transgenic animals using a Cre recombinase-expressing adeno-associated viral vector. Within four weeks from vector injection, all animals developed hyperammonemia without orotic aciduria and died. Minimal CPS1 protein was detectable in livers. To investigate the efficacy of gene therapy for CPS deficiency following knock-down of hepatic endogenous CPS1 expression, we injected these mice with a helper-dependent adenoviral vector (HDAd) expressing the large murine CPS1 cDNA under control of the phosphoenolpyruvate carboxykinase promoter. Liver-directed HDAd-mediated gene therapy resulted in survival, normalization of plasma ammonia and glutamine, and 13% of normal Cps1 expression. A gender difference in survival suggests that female mice may require higher hepatic CPS1 expression. We conclude that this conditional murine model recapitulates the clinical and biochemical phenotype detected in human patients with CPS1 deficiency and will be useful to investigate ammonia-mediated neurotoxicity and for the development of cell- and gene-based therapeutic approaches.

Inflammation and vascular leakage are prevalent in asthma. This study aimed to elucidate the mechanisms involved in serum potentiation of cytokine-induced granulocyte macrophage colony stimulating factor (GM-CSF) production by human airway smooth muscle cells and to identify possible factors responsible. Serum-deprived cells at low density were stimulated with TNF-α and IL-1β for 24 h. Human AB serum (10%), inhibitors of RNA and protein synthesis or specific signaling molecules, or known smooth muscle mitogens were then added for 24 h. Culture supernatants were analyzed for GM-CSF levels, and cells were harvested to assess viability, cell cycle progression, GM-CSF-specific mRNA content, and p38 phosphorylation. Serum potentiated GM-CSF release when added before, together with (maximal), or after the cytokines. The potentiation involved both new GM-CSF-specific mRNA production and protein synthesis. The mitogens IGF, PDGF, and thrombin all potentiated GM-CSF release, and neutralizing antibodies for EGF, IGF, and PDGF reduced the serum potentiation. Inhibitor studies ruled as unlikely the involvement of p70S6kinase and the MAPK p42/p44, two signaling pathways implicated in proliferation, and the involvement of the MAPK JNK, while establishing roles for p38 MAPK and NF-κB in the potentiation of GM-CSF release. Detection of significant p38 phosphorylation in response to serum stimulation, through Western blotting, further demonstrated the involvement of p38. These studies have provided evidence to support p38 being targeted to interrupt the cycle of inflammation, vascular leakage and cytokine production in asthma.

Liver disease affects large numbers of patients, yet there are limited treatments available to replace absent or ineffective cellular function of this crucial organ. Donor scarcity and the necessity for immunosuppression limit one effective therapy, orthotopic liver transplantation. But in some conditions such as inborn errors of metabolism or transient states of liver insufficiency, patients may be salvaged by providing partial quantities of functional liver tissue. After transplanting multicellular liver organoid units composed of a heterogeneous cellular population that includes adult stem and progenitor cells, both mouse and human tissue‐engineered liver (TELi) form in vivo. TELi contains normal liver components such as hepatocytes with albumin expression, CK19‐expressing bile ducts and vascular structures with α‐smooth muscle actin expression, desmin‐expressing stellate cells, and CD31‐expressing endothelial cells. At 4 weeks, TELi contains proliferating albumin‐expressing cells and identification of β2‐microglobulin‐expressing cells demonstrates that the majority of human TELi is composed of transplanted human cells. Human albumin is detected in the host mouse serum, indicating in vivo secretory function. Liquid chromatography/mass spectrometric analysis of mouse serum after debrisoquine administration is followed by a significant increase in the level of the human metabolite, 4‐OH‐debrisoquine, which supports the metabolic and xenobiotic capability of human TELi in vivo. Implanted TELi grew in a mouse model of inducible liver failure. Stem Cells Translational Medicine 2017;6:238–248