Dataset Information

AAV2/8-mediated correction of OTC deficiency is robust in adult but not neonatal Spf(ash) mice.

ABSTRACT: 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.

SUBMITTER: Cunningham SC

PROVIDER: S-EPMC2835243 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

AAV2/8-mediated correction of OTC deficiency is robust in adult but not neonatal Spf(ash) mice.

Cunningham Sharon C SC Spinoulas Afroditi A Carpenter Kevin H KH Wilcken Bridget B Kuchel Philip W PW Alexander Ian E IE

Molecular therapy : the journal of the American Society of Gene Therapy 20090421 8

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 ...[more]

PMID: 19384294

Similar Datasets

Project description:BackgroundAberrant 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.MethodsCreation 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.ResultsComparative 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.ConclusionsSubtle 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.

Project description:Hematopoiesis, a process that results in the differentiation of all blood lineages, is essential throughout life. The production of 1x1012 blood cells per day, including 200x109 erythrocytes, is highly dependent on nutrient consumption. Notably though, the relative requirements for micronutrients during the perinatal period, a critical developmental window for immune cell and erythrocyte differentiation, have not been extensively studied. More specifically, the impact of the vitamin C/ascorbate micronutrient on perinatal as compared to adult hematopoiesis has been difficult to assess in animal models. Even though humans cannot synthesize ascorbate, due to a pseudogenization of the L-gulono-γ-lactone oxidase (GULO) gene, its generation from glucose is an ancestral mammalian trait. Taking advantage of a Gulo-/- mouse model, we show that ascorbic acid deficiency profoundly impacts perinatal hematopoiesis, resulting in a hypocellular bone marrow (BM) with a significant reduction in hematopoietic stem cells, multipotent progenitors, and hematopoietic progenitors. Furthermore, myeloid progenitors exhibited differential sensitivity to vitamin C levels; common myeloid progenitors and megakaryocyte-erythrocyte progenitors were markedly reduced in Gulo-/- pups following vitamin C depletion in the dams, whereas granulocyte-myeloid progenitors were spared, and their frequency was even augmented. Notably, hematopoietic cell subsets were rescued by vitamin C repletion. Consistent with these data, peripheral myeloid cells were maintained in ascorbate-deficient Gulo-/- pups while other lineage-committed hematopoietic cells were decreased. A reduction in B cell numbers was associated with a significantly reduced humoral immune response in ascorbate-depleted Gulo-/- pups but not adult mice. Erythropoiesis was particularly sensitive to vitamin C deprivation during both the perinatal and adult periods, with ascorbate-deficient Gulo-/- pups as well as adult mice exhibiting compensatory splenic differentiation. Furthermore, in the pathological context of hemolytic anemia, vitamin C-deficient adult Gulo-/- mice were not able to sufficiently increase their erythropoietic activity, resulting in a sustained anemia. Thus, vitamin C plays a pivotal role in the maintenance and differentiation of hematopoietic progenitors during the neonatal period and is required throughout life to sustain erythroid differentiation under stress conditions.

Dataset Information

AAV2/8-mediated correction of OTC deficiency is robust in adult but not neonatal Spf(ash) mice.

Publications

AAV2/8-mediated correction of OTC deficiency is robust in adult but not neonatal Spf(ash) mice.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets