Sustained correction of OTC deficiency in spf( ash) mice using optimized self-complementary AAV2/8 vectors.

Ornithine transcarbamylase deficiency (OTCD) is the most common inborn error of urea synthesis. Complete OTCD can result in hyperammonemic coma in the neonatal period, which can rapidly become fatal. Current acute therapy involves dialysis; chronic therapy involves the stimulation of alternate nitrogen clearance pathways; and the only curative approach is liver transplantation. Adeno-associated virus (AAV) vector-based gene therapy would add to current treatment options provided the vector delivers high level and stable transgene expression in liver without dose-limiting toxicity. In this study, we employed an AAV2/8-based self-complementary (sc) vector expressing the murine OTC (mOTC) gene under a liver-specific thyroxine-binding globulin promoter and examined the therapeutic effects in a mouse model of OTCD, the spf (ash) mouse. Seven days after a single intravenous injection of vector, treated mice showed complete normalization of urinary orotic acid, a measure of OTC activity. We further improved vector efficacy by incorporating a Kozak or Kozak-like sequence into mOTC complementary DNA, which increased the OTC activity by five or twofold and achieved sustained correction of orotic aciduria for up to 7 months. Our results demonstrate that vector optimizations can significantly improve the efficacy of gene therapy.

Preclinical evaluation of a clinical candidate AAV8 vector for ornithine transcarbamylase (OTC) deficiency reveals functional enzyme from each persisting vector genome.

Ornithine transcarbamylase deficiency (OTCD), the most common and severe urea cycle disorder, is an excellent model for developing liver-directed gene therapy. No curative therapy exists except for liver transplantation which is limited by available donors and carries significant risk of mortality and morbidity. Adeno-associated virus 8 (AAV8) has been shown to be the most efficient vector for liver-directed gene transfer and is currently being evaluated in a clinical trial for treating hemophilia B. In this study, we generated a clinical candidate vector for a proposed OTC gene therapy trial in humans based on a self-complementary AAV8 vector expressing codon-optimized human OTC (hOTCco) under the control of a liver-specific promoter. Codon-optimization dramatically improved the efficacy of OTC gene therapy. Supraphysiological expression levels and activity of hOTC were achieved in adult spf(ash) mice following a single intravenous injection of hOTCco vector. Vector doses as low as 1×10(10) genome copies (GC) achieved robust and sustained correction of the OTCD biomarker orotic aciduria and clinical protection against an ammonia challenge. Functional expression of hOTC in 40% of liver areas was found in mice treated with a low vector dose of 1×10(9) GC. We suggest that the clinical candidate vector we have developed has the potential to achieve therapeutic effects in OTCD patients.

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

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.

Interaction between murine spf-ash mutation and genetic background yields different metabolic phenotypes.

The spf-ash mutation in mice results in reduced hepatic and intestinal ornithine transcarbamylase. However, a reduction in enzyme activity only translates in reduced ureagenesis and hyperammonemia when an unbalanced nitrogen load is imposed. Six-week-old wild-type control and spf-ash mutant male mice from different genetic backgrounds (B6 and ICR) were infused intravenously with [(13)C(18)O]urea, l-[(15)N(2)]arginine, l-[5,5 D(2)]ornithine, l-[6-(13)C, 4,4,5,5, D(4)]citrulline, and l-[ring-D(5)]phenylalanine to investigate the interaction between genetic background and spf-ash mutation on ureagenesis, arginine metabolism, and nitric oxide production. ICR(spf-ash) mice maintained ureagenesis (5.5 +/- 0.3 mmol.kg(-1).h(-1)) and developed mild hyperammonemia (145 +/- 19 micromol/l) when an unbalanced nitrogen load was imposed; however, B6(spf-ash) mice became hyperammonemic (671 +/- 15 micromol/l) due to compromised ureagenesis (3.4 +/- 0.1 mmol.kg(-1).h(-1)). Ornithine supplementation restored ureagenesis and mitigated hyperammonemia. A reduction in citrulline entry rate was observed due to the mutation in both genetic backgrounds (wild-type: 128, spf-ash: 60; SE 4.0 micromol.kg(-1).h(-1)). Arginine entry rate was only reduced in B6(spf-ash) mice (B6(spf-ash): 332, ICR(spf-ash): 453; SE 20.6 micromol.kg(-1).h(-1)). Genetic background and mutation had an effect on nitric oxide production (B6: 3.4, B6(spf-ash): 2.8, ICR: 9.0, ICR(spf-ash): 4.6, SE 0.7 micromol.kg(-1).h(-1)). Protein breakdown was the main source of arginine during the postabsorptive state and was higher in ICR(spf-ash) than in B6(spf-ash) mice (phenylalanine entry rate 479 and 327, respectively; SE 18 micromol.kg(-1).h(-1)). Our results highlight the importance of the interaction between mutation and genetic background on ureagenesis, arginine metabolism, and nitric oxide production. These observations help explain the wide phenotypic variation of ornithine transcarbamylase deficiency in the human population.

An integrated approach to the diagnosis and prospective management of partial ornithine transcarbamylase deficiency.

Ornithine transcarbamylase deficiency (OTCD) is the most common inherited urea cycle disorder, and is transmitted as an X-linked trait. Female OTCD heterozygotes exhibit wide clinical severities, ranging from being apparently asymptomatic to having the profound neurologic impairment observed in affected males. However, clinical and laboratory diagnosis of partial OTCD during asymptomatic periods is difficult, and correlation of phenotypic severity with either DNA mutation and/or in vitro enzyme activity is imprecise. Provocative testing, including protein load and allopurinol challenge used in the diagnosis of OTCD females, is not without risk and subject to both false positives and negatives. Although definitive when successful, DNA-based diagnosis is unable to detect mutations in all cases. We have previously used the ratio of isotopic enrichments of [(15)N]urea/[(15)N]glutamine ((15)N-U/G) derived from physiologic measurements of ureagenesis by stable isotope infusion as a sensitive index of in vivo urea cycle activity. We have now applied this method in combination with traditional biochemical testing to aid in the diagnosis of a symptomatic OTCD female in whom mutation in the ornithine transcarbamylase (OTC) gene was not found. The (15)N-U/G ratio in this patient showed that she had severe reduction of in vivo urea cycle activity on par with affected male subjects. This was correlated with partially deficient OTC activity in her liver, degree of orotic aciduria, and history of suspected recurrent hyperammonemic episodes before age 3. The measurement of in vivo urea cycle activity in combination with traditional biochemical indices optimizes a diagnostic approach to the at-risk partial OTCD patient, especially in those in whom molecular testing is unproductive. Together they contribute to the risk versus benefit considerations regarding the pursuit of medical therapy versus surgical, ie, orthotopic liver transplantation (OLT) therapy. The decision to resort to OLT in females with partial OTC activity is controversial, requiring consideration of phenotypic severity, failure of medical therapy, access to tertiary care centers experienced in the management of acute hyperammonemia, and social factors. In this patient, the use of in vivo and in vitro measures of urea cycle activity in conjunction with a consideration of her clinical history and medical-social situation led to a decision for OLT.