Stable transduction of the neonatal mouse liver using a hybrid rAAV/sleeping beauty transposon gene delivery system.

BACKGROUND: Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty, has been widely used for ex vivo gene delivery; however liver-targeted delivery using a hybrid rAAV/Sleeping Beauty approach remains relatively unexplored. METHODS: We investigated the capacity of a Sleeping Beauty (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC). RESULTS: At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient Spfash mouse following elimination of residual endogenous murine OTC. CONCLUSIONS: The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset.

Recapitulation of Skewed X-Inactivation in Female Ornithine Transcarbamylase-Deficient Primary Human Hepatocytes in the FRG Mouse: A Novel System for Developing Epigenetic Therapies.

Realization of the immense therapeutic potential of epigenetic editing requires development of clinically predictive model systems that faithfully recapitulate relevant aspects of the target disease pathophysiology. In female patients with ornithine transcarbamylase (OTC) deficiency, an X-linked condition, skewed inactivation of the X chromosome carrying the wild-type OTC allele is associated with increased disease severity. The majority of affected female patients can be managed medically, but a proportion require liver transplantation. With rapid development of epigenetic editing technology, reactivation of silenced wild-type OTC alleles is becoming an increasingly plausible therapeutic approach. Toward this end, privileged access to explanted diseased livers from two affected female infants provided the opportunity to explore whether engraftment and expansion of dissociated patient-derived hepatocytes in the FRG mouse might produce a relevant model for evaluation of epigenetic interventions. Hepatocytes from both infants were successfully used to generate chimeric mouse-human livers, in which clusters of primary human hepatocytes were either OTC positive or negative by immunohistochemistry (IHC), consistent with clonal expansion from individual hepatocytes in which the mutant or wild-type OTC allele was inactivated, respectively. Enumeration of the proportion of OTC-positive or -negative human hepatocyte clusters was consistent with dramatic skewing in one infant and minimal to modest skewing in the other. Importantly, IHC and fluorescence-activated cell sorting analysis of intact and dissociated liver samples from both infants showed qualitatively similar patterns, confirming that the chimeric mouse-human liver model recapitulated the native state in each infant. Also of importance was the induction of a treatable metabolic phenotype, orotic aciduria, in mice, which correlated with the presence of clonally expanded OTC-negative primary human hepatocytes. We are currently using this unique model to explore CRISPR-dCas9-based epigenetic targeting strategies in combination with efficient adeno-associated virus (AAV) gene delivery to reactivate the silenced functional OTC gene on the inactive X chromosome.

OTC intron 4 variations mediate pathogenic splicing patterns caused by the c.386G>A mutation in humans and spfash mice, and govern susceptibility to RNA-based therapies.

BACKGROUND: Aberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context. Therefore, the evaluation of the functional, and potentially pathological, role of nucleotide changes remains one of the major challenges in the modern genomic era. This aspect has also to be taken into account during the pre-clinical evaluation of innovative therapeutic approaches in animal models of human diseases. This is of particular relevance when developing therapeutics acting on splicing, an intriguing and expanding research area for several disorders. Here, we addressed species-specific splicing mechanisms triggered by the OTC c.386G>A mutation, relatively frequent in humans, leading to Ornithine TransCarbamylase Deficiency (OTCD) in patients and spfash mice, and its differential susceptibility to RNA therapeutics based on engineered U1snRNA. METHODS: Creation and co-expression of engineered U1snRNAs with human and mouse minigenes, either wild-type or harbouring different nucleotide changes, in human (HepG2) and mouse (Hepa1-6) hepatoma cells followed by analysis of splicing pattern. RNA pulldown studies to evaluate binding of specific splicing factors. RESULTS: Comparative nucleotide analysis suggested a role for the intronic +10-11 nucleotides, and pull-down assays showed that they confer preferential binding to the TIA1 splicing factor in the mouse context, where TIA1 overexpression further increases correct splicing. Consistently, the splicing profile of the human minigene with mouse +10-11 nucleotides overlapped that of mouse minigene, and restored responsiveness to TIA1 overexpression and to compensatory U1snRNA. Swapping the human +10-11 nucleotides into the mouse context had opposite effects. Moreover, the interplay between the authentic and the adjacent cryptic 5'ss in the human OTC dictates pathogenic mechanisms of several OTCD-causing 5'ss mutations, and only the c.386+5G>A change, abrogating the cryptic 5'ss, was rescuable by engineered U1snRNA. CONCLUSIONS: Subtle intronic variations explain species-specific OTC splicing patterns driven by the c.386G>A mutation, and the responsiveness to engineered U1snRNAs, which suggests careful elucidation of molecular mechanisms before proposing translation of tailored therapeutics from animal models to humans.

Circadian oscillation expression of ornithine carbamoyltransferase and its significance in sleep disturbance.

Ornithine transcarbamylases (OTC), a key enzyme in urea cycle, is an important marker for some liver injury or diseases. However, whether OTC could be a sensitive indicator for liver dysfunction under sleep disturbance condition remains unknown. The present study aimed to explore the circadian oscillation expression of OTC and its significance in disturbed sleep condition. Sleep disturbance was conducted by a sleep deprivation (SD) instrument. Our results found that SD for 72h induced abnormal increasing of OTC levels in serum and liver of rats. And, serum OTC concentration and liver OTC expression could return to normal levels after recovery sleep following SD. Moreover, hepatic OTC expression showed circadian oscillation in day and night, characterized with occurrence of a peak between ZT 22 and ZT 2, and a nadir between ZT 14 and ZT 18. Further analysis suggested the existence of ROR response element (RORE) for potential RORɑ binding sites in OTC promoter region, and elevated RORɑ expression in rat livers under sleep disturbance condition. Additionally, oscillation expression of OTC induced by serum shock in HepG2 cells was characterized with a peak occurred between ZT 12 and ZT 16, and RORɑ knockdown at ZT 16 significantly lowered OTC expression. The results together indicate that OTC is closely correlated with circadian clock, and could be a sensitive indicator for sleep disturbance stress.

Enzymology of the pathway for ATP production by arginine breakdown.

In cells, the breakdown of arginine to ornithine and ammonium ion plus carbon dioxide is coupled to the generation of metabolic energy in the form of ATP. The arginine breakdown pathway is minimally composed of arginine deiminase, ornithine transcarbamoylase, carbamate kinase, and an arginine/ornithine antiporter; ammonia and carbon dioxide most likely diffuse passively across the membrane. The genes for the enzymes and transporter have been cloned and expressed, and the proteins have been purified from Lactococcus lactis IL1403 and incorporated into lipid vesicles for sustained production of ATP. Here, we study the kinetic parameters and biochemical properties of the individual enzymes and the antiporter, and we determine how the physicochemical conditions, effector composition, and effector concentration affect the enzymes. We report the KM and VMAX values for catalysis and the native oligomeric state of all proteins, and we measured the effect of pathway intermediates, pH, temperature, freeze-thaw cycles, and salts on the activity of the cytosolic enzymes. We also present data on the protein-to-lipid ratio and lipid composition dependence of the antiporter.

The otc gene of Streptococcus suis plays an important role in biofilm formation, adhesion, and virulence in a murine model.

Streptococcus suis (S. suis) is an emerging zoonotic pathogen that can cause meningitis, arthritis, pneumonia, and sepsis. It poses a serious threat to the swine industry and public health worldwide. Ornithine carbamoyltransferase (OTC) is involved in the arginine deiminase system. OTC, which is a widely distributed enzyme in microorganisms, mammals, and higher plants, catalyzes the conversion of ornithine to citrulline. The present study showed that the otc gene plays an important role in the pathogenesis of S. suis infections. The ability of an otc-deficient mutant (Δotc) to form a biofilm was significantly reduced compared to the wild-type (WT) strain, as determined by crystal violet staining. Confocal laser scanning microscopy and scanning electron microscopy observations showed that the weakening of biofilm formation by the Δotc strain is related to a decrease in the extracellular matrix. In addition, compared to the WT strain, the Δotc strain had a reduced capacity to adhere to human laryngeal epidermoid carcinoma (HEp-2) cells compared to the WT strain. A real-time PCR analysis showed that the expression of adhesion-related genes by the Δotc strain was also lower than that of the WT strain. The virulence of the Δotc strain was significantly lower than that of the WT strain in a murine infection model. In addition, a histological analysis showed that the pathogenicity of the Δotc strain was lower than that of the WT strain, causing only slight inflammatory lesions in lung, liver, spleen, and kidney tissues. No significant differences were observed between the complemented mutant (CΔotc) and WT strains with respect to biofilm formation, adhesion, gene expression, and virulence. The present study provided evidence that the otc gene plays a pivotal role in the regulation of S. suis adhesion and biofilm formation. It also suggested that the otc gene is indirectly involved in the pathogenesis of S. suis serotype 2 infections.

An Exon-Specific Small Nuclear U1 RNA (ExSpeU1) Improves Hepatic OTC Expression in a Splicing-Defective spf/ash Mouse Model of Ornithine Transcarbamylase Deficiency.

OTC splicing mutations are generally associated with the severest and early disease onset of ornithine transcarbamylase deficiency (OTCD), the most common urea cycle disorder. Noticeably, splicing defects can be rescued by spliceosomal U1snRNA variants, which showed their efficacy in cellular and animal models. Here, we challenged an U1snRNA variant in the OTCD mouse model (spf/ash) carrying the mutation c.386G > A (p.R129H), also reported in OTCD patients. It is known that the R129H change does not impair protein function but affects pre-mRNA splicing since it is located within the 5' splice site. Through in vitro studies, we identified an Exon Specific U1snRNA (ExSpeU1O3) that targets an intronic region downstream of the defective exon 4 and rescues exon inclusion. The adeno-associated virus (AAV8)-mediated delivery of the ExSpeU1O3 to mouse hepatocytes, although in the presence of a modest transduction efficiency, led to increased levels of correct OTC transcripts (from 6.1 ± 1.4% to 17.2 ± 4.5%, p = 0.0033). Consistently, this resulted in increased liver expression of OTC protein, as demonstrated by Western blotting (~3 fold increase) and immunostaining. Altogether data provide the early proof-of-principle of the efficacy of ExSpeU1 in the spf/ash mouse model and encourage further studies to assess the potential of RNA therapeutics for OTCD caused by aberrant splicing.

Metabolic engineering against the arginine microenvironment enhances CAR-T cell proliferation and therapeutic activity.

Hematological and solid cancers catabolize the semiessential amino acid arginine to drive cell proliferation. However, the resulting low arginine microenvironment also impairs chimeric antigen receptor T cells (CAR-T) cell proliferation, limiting their efficacy in clinical trials against hematological and solid malignancies. T cells are susceptible to the low arginine microenvironment because of the low expression of the arginine resynthesis enzymes argininosuccinate synthase (ASS) and ornithine transcarbamylase (OTC). We demonstrate that T cells can be reengineered to express functional ASS or OTC enzymes, in concert with different chimeric antigen receptors. Enzyme modifications increase CAR-T cell proliferation, with no loss of CAR cytotoxicity or increased exhaustion. In vivo, enzyme-modified CAR-T cells lead to enhanced clearance of leukemia or solid tumor burden, providing the first metabolic modification to enhance CAR-T cell therapies.

A mutation-independent CRISPR-Cas9-mediated gene targeting approach to treat a murine model of ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) deficiency is an X-linked urea cycle disorder associated with high mortality. Although a promising treatment for late-onset OTC deficiency, adeno-associated virus (AAV) neonatal gene therapy would only provide short-term therapeutic effects as the non-integrated genome gets lost during hepatocyte proliferation. CRISPR-Cas9-mediated homology-directed repair can correct a G-to-A mutation in 10% of OTC alleles in the livers of newborn OTC spfash mice. However, an editing vector able to correct one mutation would not be applicable for patients carrying different OTC mutations, plus expression would not be fast enough to treat a hyperammonemia crisis. Here, we describe a dual-AAV vector system that accomplishes rapid short-term expression from a non-integrated minigene and long-term expression from the site-specific integration of this minigene without any selective growth advantage for OTC-positive cells in newborns. This CRISPR-Cas9 gene-targeting approach may be applicable to all patients with OTC deficiency, irrespective of mutation and/or clinical state.

p53 regulation of ammonia metabolism through urea cycle controls polyamine biosynthesis.

Cancer cells exhibit altered and usually increased metabolic processes to meet their high biogenetic demands1,2. Under these conditions, ammonia is concomitantly produced by the increased metabolic processing. However, it is unclear how tumour cells dispose of excess ammonia and what outcomes might be caused by the accumulation of ammonia. Here we report that the tumour suppressor p53, the most frequently mutated gene in human tumours, regulates ammonia metabolism by repressing the urea cycle. Through transcriptional downregulation of CPS1, OTC and ARG1, p53 suppresses ureagenesis and elimination of ammonia in vitro and in vivo, leading to the inhibition of tumour growth. Conversely, downregulation of these genes reciprocally activates p53 by MDM2-mediated mechanism(s). Furthermore, the accumulation of ammonia causes a significant decline in mRNA translation of the polyamine biosynthetic rate-limiting enzyme ODC, thereby inhibiting the biosynthesis of polyamine and cell proliferation. Together, these findings link p53 to ureagenesis and ammonia metabolism, and further reveal a role for ammonia in controlling polyamine biosynthesis and cell proliferation.

Urea Cycle Dysregulation Generates Clinically Relevant Genomic and Biochemical Signatures.

The urea cycle (UC) is the main pathway by which mammals dispose of waste nitrogen. We find that specific alterations in the expression of most UC enzymes occur in many tumors, leading to a general metabolic hallmark termed "UC dysregulation" (UCD). UCD elicits nitrogen diversion toward carbamoyl-phosphate synthetase2, aspartate transcarbamylase, and dihydrooratase (CAD) activation and enhances pyrimidine synthesis, resulting in detectable changes in nitrogen metabolites in both patient tumors and their bio-fluids. The accompanying excess of pyrimidine versus purine nucleotides results in a genomic signature consisting of transversion mutations at the DNA, RNA, and protein levels. This mutational bias is associated with increased numbers of hydrophobic tumor antigens and a better response to immune checkpoint inhibitors independent of mutational load. Taken together, our findings demonstrate that UCD is a common feature of tumors that profoundly affects carcinogenesis, mutagenesis, and immunotherapy response.

Variable X-chromosome inactivation and enlargement of pericentral glutamine synthetase zones in the liver of heterozygous females with OTC deficiency.

Ornithine transcarbamylase (OTC) deficiency is an X-linked disorder that causes recurrent and life-threatening episodes of hyperammonemia. The clinical picture in heterozygous females is highly diverse and derives from the genotype and the degree of inactivation of the mutated X chromosome in hepatocytes. Here, we describe molecular genetic, biochemical, and histopathological findings in the livers explanted from two female patients with late-onset OTC deficiency. Analysis of X-inactivation ratios by DNA methylation-based assays showed remarkable intra-organ variation ranging from 46:54 to 82:18 (average 70:30, n = 37), in favor of the active X chromosome carrying the mutation c.583G>C (p.G195R), in the first patient and from 75:25 to 90:10 (average 82:18, n = 20) in favor of the active X chromosome carrying the splicing mutation c.663+1G>A in the second patient. The X-inactivation ratios in liver samples correlated highly with the proportions of OTC-positive hepatocytes calculated from high-resolution image analyses of the immunohistochemically detected OTC in frozen sections that was performed on total area > 5 cm2. X-inactivation ratios in blood in both female patients corresponded to the lower limit of the liver values. Our data indicate that the proportion of about 20-30% of hepatocytes expressing the functional OTC protein is not sufficient to maintain metabolic stability. X-inactivation ratios assessed in liver biopsies taken from heterozygous females with X-linked disorders should not be considered representative of the whole liver.

Optimizing combination of liver-enriched transcription factors and nuclear receptors simultaneously favors ammonia and drug metabolism in liver cells.

The HepG2 cell line is widely used in studying liver diseases because of its immortalization, but its clinical application is limited by its low expression of the urea synthesis key enzymes and cytochromes P450 (CYPs). On the basis of our previous work, we investigated the transcriptional regulation of arginase 1 (Arg1) and ornithine transcarbamylase (OTC) in HepG2 cells. We also screened for the optimal combination of liver enrichment transcription factors (LETFs) and xenobiotic nuclear receptors that can promote the expression of key urea synthases and five major CYPs in HepG2 cells. Thus, recombinant HepG2 cells were established. Results showed that C/EBPß, not C/EBPα, could upregulate expression of Arg1 and PGC1α and HNF4α cooperatively regulate the expression of OTC. The two optimal combinations C/EBPß+HNF4α+HNF6+PXR and C/EBPß+HNF4α+HNF6+CAR were selected. Compared with the control cells, the recombinant HepG2 cells modified by the two optimal combinations exhibited enhanced ammonia metabolism and CYP enzyme activity. Moreover, the HepG2/(C/EBPß+HNF4α+HNF6+PXR) cells more strongly reduced ammonia than any other combination tested in this study. The present work indicated that optimizing the combination of transcription factors will simultaneously promote hepatocyte ammonia metabolism and drug metabolism. The recombinant HepG2 liver cell line constructed by the optimal combination provided an improved alternative means for bioartificial liver applications and drug toxicity testing.

Alternative Start Sites Downstream of Non-Sense Mutations Drive Antigen Presentation and Tolerance Induction to C-Terminal Epitopes.

CTL responses to the transgene product remain an active area of concern for the gene therapy field. A patient's underlying genetic mutation may influence the qualitative nature of these potentially destructive T cell responses. Individuals with a mutation that introduces a premature termination codon (PTC) that prevents synthesis of the full-length peptide are considered more likely to mount a transgene-specific T cell response because of a lack of immune tolerance to C-terminal epitopes as a consequence of absent endogenous Ag presentation. In this article, we demonstrate that a human ornithine transcarbamylase gene containing various PTC-inducing non-sense mutations is able to generate and present epitopes downstream of the termination codon. Generation of these epitopes occurs primarily from alternative translation start sites downstream of the stop codon. Furthermore, we show that expression of these genes from adeno-associated virus vectors in C57BL/6 mice is able to induce peripheral tolerance to epitopes downstream of the PTC. These results suggest that, despite the lack of full-length endogenous protein, patients with PTC-inducing non-sense mutations may still present T cell epitopes downstream of the premature termination site that may render the subject tolerant to wild-type transgene products.

AAV gene therapy corrects OTC deficiency and prevents liver fibrosis in aged OTC-knock out heterozygous mice.

Ornithine transcarbamylase (OTC) deficiency is an X-linked disorder of the urea cycle. Hemizygous males and heterozygous females may experience life-threatening elevations of ammonia in blood and brain, leading to irreversible cognitive impairment, coma, and death. Recent evidence of acute liver failure and fibrosis/cirrhosis is also emerging in OTC-deficient patients. Here, we investigated the long-term consequences of abnormal ureagenesis in female mice heterozygous (Het) for a null mutation in the OTC gene. Two-month-old Het OTC knockout (KO) mice received a single dose of self-complementary adeno-associated virus (AAV) encoding a codon-optimized human OTC gene at 1×1010, 3×1010, or 1×1011 vector genome copies per mouse. We compared liver pathology from 18-month-old treated Het OTC-KO mice, age-matched untreated Het OTC-KO mice, and WT littermates, and assessed urinary orotic acid levels and vector genome copies in liver at 4, 10, and 16months following vector administration. Het OTC-KO female mice showed evidence of liver inflammation and the eventual development of significant fibrosis. Treatment with AAV gene therapy not only corrected the underlying metabolic abnormalities, but also prevented the development of liver fibrosis. Our study demonstrates that early treatment of OTC deficiency with gene therapy may prevent clinically relevant consequences of chronic liver damage from developing.

Effects of Self-Complementarity, Codon Optimization, Transgene, and Dose on Liver Transduction with AAV8.

Numerous methods of vector design and delivery have been employed in an attempt to increase transgene expression following AAV-based gene therapy. Here, a gene transfer study was conducted in mice to compare the effects of vector self-complementarity (double- or single-stranded DNA), codon optimization of the transgene, and vector dose on transgene expression levels in the liver. Two different reporter genes were used: human ornithine transcarbamylase (hOTC) detected by immunofluorescence, and enhanced green fluorescent protein (EGFP) detected by direct fluorescence. The AAV8 capsid was chosen for all experiments due to its strong liver tropism. While EGFP is already a codon-optimized version of the original gene, both wild-type (WT) and codon-optimized (co) versions of the hOTC transgene were compared in this study. In addition, the study evaluated which of the two hOTC modifications-codon optimization or self-complementarity-would confer the highest increase in expression levels at a given dose. Interestingly, based on morphometric image analysis, it was observed that the difference in detectable expression levels between self-complementary (sc) and single-stranded (ss) hOTCco vectors was dose dependent, with a sevenfold increase in OTC-positive area using sc vectors at a dose of 3 × 109 genome copies (GC) per mouse, but no significant difference at a dose of 1 × 1010 GC/mouse. In contrast, with EGFP as a transgene, the increases in expression levels when using the sc vector were observed at both the 3 × 109 GC/mouse and 1 × 1010 GC/mouse doses. Furthermore, codon optimization of the hOTC transgene generated a more significant improvement in expression than the use of self-complementarity did. Overall, the results demonstrate that increases in expression levels gained by using sc vectors instead of ss vectors can vary between different transgenes, and that codon optimization of the transgene can have an even more powerful effect on the resulting expression levels.

A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice.

Many genetic liver diseases in newborns cause repeated, often lethal, metabolic crises. Gene therapy using nonintegrating viruses such as adeno-associated virus (AAV) is not optimal in this setting because the nonintegrating genome is lost as developing hepatocytes proliferate. We reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR-Cas9. Here we intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylase (OTC). This resulted in reversion of the mutation in 10% (6.7-20.1%) of hepatocytes and increased survival in mice challenged with a high-protein diet, which exacerbates disease. Gene correction in adult OTC-deficient mice was lower and accompanied by larger deletions that ablated residual expression from the endogenous OTC gene, leading to diminished protein tolerance and lethal hyperammonemia on a chow diet.

Cell-sized condensed collagen microparticles for preparing microengineered composite spheroids of primary hepatocytes.

The reconstitution of extracellular matrix (ECM) components in three-dimensional (3D) cell culture environments with microscale precision is a challenging issue. ECM microparticles would potentially be useful as solid particulate scaffolds that can be incorporated into 3D cellular constructs, but technologies for transforming ECM proteins into cell-sized stable particles are currently lacking. Here, we describe new processes to produce highly condensed collagen microparticles by means of droplet microfluidics or membrane emulsification. Droplets of an aqueous solution of type I collagen were formed in a continuous phase of polar organic solvent followed by rapid dissolution of water molecules into the continuous phase because the droplets were in a non-equilibrium state. We obtained highly unique, disc-shaped condensed collagen microparticles with a final collagen concentration above 10% and examined factors affecting particle size and morphology. After testing the cell-adhesion properties on the collagen microparticles, composite multicellular spheroids comprising the particles and primary rat hepatocytes were formed using microfabricated hydrogel chambers. We found that the ratio of the cells and particles is critical in terms of improvement of hepatic functions in the composite spheroids. The presented methodology for incorporating particulate-form ECM components in multicellular spheroids would be advantageous because of the biochemical similarity with the microenvironments in vivo.

Dietary arginine requirements for growth are dependent on the rate of citrulline production in mice.

BACKGROUND: In many species, including humans, arginine is considered a semiessential amino acid because under certain conditions endogenous synthesis cannot meet its demand. The requirements of arginine for growth in mice are ill defined and seem to vary depending on the genetic background of the mice. OBJECTIVE: The objective of this study was to determine the metabolic and molecular basis for the requirement of arginine in 2 mouse strains. METHODS: Institute of Cancer Research (ICR) and C57BL/6 (BL6) male mice were fed arginine-free or arginine-sufficient diets (Expt. 1) or 1 of 7 diets with increasing arginine concentration (from 0- to 8-g/kg diet, Expt. 2) between day 24 and 42 of life to determine the arginine requirements for growth. Citrulline production and "de novo" arginine synthesis were measured with use of stable isotopes, and arginine requirements were determined by breakpoint analysis and enzyme expression by reverse transcriptase-polymerase chain reaction. RESULTS: In Expt. 1, ICR mice grew at the same rate regardless of the arginine concentration of the diet (mean ± SE: 0.66 ± 0.04 g/d, P = 0.80), but BL6 mice had a reduced growth rate when fed the arginine-free diet (0.25 ± 0.02 g/d, P < 0.001) compared to the 8-g arginine/kg diet (0.46 ± 0.03 g/d). ICR mice showed at least a 2-fold greater expression (P < 0.001) of ornithine transcarbamylase (OTC) than BL6 mice, which translated into a greater rate of citrulline (25%) and arginine synthesis (49%, P < 0.002). In Expt. 2, breakpoint analysis showed that the requirement for growth of BL6 mice was met with 2.32 ± 0.39 g arginine/kg diet; for ICR mice, however, no breakpoint was found. CONCLUSION: Our data indicate that a reduced expression of OTC in BL6 mice translates into a reduced production of citrulline and arginine compared with ICR mice, which results in a dietary arginine requirement for growth in BL6 mice, but not in ICR mice.

Fatal hyperammonemia and carbamoyl phosphate synthetase 1 (CPS1) deficiency following high-dose chemotherapy and autologous hematopoietic stem cell transplantation.

Fatal hyperammonemia secondary to chemotherapy for hematological malignancies or following bone marrow transplantation has been described in few patients so far. In these, the pathogenesis of hyperammonemia remained unclear and was suggested to be multifactorial. We observed severe hyperammonemia (maximum 475 µmol/L) in a 2-year-old male patient, who underwent high-dose chemotherapy with carboplatin, etoposide and melphalan, and autologous hematopoietic stem cell transplantation for a neuroblastoma stage IV. Despite intensive care treatment, hyperammonemia persisted and the patient died due to cerebral edema. The biochemical profile with elevations of ammonia and glutamine (maximum 1757 µmol/L) suggested urea cycle dysfunction. In liver homogenates, enzymatic activity and protein expression of the urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) were virtually absent. However, no mutation was found in CPS1 cDNA from liver and CPS1 mRNA expression was only slightly decreased. We therefore hypothesized that the acute onset of hyperammonemia was due to an acquired, chemotherapy-induced (posttranscriptional) CPS1 deficiency. This was further supported by in vitro experiments in HepG2 cells treated with carboplatin and etoposide showing a dose-dependent decrease in CPS1 protein expression. Due to severe hyperlactatemia, we analysed oxidative phosphorylation complexes in liver tissue and found reduced activities of complexes I and V, which suggested a more general mitochondrial dysfunction. This study adds to the understanding of chemotherapy-induced hyperammonemia as drug-induced CPS1 deficiency is suggested. Moreover, we highlight the need for urgent diagnostic and therapeutic strategies addressing a possible secondary urea cycle failure in future patients with hyperammonemia during chemotherapy and stem cell transplantation.