Patient selection may affect gene therapy success. Dominant negative effects observed for ornithine transcarbamylase in mouse and human hepatocytes.

Abstract

We have achieved significant improvement of ornithine transcarbamylase deficiency (OTCD) in a mouse model through adenoviral-mediated gene transfer of the human ornithine transcarbamylase cDNA. Substantial reduction in orotic aciduria was observed within 24 h of treatment. Metabolic correction was later associated with phenotypic correction and moderate increase in enzymatic activity. In an effort to identify the level of gene expression required to achieve wild-type levels of enzyme activity we uncovered a dominant negative effect of the endogenous mutant protein on the activity of the delivered recombinant wild-type protein. This phenomenon is relevant to homomultimeric protein defects such as OTCD, which represent a challenging category of disorders for gene therapy. Thus, although our findings indicate that adenoviral-mediated gene transfer may have potential as a short-term treatment for OTCD in humans and may be effective especially during catabolic crisis, the observations in this study suggest that careful patient selection based on mutation class may be essential for initial OTCD gene therapy trials and, perhaps, for other homomultimeric enzyme deficiencies being considered as gene therapy targets. IntroductionOrnithine transcarbamylase (OTC) 1 deficiency (OTCD) is the most common and severe of the urea cycle disorders and is inherited as an X-linked recessive, with frequent new mutation occurrences (1, 2). Mutations range from deletions of the gene to point mutations that affect the activity and/or stability of the protein (1). The OTC protein is predominantly expressed in the liver and intestine with trace amounts found in kidney and brain (1). OTC is targeted to the mitochondria and assumes a homotrimer configuration in its active form (1). The deficiency is associated with citrulline and arginine depletion, orotic aciduria, hyperammonemia, and coma (3-6). Patients deteriorate rapidly if the hyperammonemia is not corrected within 24-48 h (2). Despite dietary and pharmacological intervention, the therapeutic outcome is inadequate and frequently associated with recurrent acute episodes resulting in mental retardation or, in the majority of cases, death (1-7). Liver transplantation is an option for qualified patients but can be associated with high morbidity and mortality (8, 9). Because of the severity of OTCD, the high frequency of new mutations, the limited organ target needed for enzymatic correction, and the currently inadequate therapy, we elected to develop a gene transfer method for treatment.There are two mouse models for OTC deficiency: the sparse fur ( spf ) and the sparse fur-abnormal skin and hair ( spf ash ) mutants. The molecular defect in the spf model is a missense point mutation in exon 4 which alters the pH optimum for the enzyme activity, resulting in only 20% wild-type activity at physiological pH despite high levels of expressed protein (10, 11). The spf ash OTC gene has a splice junction mutation, leading to expression of 5-15% wild-type levels of active enzyme (12). The ...

Genetic manipulations of adenovirus type 5 fiber resulting in liver tropism attenuation

“…18 Systemic delivery of Ad5-based vectors in mice invariably leads to preferential transduction of hepatocytes, which are known to express CAR to high levels. 10,14,55,56 The pathway involved in this uptake is however complex and remains largely unknown. On the one hand, several reports suggest that fiber-CAR interactions are primarily responsible for the natural tropism of Ad5-based vectors for the liver.…”

In vivo assessment of mutations in OTC for dominant-negative effects following rAAV2/8-mediated gene delivery to the mouse liver

“…Evidence for this phenomenon has been reported in vitro. 3 The OTC protein is predominantly expressed in the liver and intestinal mucosa as an inactive precursor polypeptide containing an N-terminal leader sequence. On transport to the mitochondria the leader sequence is cleaved and the protein assembled into an active homotrimeric form that catalyses conversion of ornithine and carbamyl phosphate to citrulline and inorganic phosphate.…”

“…7 Studies by other researchers have shown that co-expression of both wild-type and mutant OTC substantially inhibited wild-type enzyme activity, suggesting the R92Q mutation exerts a dominant-negative effect, at least in vitro. 3 The next two mutations result in amino acid substitutions R141Q or K88N and produce a severe neonatal 8 or late presenting phenotype, 9 respectively. Mutation R141Q affects the carbamyl phosphate-binding site without altering the tertiary structure of the protein 10 whereas K88N alters a lysine residue in the substrate-binding site of OTC.…”

“…3 In this earlier study, human hepatocytes established from a patient carrying the R92Q mutation were transduced with an adenoviral vector expressing wild-type OTC and the levels of activity compared with another patient-derived line, transduced with the same vector. The authors found that enzyme activity did not correlate with protein levels and proposed a dominantnegative effect for the R92Q mutation.…”

Expression of Wild-Type and Mutant Human Ornithine Transcarbamylase Genes in Chinese Hamster Ovary Cells and Lack of Dominant Negative Effect of R141Q and R40H Mutants

“…If a wild-type OTC gene is expressed within the same cell, it is unknown whether the mutant polypeptide that is being simultaneously synthesized by the cell will interfere with the assembly of a wild-type trimer and thus exert a dominant negative effect. One previous study suggested that such a phenomenon may occur in the OTC protein (10).…”