RESUMO
Ornithine transcarbamylase (OTC) deficiency, the most common urea cycle disorder, is associated with severe hyperammonemia accompanied by a high risk of neurological damage and death in patients presenting with the neonatal-onset form. Contemporary therapies, including liver transplantation, remain inadequate with considerable morbidity, justifying vigorous investigation of alternate therapies. Clinical evidence suggests that as little as 3% normal enzyme activity is sufficient to ameliorate the severe neonatal phenotype, making OTC deficiency an ideal model for the development of liver-targeted gene therapy. In this study, we investigated metabolic correction in neonatal and adult male OTC-deficient Spf(ash) mice following adeno-associated virus (AAV)2/8-mediated delivery of the murine OTC complementary DNA under the transcriptional control of a liver-specific promoter. Substantially supraphysiological levels of OTC enzymatic activity were readily achieved in both adult and neonatal mice following a single intraperitoneal (i.p.) injection, with metabolic correction in adults being robust and life-long. In the neonates, however, full metabolic correction was transient, although modest levels of OTC expression persisted into adulthood. Although not directly testable in Spf(ash) mice, these levels were theoretically sufficient to prevent hyperammonemia in a null phenotype. This loss of expression in the neonatal liver is the consequence of hepatocellular proliferation and presents an added challenge to human therapy.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Doença da Deficiência de Ornitina Carbomoiltransferase/metabolismo , Doença da Deficiência de Ornitina Carbomoiltransferase/terapia , Animais , Animais Recém-Nascidos , Western Blotting , Linhagem Celular , DNA Complementar/genética , Feminino , Humanos , Imuno-Histoquímica , Fígado/metabolismo , Masculino , Camundongos , Ornitina Carbamoiltransferase/genética , Ornitina Carbamoiltransferase/metabolismo , Ornitina Carbamoiltransferase/fisiologia , Doença da Deficiência de Ornitina Carbomoiltransferase/genética , Doença da Deficiência de Ornitina Carbomoiltransferase/urina , Ácido Orótico/urinaRESUMO
We disrupted the Aspergillus niger gene argB, encoding ornithine transcarbamylase. Full characterisation of the argB deletion was performed by Southern blot analysis, growth tests and by means of mitotic recombination, complementation and transformation. The argB locus was found to be physically removed, thus creating an auxotrophic mutation. The latter can be supplemented by addition of arginine into the culture medium. The argB gene and its disruption do not correlate to the argI13 (formerly argB13) allele described. The delta argB is on chromosome I whereas argI13 is on V. In addition, the argI13 mutation can only be complemented by the A. nidulans argB gene, whereas the new argB deletion can be complemented by both the A. niger and A. nidulans argB genes. The delta argB strain has been used to generate several strains in a breeding programme and to study the expression of important genes, such as areA and kexB.
Assuntos
Arginina/genética , Aspergillus niger/genética , Genes Fúngicos , Ornitina Carbamoiltransferase/genética , Sequência de Aminoácidos , Arginina/biossíntese , Aspergillus niger/fisiologia , Southern Blotting , Cromossomos Fúngicos , Deleção de Genes , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Ornitina Carbamoiltransferase/fisiologia , Plasmídeos , Recombinação Genética , Homologia de Sequência de AminoácidosRESUMO
Sparse-fur (spf) mutant mice with X-linked ornithine transcarbamylase deficiency were used to study the effect of L-carnitine on energy metabolites in congenital hyperammonemia. L-Carnitine was used at doses of 2, 4, 8, or 16 mmol/kg body weight (BW), and levels of ammonia, glutamine, glutamate, and some intermediates of energy metabolism were measured in brain and liver of spf/Y mice. Cerebral and hepatic levels of ammonia were decreased with 4 mmol L-carnitine (P < .001), whereas other doses did not seem to have any effect on this metabolite. Cerebral levels of glutamine were decreased following administration of L-carnitine at doses of up to 4 mmol/kg BW, whereas hepatic glutamine levels remained unaltered at all doses of L-carnitine. Both cerebral and hepatic levels of pyruvate, lactate, and alpha-ketoglutarate were decreased at doses of up to 8 mmol L-carnitine/kg BW. L-Carnitine treatment elevated adenosine triphosphate (ATP), free coenzyme A (CoA), and acetyl CoA levels in both brain and liver of spf/Y mice. Cytosolic and mitochondrial redox ratios of spf/Y mice, which were altered by congenital chronic hyperammonemia, were partially corrected by L-carnitine administration. L-Carnitine supplementation to spf/Y mice during sodium benzoate therapy also restored the availability of free CoA and ATP, thus counteracting the adverse effects of higher doses of sodium benzoate. These changes in free CoA and acetyl CoA levels could be due to the deinhibition of pantothenate kinase and stimulation of fatty acid oxidation by L-carnitine.(ABSTRACT TRUNCATED AT 250 WORDS)