Large-scale Pan-cancer Cell Line Screening Identifies Actionable and Effective Drug Combinations.

Oncology drug combinations can improve therapeutic responses and increase treatment options for patients. The number of possible combinations is vast and responses can be context-specific. Systematic screens can identify clinically relevant, actionable combinations in defined patient subtypes. We present data for 109 anticancer drug combinations from AstraZeneca's oncology small molecule portfolio screened in 755 pan-cancer cell lines. Combinations were screened in a 7 × 7 concentration matrix, with more than 4 million measurements of sensitivity, producing an exceptionally data-rich resource. We implement a new approach using combination Emax (viability effect) and highest single agent (HSA) to assess combination benefit. We designed a clinical translatability workflow to identify combinations with clearly defined patient populations, rationale for tolerability based on tumor type and combination-specific "emergent" biomarkers, and exposures relevant to clinical doses. We describe three actionable combinations in defined cancer types, confirmed in vitro and in vivo, with a focus on hematologic cancers and apoptotic targets. SIGNIFICANCE: We present the largest cancer drug combination screen published to date with 7 × 7 concentration response matrices for 109 combinations in more than 750 cell lines, complemented by multi-omics predictors of response and identification of "emergent" combination biomarkers. We prioritize hits to optimize clinical translatability, and experimentally validate novel combination hypotheses. This article is featured in Selected Articles from This Issue, p. 695.

Capivasertib combines with docetaxel to enhance anti-tumour activity through inhibition of AKT-mediated survival mechanisms in prostate cancer.

BACKGROUND/OBJECTIVE: To explore the anti-tumour activity of combining AKT inhibition and docetaxel in PTEN protein null and WT prostate tumours. METHODS: Mechanisms associated with docetaxel capivasertib treatment activity in prostate cancer were examined using a panel of in vivo tumour models and cell lines. RESULTS: Combining docetaxel and capivasertib had increased activity in PTEN null and WT prostate tumour models in vivo. In vitro short-term docetaxel treatment caused cell cycle arrest in the majority of cells. However, a sub-population of docetaxel-persister cells did not undergo G2/M arrest but upregulated phosphorylation of PI3K/AKT pathway effectors GSK3ß, p70S6K, 4E-BP1, but to a lesser extent AKT. In vivo acute docetaxel treatment induced p70S6K and 4E-BP1 phosphorylation. Treating PTEN null and WT docetaxel-persister cells with capivasertib reduced PI3K/AKT pathway activation and cell cycle progression. In vitro and in vivo it reduced proliferation and increased apoptosis or DNA damage though effects were more marked in PTEN null cells. Docetaxel-persister cells were partly reliant on GSK3ß as a GSK3ß inhibitor AZD2858 reversed capivasertib-induced apoptosis and DNA damage. CONCLUSION: Capivasertib can enhance anti-tumour effects of docetaxel by targeting residual docetaxel-persister cells, independent of PTEN status, to induce apoptosis and DNA damage in part through GSK3ß.

Cells transiently expressing periostin are required for intramedullary intramembranous bone regeneration.

Intramembranous bone regeneration plays an important role in fixation of intramedullary implants used in joint replacement and dental implants used in tooth replacement. Despite widespread recognition of the importance of intramembranous bone regeneration in these clinical procedures, the underlying mechanisms have not been well explored. A previous study that examined transcriptomic profiles of regenerating bone from the marrow space showed that increased periostin gene expression preceded increases in several osteogenic genes. We therefore sought to determine the role of cells transiently expressing periostin in intramedullary intramembranous bone regeneration. We used a genetic mouse model that allows tamoxifen-inducible fluorescent labeling of periostin expressing cells. These mice underwent ablation of the bone marrow cavity through surgical disruption, a well-established intramembranous bone regeneration model. We found that in intact bones, fluorescently labeled cells were largely restricted to the periosteal surface of cortical bone and were absent in bone marrow. However, following surgical disruption of the bone marrow cavity, cells transiently expressing periostin were found within the regenerating tissue of the bone marrow compartment even though the cortical bone remained intact. The source of these cells is likely heterogenous, including cells occupying the periosteal surface as well as pericytes and endothelial cells within the marrow cavity. We also found that diphtheria toxin-mediated depletion of cells transiently expressing periostin at the time of surgery impaired intramembranous bone regeneration in mice. These data suggest a critical role of periostin expressing cells in intramedullary intramembranous bone regeneration and may lead to novel therapeutic interventions to accelerate or enhance implant fixation.

Combining the AKT inhibitor capivasertib and SERD fulvestrant is effective in palbociclib-resistant ER+ breast cancer preclinical models.

Combining the selective AKT inhibitor, capivasertib, and SERD, fulvestrant improved PFS in a Phase III clinical trial (CAPItello-291), treating HR+ breast cancer patients following aromatase inhibitors, with or without CDK4/6 inhibitors. However, clinical data suggests CDK4/6 treatment may reduce response to subsequent monotherapy endocrine treatment. To support understanding of trials such as CAPItello-291 and gain insight into this emerging population of patients, we explored how CDK4/6 inhibitor treatment influences ER+ breast tumour cell function and response to fulvestrant and capivasertib after CDK4/6 inhibitor treatment. In RB+, RB- T47D and MCF7 palbociclib-resistant cells ER pathway ER and Greb-1 expression were reduced versus naïve cells. PI3K-AKT pathway activation was also modified in RB+ cells, with capivasertib less effective at reducing pS6 in RB+ cells compared to parental cells. Expression profiling of parental versus palbociclib-resistant cells confirmed capivasertib, fulvestrant and the combination differentially impacted gene expression modulation in resistant cells, with different responses seen in T47D and MCF7 cells. Fulvestrant inhibition of ER-dependent genes was reduced. In resistant cells, the combination was less effective at reducing cell cycle genes, but a consistent reduction in cell fraction in S-phase was observed in naïve and resistant cells. Despite modified signalling responses, both RB+ and RB- resistant cells responded to combination treatment despite some reduction in relative efficacy and was effective in vivo in palbociclib-resistant PDX models. Collectively these findings demonstrate that simultaneous inhibition of AKT and ER signalling can be effective in models representing palbociclib resistance despite changes in pathway dependency.

Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity.

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for "correcting" variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.

mTOR inhibition amplifies the anti-lymphoma effect of PI3Kß/δ blockage in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease that exhibits constitutive activation of phosphoinositide 3-kinase (PI3K) driven by chronic B-cell receptor signaling or PTEN deficiency. Since pan-PI3K inhibitors cause severe side effects, we investigated the anti-lymphoma efficacy of the specific PI3Kß/δ inhibitor AZD8186. We identified a subset of DLBCL models within activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL that were sensitive to AZD8186 treatment. On the molecular level, PI3Kß/δ inhibition decreased the pro-survival NF-κB and AP-1 activity or led to downregulation of the oncogenic transcription factor MYC. In AZD8186-resistant models, we detected a feedback activation of the PI3K/AKT/mTOR pathway following PI3Kß/δ inhibition, which limited AZD8186 efficacy. The combined treatment with AZD8186 and the mTOR inhibitor AZD2014 overcame resistance to PI3Kß/δ inhibition and completely prevented outgrowth of lymphoma cells in vivo in cell line- and patient-derived xenograft mouse models. Collectively, our study reveals that subsets of DLBCLs are addicted to PI3Kß/δ signaling and thus identifies a previously unappreciated role of the PI3Kß isoform in DLBCL survival. Furthermore, our data demonstrate that combined targeting of PI3Kß/δ and mTOR is effective in all major DLBCL subtypes supporting the evaluation of this strategy in a clinical trial setting.

A novel DPH5-related diphthamide-deficiency syndrome causing embryonic lethality or profound neurodevelopmental disorder.

PURPOSE: Diphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs). METHODS: Molecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5em1Mbp/Mmucd) was created for a DPH5 p.His260Arg homozygous variant identified in 1 family. Adenosine diphosphate-ribosylation assays in DPH5-knockout human and yeast cells and in silico modeling were performed for the identified DPH5 potential pathogenic variants. RESULTS: DPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate-ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2. CONCLUSION: We provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies.

Experiments from unfinished Registered Reports in the Reproducibility Project: Cancer Biology.

As part of the Reproducibility Project: Cancer Biology, we published Registered Reports that described how we intended to replicate selected experiments from 29 high-impact preclinical cancer biology papers published between 2010 and 2012. Replication experiments were completed and Replication Studies reporting the results were submitted for 18 papers, of which 17 were accepted and published by eLife with the rejected paper posted as a preprint. Here, we report the status and outcomes obtained for the remaining 11 papers. Four papers initiated experimental work but were stopped without any experimental outcomes. Two papers resulted in incomplete outcomes due to unanticipated challenges when conducting the experiments. For the remaining five papers only some of the experiments were completed with the other experiments incomplete due to mundane technical or unanticipated methodological challenges. The experiments from these papers, along with the other experiments attempted as part of the Reproducibility Project: Cancer Biology, provides evidence about the challenges of repeating preclinical cancer biology experiments and the replicability of the completed experiments.

Loss of miR-29a/b1 promotes inflammation and fibrosis in acute pancreatitis.

MicroRNA-29 (miR-29) is a critical regulator of fibroinflammatory processes in human diseases. In this study, we found a decrease in miR-29a in experimental and human chronic pancreatitis, leading us to investigate the regulatory role of the miR-29a/b1 cluster in acute pancreatitis (AP) utilizing a conditional miR-29a/b1-KO mouse model. miR-29a/b1-sufficient (WT) and -deficient (KO) mice were administered supramaximal caerulein to induce AP and characterized at different time points, utilizing an array of IHC and biochemical analyses for AP parameters. In caerulein-induced WT mice, miR-29a remained dramatically downregulated at injury. Despite high-inflammatory milieu, fibrosis, and parenchymal disarray in the WT mice during early AP, the pancreata fully restored during recovery. miR-29a/b1-KO mice showed significantly greater inflammation, lymphocyte infiltration, macrophage polarization, and ECM deposition, continuing until late recovery with persistent parenchymal disorganization. The increased pancreatic fibrosis was accompanied by enhanced TGFß1 coupled with persistent αSMA+ PSC activation. Additionally, these mice exhibited higher circulating IL-6 and inflammation in lung parenchyma. Together, this collection of studies indicates that depletion of miR-29a/b1 cluster impacts the fibroinflammatory mechanisms of AP, resulting in (a) aggravated pathogenesis and (b) delayed recovery from the disease, suggesting a protective role of the molecule against AP.

A phase 1/2 study of the combination of acalabrutinib and vistusertib in patients with relapsed/refractory B-cell malignancies.

In a phase 1b study of acalabrutinib (a covalent Bruton tyrosine kinase (BTK) inhibitor) in combination with vistusertib (a dual mTORC1/2 inhibitor) in patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL), multiple ascending doses of the combination as intermittent or continuous schedules of vistusertib were evaluated. The overall response rate was 12% (3/25). The pharmacodynamic (PD) profile for acalabrutinib showed that BTK occupancy in all patients was >95%. In contrast, PD analysis for vistusertib showed variable inhibition of phosphorylated 4EBP1 (p4EBP1) without modulation of AKT phosphorylation (pAKT). The pharmacokinetic (PK)/PD relationship of vistusertib was direct for TORC1 inhibition (p4EBP1) but did not correlate with TORC2 inhibition (pAKT). Cell-of-origin subtyping or next-generation sequencing did not identify a subset of DLBCL patients with clinical benefit; however, circulating tumor DNA dynamics correlated with radiographic response. These data suggest that vistusertib does not modulate targets sufficiently to add to the clinical activity of acalabrutinib monotherapy. Clinicaltrials.gov identifier: NCT03205046.

Specific targeting of the KRAS mutational landscape in myeloma as a tool to unveil the elicited antitumor activity.

Alterations in KRAS have been identified as the most recurring somatic variants in the multiple myeloma (MM) mutational landscape. Combining DNA and RNA sequencing, we studied 756 patients and observed KRAS as the most frequently mutated gene in patients at diagnosis; in addition, we demonstrated the persistence or de novo occurrence of the KRAS aberration at disease relapse. Small-molecule inhibitors targeting KRAS have been developed; however, they are selective for tumors carrying the KRASG12C mutation. Therefore, there is still a need to develop novel therapeutic approaches to target the KRAS mutational events found in other tumor types, including MM. We used AZD4785, a potent and selective antisense oligonucleotide that selectively targets and downregulates all KRAS isoforms, as a tool to dissect the functional sequelae secondary to KRAS silencing in MM within the context of the bone marrow niche and demonstrated its ability to significantly silence KRAS, leading to inhibition of MM tumor growth, both in vitro and in vivo, and confirming KRAS as a driver and therapeutic target in MM.

Metabolic physiology and skeletal muscle phenotypes in male and female myoglobin knockout mice.

Myoglobin (Mb) is a regulator of O2 bioavailability in type I muscle and heart, at least when tissue O2 levels drop. Mb also plays a role in regulating cellular nitric oxide (NO) pools. Robust binding of long-chain fatty acids and long-chain acylcarnitines to Mb, and enhanced glucose metabolism in hearts of Mb knockout (KO) mice, suggest additional roles in muscle intermediary metabolism and fuel selection. To evaluate this hypothesis, we measured energy expenditure (EE), respiratory exchange ratio (RER), body weight gain and adiposity, glucose tolerance, and insulin sensitivity in Mb knockout (Mb-/-) and wild-type (WT) mice challenged with a high-fat diet (HFD, 45% of calories). In males (n = 10/genotype) and females (n = 9/genotype) tested at 5-6, 11-12, and 17-18 wk, there were no genotype effects on RER, EE, or food intake. RER and EE during cold (10°C, 72 h), and glucose and insulin tolerance, were not different compared with within-sex WT controls. At ∼18 and ∼19 wk of age, female Mb-/- adiposity was ∼42%-48% higher versus WT females (P = 0.1). Transcriptomics analyses (whole gastrocnemius, soleus) revealed few consistent changes, with the notable exception of a 20% drop in soleus transferrin receptor (Tfrc) mRNA. Capillarity indices were significantly increased in Mb-/-, specifically in Mb-rich soleus and deep gastrocnemius. The results indicate that Mb loss does not have a major impact on whole body glucose homeostasis, EE, RER, or response to a cold challenge in mice. However, the greater adiposity in female Mb-/- mice indicates a sex-specific effect of Mb KO on fat storage and feed efficiency.NEW & NOTEWORTHY The roles of myoglobin remain to be elaborated. We address sexual dimorphism in terms of outcomes in response to the loss of myoglobin in knockout mice and perform, for the first time, a series of comprehensive metabolic studies under conditions in which fat is mobilized (high-fat diet, cold). The results highlight that myoglobin is not necessary and sufficient for maintaining oxidative metabolism and point to alternative roles for this protein in muscle and heart.

On the potential role of globins in brown adipose tissue: a novel conceptual model and studies in myoglobin knockout mice.

Myoglobin (Mb) regulates O2 bioavailability in muscle and heart as the partial pressure of O2 (Po2) drops with increased tissue workload. Globin proteins also modulate cellular NO pools, "scavenging" NO at higher Po2 and converting NO2- to NO as Po2 falls. Myoglobin binding of fatty acids may also signal a role in fat metabolism. Interestingly, Mb is expressed in brown adipose tissue (BAT), but its function is unknown. Herein, we present a new conceptual model that proposes links between BAT thermogenic activation, concurrently reduced Po2, and NO pools regulated by deoxy/oxy-globin toggling and xanthine oxidoreductase (XOR). We describe the effect of Mb knockout (Mb-/-) on BAT phenotype [lipid droplets, mitochondrial markers uncoupling protein 1 (UCP1) and cytochrome C oxidase 4 (Cox4), transcriptomics] in male and female mice fed a high-fat diet (HFD, 45% of energy, ∼13 wk), and examine Mb expression during brown adipocyte differentiation. Interscapular BAT weights did not differ by genotype, but there was a higher prevalence of mid-large sized droplets in Mb-/-. COX4 protein expression was significantly reduced in Mb-/- BAT, and a suite of metabolic/NO/stress/hypoxia transcripts were lower. All of these Mb-/--associated differences were most apparent in females. The new conceptual model, and results derived from Mb-/- mice, suggest a role for Mb in BAT metabolic regulation, in part through sexually dimorphic systems and NO signaling. This possibility requires further validation in light of significant mouse-to-mouse variability of BAT Mb mRNA and protein abundances in wild-type mice and lower expression relative to muscle and heart.NEW & NOTEWORTHY Myoglobin confers the distinct red color to muscle and heart, serving as an oxygen-binding protein in oxidative fibers. Less attention has been paid to brown fat, a thermogenic tissue that also expresses myoglobin. In a mouse knockout model lacking myoglobin, brown fat had larger fat droplets and lower markers of mitochondrial oxidative metabolism, especially in females. Gene expression patterns suggest a role for myoglobin as an oxygen/nitric oxide-sensor that regulates cellular metabolic and signaling pathways.

Biallelic UBE4A loss-of-function variants cause intellectual disability and global developmental delay.

PURPOSE: To identify novel genes associated with intellectual disability (ID) in four unrelated families. METHODS: Here, through exome sequencing and international collaboration, we report eight individuals from four unrelated families of diverse geographic origin with biallelic loss-of-function variants in UBE4A. RESULTS: Eight evaluated individuals presented with syndromic intellectual disability and global developmental delay. Other clinical features included hypotonia, short stature, seizures, and behavior disorder. Characteristic features were appreciated in some individuals but not all; in some cases, features became more apparent with age. We demonstrated that UBE4A loss-of-function variants reduced RNA expression and protein levels in clinical samples. Mice generated to mimic patient-specific Ube4a loss-of-function variant exhibited muscular and neurological/behavioral abnormalities, some of which are suggestive of the clinical abnormalities seen in the affected individuals. CONCLUSION: These data indicate that biallelic loss-of-function variants in UBE4A cause a novel intellectual disability syndrome, suggesting that UBE4A enzyme activity is required for normal development and neurological function.

Generation of desminopathy in rats using CRISPR-Cas9.

BACKGROUND: Desminopathy is a clinically heterogeneous muscle disease caused by over 60 different mutations in desmin. The most common mutation with a clinical phenotype in humans is an exchange of arginine to proline at position 350 of desmin leading to p.R350P. We created the first CRISPR-Cas9 engineered rat model for a muscle disease by mirroring the R350P mutation in humans. METHODS: Using CRISPR-Cas9 technology, Des c.1045-1046 (AGG > CCG) was introduced into exon 6 of the rat genome causing p.R349P. The genotype of each animal was confirmed via quantitative PCR. Six male rats with a mutation in desmin (n = 6) between the age of 120-150 days and an equal number of wild type littermates (n = 6) were used for experiments. Maximal plantar flexion force was measured in vivo and combined with the collection of muscle weights, immunoblotting, and histological analysis. In addition to the baseline phenotyping, we performed a synergist ablation study in the same animals. RESULTS: We found a difference in the number of central nuclei between desmin mutants (1 ± 0.4%) and wild type littermates (0.2 ± 0.1%; P < 0.05). While muscle weights did not differ, we found the levels of many structural proteins to be altered in mutant animals. Dystrophin and syntrophin were increased 54% and 45% in desmin mutants, respectively (P < 0.05). Dysferlin and Annexin A2, proteins associated with membrane repair, were increased two-fold and 32%, respectively, in mutants (P < 0.05). Synergist ablation caused similar increases in muscle weight between mutant and wild type animals, but changes in fibre diameter revealed that fibre hypertrophy in desmin mutants was hampered compared with wild type animals (P < 0.05). CONCLUSIONS: We created a novel animal model for desminopathy that will be a useful tool in furthering our understanding of the disease. While mutant animals at an age corresponding to a preclinical age in humans show no macroscopic differences, microscopic and molecular changes are already present. Future studies should aim to further decipher those biological changes that precede the clinical progression of disease and test therapeutic approaches to delay disease progression.

A constitutive knockout of murine carbamoyl phosphate synthetase 1 results in death with marked hyperglutaminemia and hyperammonemia.

The enzyme carbamoyl phosphate synthetase 1 (CPS1; EC 6.3.4.16) forms carbamoyl phosphate from bicarbonate, ammonia, and adenosine triphosphate (ATP) and is activated allosterically by N-acetylglutamate. The neonatal presentation of bi-allelic mutations of CPS1 results in hyperammonemia with reduced citrulline and is reported as the most challenging nitrogen metabolism disorder to treat. As therapeutic interventions are limited, patients often develop neurological injury or die from hyperammonemia. Survivors remain vulnerable to nitrogen overload, being at risk for repetitive neurological injury. With transgenic technology, our lab developed a constitutive Cps1 mutant mouse and reports its characterization herein. Within 24 hours of birth, all Cps1 -/- mice developed hyperammonemia and expired. No CPS1 protein by Western blot or immunostaining was detected in livers nor was Cps1 mRNA present. CPS1 enzymatic activity was markedly decreased in knockout livers and reduced in Cps1+/- mice. Plasma analysis found markedly reduced citrulline and arginine and markedly increased glutamine and alanine, both intermolecular carriers of nitrogen, along with elevated ammonia, taurine, and lysine. Derangements in multiple other amino acids were also detected. While hepatic amino acids also demonstrated markedly reduced citrulline, arginine, while decreased, was not statistically significant; alanine and lysine were markedly increased while glutamine was trending towards significance. In conclusion we have determined that this constitutive neonatal mouse model of CPS1 deficiency replicates the neonatal human phenotype and demonstrates the key biochemical features of the disorder. These mice will be integral for addressing the challenges of developing new therapeutic approaches for this, at present, poorly treated disorder.

Conditional disruption of hepatic carbamoyl phosphate synthetase 1 in mice results in hyperammonemia without orotic aciduria and can be corrected by liver-directed gene therapy.

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.