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.

Differences in the human and mouse amino-terminal leader peptides of ornithine transcarbamylase affect mitochondrial import and efficacy of adenoviral vectors.

Mouse models of ornithine transcarbamylase (OTC) deficiency are being used to test the efficacy of viral vectors as possible vehicles for gene therapy. However, it has been demonstrated that virus containing the human OTC cDNA failed to express functional OTC enzyme in the recipient animals. Because functional OTC is assembled as a homotrimer in the mitochondria, there are at least two possible explanations for these results. Either endogenous mutant protein coassembles with the human OTC and has a "dominant-negative effect," or the human version of the protein is not appropriately imported or processed in the mouse mitochondria. To test the importance of processing, which in rodents is thought to depend on the leader peptide, adenoviral vectors containing chimeric OTC cDNAs were prepared. These vectors were evaluated in the OTC-deficient sparse fur mouse models. Although comparable levels of transgene expression were observed in all groups of mice, the only mice that had high levels of OTC activity and mitochondrial OTC immunoreactivity were those mice injected with the vectors containing the mouse leader peptide (mouse OTC and a mouse-human chimera of OTC). To address possible dominant-negative effects, adenoviruses containing mutant human or mouse OTC cDNAs were prepared and evaluated in cell lines or normal C3H mice, respectively. No inhibition of normal OTC activity was observed in either model system. Together, these studies provide no evidence of a dominant-negative effect and suggest that the human and rodent enzymes responsible for transporting of OTC and possibly other mitochondrial proteins have different specificity.

Prospects for prenatal gene therapy in disorders causing mental retardation.

Advances in understanding the genetics and pathogenesis of disease and in prenatal diagnosis have lead to an exploration of ways to intervene earlier and earlier in the disease process. The possibility of prenatal gene therapy for severe genetic and developmental disorders has sparked new research and debate as to its feasibility, reliability, and ethics as a therapeutic option. Recent animal studies have demonstrated the feasibility of introducing a vector into the developing fetus. The optimal timing and best mode of delivery, however, have yet to be defined. Whether or not this research should be pursued also has been the subject of recent bioethical debates. There is additional concern with the possibility of in utero gene transfer inducing mutagenesis and subsequent tumor formation. This review will provide a summary of the current state of knowledge in the field of prenatal gene therapy and possible directions for the future research.

Alternative pathway therapy for urea cycle disorders: twenty years later.

Alternative pathway therapy is currently an accepted treatment approach for inborn errors of the urea cycle. This involves the long-term use of oral sodium phenylbutyrate, arginine supplements, or both, depending on the specific enzyme deficiency, and treatment of acute hyperammonemic crises with intravenous sodium benzoate/sodium phenylacetate plus arginine. A review of 20 years of experience with this approach illustrates the strengths and limitations of this treatment. It has clearly decreased the mortality and morbidity from these disorders, but they remain unacceptably high. The medications are generally well tolerated, but severe accidental overdosage has been reported because of the infrequent use of the medication. There is also a difference in their metabolism between newborns and older children that must be addressed in determining dosage. To avoid these complications it is recommended that drug levels in blood be monitored routinely and that very specific treatment protocols and oversight be followed to avoid overdoses. Finally, it must be acknowledged that alternative pathway therapy has limited effectiveness in preventing hyperammonemia and must be combined with effective dietary management. Therefore in children with neonatal-onset disease or in those with very poor metabolic control, liver transplantation should be considered. There should also be the continued search for innovative therapies that may offer a more permanent and complete correction, such as gene therapy.

Transient depletion of CD4 lymphocyte improves efficacy of repeated administration of recombinant adenovirus in the ornithine transcarbamylase deficient sparse fur mouse.

One of the current limitations of adenoviral gene therapy is a vector-induced humoral immune response that blocks effective re-administration of the vector. In an animal model of the inborn error of urea synthesis ornithine transcarbamylase (OTC) deficiency, the sparse fur (spf/y) mouse, we tested a strategy to transiently block the CD4 mediated immune response at the time of virus administration using an anti-CD4 monoclonal antibody (GK1.5). The co-administration of GK1.5 resulted in a significantly diminished production of neutralizing antibody to the adenovirus vector, but minimally prolonged metabolic correction. A second infusion of the same virus in GK1.5 treated spf/y mice led to a complete normalization of liver OTC activity at day 3 after infection and a significant metabolic correction of urinary orotate and plasma glutamine. In contrast, there was no evidence of enhanced OTC expression or metabolic correction (measured by normalization of plasma glutamine and urinary orotate) after the second infusion of virus in spf/y mice not treated with GK1.5. Furthermore, when co-administered with two consecutive doses of adenovirus, the anti-CD4 treatment allowed improved transgene expression upon a third administration of virus and a partial normalization of the metabolic abnormalities, compared with mice that did not receive anti-CD4 treatment. The level of OTC expression from the third viral infusion, however, was lower than that from the second viral infusion. Passive transfer experiments suggest that low levels of neutralizing antibodies developing over repeated viral administration was the likely cause of the reduced transgene expression. Together, these findings demonstrated that the host immune system can be modulated to permit effective transgene expression at therapeutic levels by re-administered adenoviral vectors.

Correction of argininosuccinate synthetase (AS) deficiency in a murine model of citrullinemia with recombinant adenovirus carrying human AS cDNA.

Citrullinemia is an autosomal recessive disorder caused by the deficiency of argininosuccinate synthetase (AS). It is characterized by elevated levels of blood citrulline and ammonia, which often results in hyperammonemic coma and early neonatal death in affected children. We have explored the use of adenoviral vectors as a treatment modality in a murine model of citrullinemia, the Ass mouse. The Ass mouse has no endogenous AS activity due to a targeted interruption of the AS gene. Homozygous mutant animals develop high levels of blood citrulline, become hyperammonemic, and die within 24-48 h after birth. We demonstrated that the neonatal crisis in Ass mice can be ameliorated by the injection of a recombinant adenovirus carrying human AS cDNA (Ad.CMVhAS) within hours after birth. The average life span of the virus-treated animals was extended from 30 +/- 9.5 h to 16.1 +/- 1.6 days. A second viral infusion 14 days after the first dose further prolonged the life span to an average of 36.2 +/- 7.0 days, and to 40.7 +/- 3.3 days with a concurrent daily injection of arginine and sodium benzoate. Significantly increased liver AS activity (47.3 +/- 7.9% of normal) was detected 24 h after viral infusion, which reached peak levels (80-90% of normal) at day 7 and decreased to about 20% of normal within 2-3 weeks after viral infusion. Southern blot analysis of liver DNA revealed a transduction efficiency of about one viral genome per hepatocyte 7 days after viral infusion and a gradual decrease of viral genome per cell parallel to the loss of liver AS activity. Plasma glutamine levels were partially normalized in virus-treated animals and were completely normalized in animals receiving Ad.CMVhAS concurrently with alternative pathway therapy. Plasma arginine levels were also partially normalized. Together, these results demonstrated that the recombinant adenovirus was capable of conferring AS activity in the liver of the recipient animals within 24 h, and the neonatal crisis of hyperammonemia could be averted by acute treatment with the AS containing adenovirus.

Genotype spectrum of ornithine transcarbamylase deficiency: correlation with the clinical and biochemical phenotype.

Ornithine transcarbamylase (OTC) deficiency, a partially dominant X-linked disorder, is the most common inherited defect of the urea cycle. Previous reports suggested a variable phenotypic spectrum, and several studies documented different "private" mutations in the OTC genes of patients. Our laboratory identified disease-causing mutations in 157 families with OTC deficiency, 100 of which came to medical attention through a hemizygous propositus and in 57 the index case was a heterozygous female. We correlated the genotype with age of onset, liver OTC activity, incorporation of nitrogen into urea, and peak plasma ammonia levels. The "neonatal onset" group has a homogeneous clinical and biochemical phenotype, whereas the "late onset" group shows an extremely wide phenotype; 60% of the mutations are associated exclusively with acute neonatal hyperammonemic coma. The remaining mutations caused a nonuniform phenotype ranging from severe disease to no symptoms; 31% of the mutations in the OTC gene occur in CpG dinucleotides (methylation-mediated deamination), and none of them accounted for more than 4% of the total. Eighty-six percent of the mutations represented single-base substitutions and 68% of the substitutions were transitions. G-to-A and C-to-T transitions were the most frequent substitutions (34 and 21%, respectively) whereas C-to-A, A-to-C, C-to-G, and T-to-A transversions were the least common (1.5-3%). Twenty percent of propositi and 77% of propositae carried new mutations. Forty percent of female germinal mutations were in CpG dinucleotides whereas this number appears much smaller in male germinal mutations. These data allow classification of patients with OTC deficiency into at least two groups who have discordant disease course and prognoses. In addition, they improve our understanding on the origin of mutations in the OTC gene and allow better counseling of affected families.

Long-term gene transfer to mouse fetuses with recombinant adenovirus and adeno-associated virus (AAV) vectors.

We have developed a micro-injection technique to deliver recombinant adenovirus and AAV to mouse fetuses at day 15 after conception. Several routes of delivery, including injections to the amniotic fluid, the front limb, the placenta, the liver, and the retro-orbital venus plexus, were tested using an E1-deleted recombinant adenovirus (Ad.CBlacZ) or a recombinant adeno-associated virus (AAV.CMVlacZ) carrying a beta-galactosidase (lacZ) gene. Injection of Ad.CBlacZ into the amniotic cavity led to transgene expression in the skin and in the digestive tract of the fetuses. Injection of Ad.CBlacZ in the front limb resulted in LacZ expression in all major muscle groups around the injection site and at low levels in the liver. The other three routes of delivery, ie intra-placental, intra-hepatic and retro-orbital injections of Ad.CBlacZ, all led to lacZ expression predominantly in the liver. Further studies revealed a maximal tolerant dose (defined as the highest viral dose with < or =20% mortality in the injected fetuses) of 1 x 10(9) particles per fetus for intra- hepatic injections, 3 x 10(9) particles per fetus for intra-placental injection, 1 x 1010 particles per fetus for retro-orbital and intra-amniotic injections, and 2 x 10(10) particle per fetus for intra-muscular injection. The adenovirus-mediated lacZ expression in liver and muscle persisted for at least 6 weeks. Intra-muscular injection of AAV.CMVlacZ also resulted in lacZ expression in the muscle up to 3 months after birth with no indication of cellular immune response at the injection site. Taken together, our results demonstrated that prolonged transgene expression can be achieved by in utero gene transfer using either adenoviral or AAV vectors. The distribution of virus-mediated gene transfer appeared to determined mostly by the route of viral administration.

Correction of ureagenesis after gene transfer in an animal model and after liver transplantation in humans with ornithine transcarbamylase deficiency.

We report effects of gene transfer and liver transplantation on urea synthesis in ornithine transcarbamylase deficiency (OTCD). We measured the formation of [15N] urea after oral administration of 15NH4Cl in two girls with partial OTCD before and after liver transplantation. Ureagenesis was less than 20% of that observed in controls before transplantation, and was normalized afterward. Studies performed on the OTCD sparse fur (spf/Y) mouse showed discordance between OTC enzyme activity and ureagenesis with modest increases in OTC enzyme activity after gene transfer resulting in significant improvement in ureagenesis. This study suggests that both liver transplantation and gene therapy may be effective in improving ureagenesis in OTCD.

Cognitive and behavior profile of preschool children with chromosome 22q11.2 deletion.

A microscopic deletion of chromosome 22q11.2 has been identified in most patients with the DiGeorge, velocardiofacial syndrome, conotruncal anomaly face syndrome, and in some patients with isolated conotruncal cardiac anomalies. This study presents the neurodevelopmental outcome, including cognitive development, language development, speech, neuromuscular development, and behavioral characteristics of 40 preschool children (ages 13 to 63 months) who have been diagnosed with the 22q11.2 deletion. The impact of cardiac disease, cardiac surgery, and the palatal anomalies on this population was also studied. In the preschool years, children with a 22q11.2 deletion are most commonly found to be developmentally delayed, have mild hypotonia, and language and speech delays. The more significantly delayed children are at high risk to be subsequently diagnosed with mild or moderate mental retardation. The global delays and the variations in intelligence found are directly associated with the 22q11.2 deletion and are not explained by physical anomalies such as palatal defects or cardiac defects, or therapeutic interventions such as cardiac surgery. Our findings demonstrate that there is a pattern of significant speech disorders within this population. All of the children had late onset of verbal speech. Behavioral outcomes included both inhibition and attention disorders. Early intervention services are strongly recommended beginning in infancy to address the delays in gross motor skills, speech and language, and global developmental delays.

Psychoeducational profile of the 22q11.2 microdeletion: A complex pattern.

OBJECTIVES: To examine the psychoeducational profile associated with the chromosome 22q11.2 microdeletion (DiGeorge/velocardiofacial syndrome). STUDY DESIGN: Thirty-three patients (aged 6 to 27 years) with a 22q11.2 microdeletion underwent psychoeducational testing as part of a comprehensive evaluation. Nonparametric statistics were used to compare verbal and performance IQ, academic achievement scores, and receptive versus expressive language scores. Post hoc comparisons were made of IQ subtest scores and of language versus verbal IQ. RESULTS: Full-scale IQ ranged from the normal to the moderately retarded range. Mean verbal IQ was significantly higher than mean performance IQ. In a similar manner, mean reading and spelling scores were superior to the mean mathematics score, although achievement scores typically were in the range of verbal IQ. In addition, many children showed clinically significant language impairments, with mean language scores lower than mean verbal IQ. CONCLUSIONS: The IQ and academic profiles are reminiscent of a "nonverbal learning disability," although achievement was not discrepant from IQ. The coincidence of language impairment with a relative strength in reading belies a unique neuropsychologic profile. Educational programming for these children must address both verbal and nonverbal deficits.

In vivo measurement of ureagenesis with stable isotopes.

We have utilized stable isotopes to measure in vivo rates of ureagenesis. In one testing procedure, 15NH4Cl was administered orally to controls and to heterozygotes for ornithine transcarbamylase deficiency. Controls produced [15N]urea at a rate that was greater than that of symptomatic carriers, but indistinguishable from that of asymptomatic carriers. In contrast, both symptomatic and asymptomatic heterozygotes produced [5-15N]glutamine more rapidly than the controls. Ureagenesis could also be measured by administering sodium [1-13C]acetate to a healthy adult and measuring subsequent formation of [13C]urea. The latter approach involves the use of isotope ratio mass spectrometry to determine isotopic abundance. This technique is much more sensitive than gas chromatography-mass spectrometry for the measurement of isotopic label, a consideration that makes the method more suitable for the study of subjects in whom ureagenesis is severely compromised, for example the human male neonate with a near complete deficiency of ornithine transcarbamylase.

Developing adenoviral-mediated in vivo gene therapy for ornithine transcarbamylase deficiency.

There are a number of reasons for choosing ornithine transcarbamylase (OTC) deficiency as a candidate for gene therapy: the gene has been cloned; the disorder is relatively common; the current clinical outcome is poor; and there are authentic animal models. In considering the development of gene therapy for OTC deficiency, we focused on the use of in vivo gene therapy with an adenoviral vector. Using the partially OTC-deficient sparse fur mouse we found transduction and expression could be achieved using an intravenous infusion of a recombinant adenovirus containing the OTC cDNA. The results were transient as a result of immune activation in response to the vector and vector-transduced cells. By modifying the adenoviral construct, creating an E1 deletion-E2 temperature-sensitive mutation, we blunted the cytotoxic T lymphocyte immune response and achieved correction of biochemical abnormalities for 2-3 months. We also found that transduction and expression following gene transfer occurred sufficiently rapidly to protect against acute hyperammonaemia within 24 h. Subsequent preclinical studies in mice and non-human primates demonstrated that E1-E4-deleted vectors had a substantially improved safety profile and similar efficacy. With this evidence of efficacy and safety of adenoviral vectors, we are embarking on a phase I trial of intravascular gene transfer using an E1-E4-deleted vector in adults with partial OTC deficiency.

Adenovirus-mediated in vivo gene transfer rapidly protects ornithine transcarbamylase-deficient mice from an ammonium challenge.

The purpose of this study was to determine the time of onset, duration, and the efficacy of in vivo gene transfer in protecting the ornithine transcarbamylase deficient spf/Y mouse from an acute ammonium challenge. The animals were challenged with ammonia (10 mmol/kg NH4Cl) 1, 2, 7, 14, or 28 d after the administration of a recombinant adenoviral construct deleted in E1 and with a temperature sensitive mutation in E2. Although there was no protection with the control LacZ virus, the ornithine transcarbamylase (OTC)-containing vector provided partial protection from both behavioral symptoms (ataxia, seizures, and abnormal response to sound) and biochemical abnormalities (ammonium, aspartate, alanine, and glutamine) within 24 h and complete protection by 48 h. Mortality was also decreased. Animals receiving the vector 7 and 14 d before the ammonium load were also protected, whereas those treated 28 d before the challenge were not. OTC enzyme activity in liver of untreated spf/Y mice was 5% of control C3H mice. After gene transfer, activity was increased to near control levels through 14 d but had returned to baseline by 28 d. These studies indicate that adenovirus-mediated gene transfer confers a metabolic benefit within 24 h of administration and provides protection against an acute metabolic insult for at least 2 wk.

Prolonged metabolic correction in adult ornithine transcarbamylase-deficient mice with adenoviral vectors.

A murine model of ornithine transcarbamylase (OTC) deficiency was used in this study to evaluate the efficacy of recombinant adenoviruses for correcting the metabolic defect in liver. Recombinant adenoviruses deleted in E1 and containing a human OTC cDNA expressed little functional OTC enzyme in vivo and had no observable impact on the underlying metabolic abnormalities of the OTC-deficient mouse (i.e. elevated urinary orotate and serum glutamine). E1-deleted vectors were improved through the use of the strong constitutive promoter from cytomegalovirus driving the normal murine homolog of OTC cDNA and the ablation of E2a with a temperature-sensitive mutation. Infusion of this improved vector into the mouse model was associated with a complete normalization of liver OTC enzyme activity that persisted for at least 2 months with complete but transient correction in serum glutamine and urine orotic acid. These studies illustrate the utility of improved adenoviral vectors in the treatment of liver metabolic disease.

Evidence of excitotoxicity in the brain of the ornithine carbamoyltransferase deficient sparse fur mouse.

Ornithine carbamoyltransferase deficiency (OCTD) is the most common inborn error of urea synthesis. An X-linked disorder, OCTD males commonly present with hyperammonemic coma in the newborn period. There is a high rate of mortality and morbidity, with most survivors sustaining severe brain damage and resultant developmental disabilities. Although ammonia is presumed to be the principal neurotoxin, there is evidence that other neurochemical alterations may also be involved. The OCTD sparse fur (spf/Y) mouse has proven to be a useful model of this disease with similar metabolic and neurochemical alterations to those found in the human disease. In this study, the levels of the tryptophan derived excitotoxin quinolinic acid were examined in the brains of spf/Y mice. In addition, the neuropathology was examined using both light and electron microscopic approaches. Consistent with reports in children with urea cycle disorders, the levels of tryptophan and quinolinic acid were increased two-fold in various brain regions of the spf/Y mouse. Quinolinic acid, an agonist at the N-methyl-D-aspartate (NMDA) receptors, is known to produce selective cell loss in the striatum. We found a significant loss of medium spiny neurons and increased numbers of reactive oligodendroglia and microglia in the striatum of spf/Y mice. These neurochemical and neuropathological observations are consistent with an excitotoxic influence on brain injury in OCTD. It leads us to suggest that administration of NMDA receptor antagonists may ameliorate brain damage in children with inborn errors of urea synthesis.

The sparse fur mouse as a model for gene therapy in ornithine carbamoyltransferase deficiency.

The sparse fur (spf/Y) mouse was evaluated as a model for studying gene therapy in ornithine carbamoyltransferase deficiency (OCTD), the most common inborn error of urea synthesis. Previous studies have defined a number of biochemical characteristics of this animal model that are analogous to the human disease: OCTD in liver, elevated ammonium and glutamine, low citrulline and arginine in plasma, elevated urinary orotic acid excretion, neurochemical alterations and responsiveness to alternative pathway therapy. In this study, metabolic flux, survival, behavior and learning of these animals were examined in preparation for a trial of gene therapy. We found that, as has been previously reported, OCT activity in liver ranged from 10 to 20% of control. Yet, stable isotope studies using 15N ammonium chloride to follow ureagenesis in vivo showed 55% of normal urea synthetic capacity. This suggests that partial correction with gene therapy may be sufficient to normalize urea synthesis. Although it has been suggested that liver OCTD and its consequent metabolic effects normalize without treatment by adulthood in the spf/Y mouse, we did not find this to be the case. We documented that the spf/Y mouse had a markedly decreased lifespan (< 10% of normal) and remained runted throughout life. In terms of behavior, the spf/Y mice had evidence of decreased learning in a passive avoidance task that was not attributable to alterations in activity. These clearly definable metabolic and behavioral abnormalities suggest that the spf/Y mouse should prove a useful model for studying the efficacy of gene therapy in OCTD.

Inborn errors of urea synthesis.

Inborn errors of urea synthesis can present in the newborn period as a catastrophic illness or later in childhood or adulthood with an indolent course punctuated by hyperammonemic episodes. Because symptoms mimic other neuropsychiatric disorders, it is common for there to be a delay in diagnosis, often with dire consequences. Diagnosis relies on the combination of clinical suspicion and the measurement of ammonium, lactate, and amino acids in plasma and organic acids and orotic acid in urine. Treatment involves nitrogen restriction combined with the stimulation of alternate pathways of waste nitrogen excretion. More recently liver transplantation has been performed as enzyme replacement therapy. The outcome is poor in children who survive prolonged neonatal hyperammonemic coma, with most manifesting developmental disabilities. The etiology of neuronal injury in this disorder is unclear but may involve some combination of ammonia/amino acid accumulation, neurotransmitter alterations, and excitotoxic injury. Gene therapy holds the promise of improved treatment in the future.

Quinolinic acid in children with congenital hyperammonemia.

Levels of the excitotoxin quinolinic acid (QUIN) were measured in the cerebrospinal fluid of infants and children with congenital hyperammonemia. Twofold to tenfold elevations of QUIN were found in 4 neonates in hyperammonemic coma (QUIN range, 250-990 nM; control mean, 110 +/- 90 nM; p < 0.005). Similar elevations of neopterin were found (range, 24-75 nM; control mean, 9.0 +/- 4.9 nM; p < 0.005). In addition, significant elevations of QUIN were found in 14 older children with congenital hyperammonemia (mean, 50 +/- 20 vs 17 +/- 6 nM; p < 0.05). Neopterin levels were not elevated in these children. The QUIN may originate from an increase in tryptophan transport across the blood-brain barrier or from induction of indolamine-2,3-dioxygenase activity. These findings support a role for QUIN in the neuropathology of congenital hyperammonemia. They also suggest the potential utility of N-methyl-D-aspartate receptor-blocking agents or inhibitors of QUIN synthesis in the treatment of hyperammonemic coma.