Phenylketonuria Mice Research Articles

177. Improvement of Monoamine Metabolism in Phenylketonuria Mouse Brain Treated with a Self-Complementary Adeno-Associated Virus Vector

177. Improvement of Monoamine Metabolism in Phenylketonuria Mouse Brain Treated with a Self-Complementary Adeno-Associated Virus Vector

Carcinogenic Effects in a Phenylketonuria Mouse Model

Phenylketonuria (PKU) is a metabolic disorder caused by impaired phenylalanine hydroxylase (PAH). This condition results in hyperphenylalaninemia and elevated levels of abnormal phenylalanine metabolites, among which is phenylacetic acid/phenylacetate (PA). In recent years, PA and its analogs were found to have anticancer activity against a variety of malignancies suggesting the possibility that PKU may offer protection against cancer through chronically elevated levels of PA. We tested this hypothesis in a genetic mouse model of PKU (PAHenu2) which has a biochemical profile that closely resembles that of human PKU. Plasma levels of phenylalanine in homozygous (HMZ) PAHenu2 mice were >12-fold those of heterozygous (HTZ) littermates while tyrosine levels were reduced. Phenylketones, including PA, were also markedly elevated to the range seen in the human disease. Mice were subjected to 7,12 dimethylbenz[a]anthracene (DMBA) carcinogenesis, a model which is sensitive to the anticancer effects of the PA derivative 4-chlorophenylacetate (4-CPA). Tumor induction by DMBA was not significantly different between the HTZ and HMZ mice, either in total tumor development or in the type of cancers that arose. HMZ mice were then treated with 4-CPA as positive controls for the anticancer effects of PA and to evaluate its possible effects on phenylalanine metabolism in PKU mice. 4-CPA had no effect on the plasma concentrations of phenylalanine, phenylketones, or tyrosine. Surprisingly, the HMZ mice treated with 4-CPA developed an unexplained neuromuscular syndrome which precluded its use in these animals as an anticancer agent. Together, these studies support the use of PAHenu2 mice as a model for studying human PKU. Chronically elevated levels of PA in the PAHenu2 mice were not protective against cancer.

Altered brain gene expression profiles associated with the pathogenesis of phenylketonuria in a mouse model

Background Phenylketonuria (PKU) is an autosomal recessive disorder caused by a deficiency of phenylalanine hydroxylase (PAH), which catalyzes the conversion of phenylalanine to tyrosine. The resultant hyperphenylalaninemia causes mental retardation, seizure, and abnormalities in behavior and movement. Methods We analyzed gene expression profiles in brain tissues of Pah enu2 mice to elucidate the mechanisms involved in phenylalanine-induced neurological damage. The altered gene expression was confirmed by real-time PCR and Western blotting. To identify markers associated with neurological damage, we examined TTR expression in serum by Western blotting. Results Gene expression profiling of brain tissue from a mouse model of PKU revealed overexpression of transthyretin ( Ttr), sclerostin domain containing 1 ( Sostdc1), α-Klotho ( Kl), prolactin receptor ( Prlr), and early growth response 2 ( Egr2). In contrast to its overexpression in the brain, TTR expression was low in the sera of PKU mice offered unrestricted access to a diet containing phenylalanine. Expression of TTR decreased in a time-dependent manner in phenylalanine-treated HepG2 cells. Conclusions These findings indicate that Ttr, Sostdc1, Kl, Prlr, and Egr2 can be involved in the pathogenesis of PKU and that phenylalanine might have a direct effect on the level of TTR in serum.

Nutritional management of PKU with glycomacropeptide from cheese whey

Individuals with phenylketonuria (PKU) must follow a lifelong low-phenylalanine (Phe) diet to prevent neurological impairment. Compliance with the low-Phe diet is often poor owing to restriction in natural foods and the requirement for consumption of a Phe-free amino acid formula or medical food. Glycomacropeptide (GMP), a natural protein produced during cheese-making, is uniquely suited to a low-Phe diet because when isolated from cheese whey it contains minimal Phe (2.5-5 mg Phe/g protein). This paper reviews progress in evaluating the safety, acceptability and efficacy of GMP in the nutritional management of PKU. A variety of foods and beverages can be made with GMP to improve the taste, variety and convenience of the PKU diet. Sensory studies in individuals with PKU demonstrate that GMP foods are acceptable alternatives to amino acid medical foods. Studies in the PKU mouse model demonstrate that GMP supplemented with limiting indispensable amino acids provides a nutritionally adequate source of protein and improves the metabolic phenotype by reducing concentrations of Phe in plasma and brain. A case report in an adult with classical PKU who followed the GMP diet for 10 weeks at home indicates safety, acceptability of GMP food products, a 13-14% reduction in blood Phe levels (p<0.05) and improved distribution of dietary protein throughout the day compared with the amino acid diet. In summary, food products made with GMP that is supplemented with limiting indispensable amino acids provide a palatable alternative source of protein that may improve dietary compliance and metabolic control of PKU.

871. Complete Restoration of In Vivo Oxidative Capacity for Phenylalanine in Self-Complementary AAV Vector-Treated Phenylketonuria Mice

871. Complete Restoration of In Vivo Oxidative Capacity for Phenylalanine in Self-Complementary AAV Vector-Treated Phenylketonuria Mice

Dietary Glycomacropeptide Supports Growth and Reduces the Concentrations of Phenylalanine in Plasma and Brain in a Murine Model of Phenylketonuria ,

Phenylketonuria (PKU) is a genetic disorder caused by deficiency of phenylalanine hydroxylase (PAH) that requires life-long adherence to a low-phenylalanine (Phe) diet. Glycomacropeptide (GMP) is uniquely suited to the nutritional management of PKU, because pure GMP contains no Phe. Our aim was to assess how ingestion of diets containing GMP support growth and affect the concentrations of amino acids in plasma and brains of mice with a deficiency of PAH, the Pahenu2 mouse (PKU mouse). Experiments were conducted in 4- to 6-wk-old wild-type (WT) (C57Bl/6) and PKU mice fed diets containing 20% protein from casein, amino acids, or GMP supplemented with limiting indispensable amino acids (IAA). PKU mice fed the GMP diet showed gains in body weight, feed efficiency, and a protein efficiency ratio that did not differ from the amino acid diet. The concentrations of isoleucine and threonine in plasma showed a significant 2- to 3-fold increase for WT and PKU mice fed GMP compared with casein or amino acid diets, respectively. PKU mice fed the GMP diet had decreased concentrations of Phe in plasma (11% decrease) and in 5 regions of the brain (20% decrease) compared with the amino acid diet. The concentration of Phe in the brain was inversely correlated with the concentrations of isoleucine, threonine, and valine in plasma (R2 = 0.74; P < 0.0001), suggesting competitive inhibition of Phe transport into the brain. In summary, PKU mice fed GMP showed comparable growth and reduced concentrations of Phe in plasma and the brain compared with an amino acid diet. These data support the use of GMP supplemented with IAA as an alternative source of dietary protein for individuals with PKU.

Protective Effect of Recombinant Adeno-Associated Virus 2/8-Mediated Gene Therapy from the Maternal Hyperphenylalaninemia in Offsprings of a Mouse Model of Phenylketonuria

Phenylketonuria (PKU) is an autosomal recessively inherited metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH). The accumulation of phenylalanine leads to severe mental and psychomotor retardation, and the fetus of an uncontrolled pregnant female patient presents with maternal PKU syndrome. We have reported previously on the cognitive outcome of biochemical and phenotypic reversal of PKU in a mouse model, Pahenu2, by the AAV serotype 2-mediated gene delivery of a human PAH transgene. However, the therapeutic efficacy had been limited to only male PKU mice. In this study, we generated a pseudotyped recombinant AAV2/8-hPAH vector and infused it into female PKU mice through the hepatic portal vein or tail vein. Two weeks after injection, complete fur color change to black was observed in female PKU, as in males. The PAH activity in the liver increased to 65-70% of the wild-type activity in female PKU mice and to 90% in male PKU mice. Plasma phenylalanine concentration in female PKU mice decreased to the normal value. In addition, the offsprings of the treated female PKU mice can rescue from the harmful effect of maternal hyperphenylalaninemia. These results indicate that recombinant AAV2/8-mediated gene therapy is a potential therapeutic strategy for PKU.

Correction in Female PKU Mice by Repeated Administration of mPAH cDNA Using phiBT1 Integration System

Phenylketonuria (PKU) is a metabolic disorder secondary to a hepatic deficiency of phenylalanine hydroxylase (PAH) that predisposes affected children to develop severe and irreversible mental retardation. We have previously reported the complete and permanent correction of the hyperphenylalaninemic and hypopigmentation phenotypes in male, but not female, PKU mice after genome-targeted delivery of murine PAH (mPAH) complementary DNA (cDNA) in a phiBT1 bacteriophage integration system. Here we show that sequential administration of green fluorescent protein (GFP)- and red fluorescent protein (RFP)-expressing cassettes in the phiBT1 integration system led to distinct and non-overlapping populations of green and red fluorescent hepatocytes in vivo. The hyperphenylalaninemic and hypopigmentation phenotypes of female PKU mice were completely corrected after 10 weekly administrations of mPAH cDNA. Importantly, there was no apparent liver pathology in mice even after 10 consecutive administrations of the phiBT1 integration system. The results indicate that repeated administration of transgenes in the phiBT1 integration system can lead to their genome-targeted integration in a diverse population of hepatocytes and result in the elevation of transgene expression levels in a cumulative manner, which can be utilized to overcome insufficient transgene expression owing to low genome integration frequencies in a gene therapy paradigm for metabolic disorders.

Metabolic Basis of Sexual Dimorphism in PKU Mice After Genome-targeted PAH Gene Therapy

We have previously reported a transgene delivery system based on phiBT1 bacteriophage integrase that results in targeted insertion of transgenes into mammalian genomes, and its use in the delivery of murine phenylalanine hydroxylase (PAH) complementary DNA (cDNA) into the hepatocytes of male phenylketonuria (PKU) mice, leading to a complete and permanent correction of their hyperphenylalaninemic phenotype. In this study, we report only partial phenotypic correction in female PKU mice, even though hepatic PAH activities in both sexes after gene treatment were similar. Daily injections of tetrahydrobiopterin (BH4), an essential co-factor for phenylalanine hydroxylation, in the gene-treated females led to complete correction of their PKU phenotype. After gonadectomy, serum phenylalanine levels in the gene-treated females were reduced to normal, whereas those in the gene-treated males remained unchanged. The sterile gene-treated PKU mice were subjected to daily sex hormone injections. Whereas the estradiol-treated sterile males developed hyperphenylalaninemia, the dihydrotestosterone-treated sterile females remained normal phenylalaninemic. The results indicate that it is estrogen that suppresses the steady-state levels of BH4 in mouse hepatocytes that became limiting, which is the underlying mechanism for the observed sexual dimorphism in PKU mice after PAH gene treatment. Livers of the PAH gene-corrected PKU mice also appeared normal and without apparent pathologies.

Epilepsy in Phenylketonuria: A Complex Dependence on Serum Phenylalanine Levels

Phenylketonuria (PKU) is a disorder of phenylalanine (Phe) metabolism that frequently results in epilepsy if a low Phe diet was not implemented at birth. The mechanisms by which Phe affects the brain are poorly understood. Audiogenic seizures (AGS) were studied in female homozygous Pah(enu2) BTBR (PKU) mice. Adult PKU mice, 18-20 weeks of age, in contrast to wild-type and heterozygous counterparts, exhibited a full range of AGS. Younger PKU mice, 5-7 weeks of age, had higher serum Phe levels (2.22 +/- 0.20 mM) in comparison with the adult animals (1.72 +/- 0.05 mM) and were not susceptible to AGS. Among adult mice, animals susceptible to AGS had significantly lower serum Phe levels (1.62 +/- 0.06 mM) in comparison with those resistant to AGS (1.86 +/- 0.07 mM). Susceptibility to AGS tended to increase in the afternoon when serum Phe concentration decreased in comparison to evening and morning. Normalization of serum Phe level by instituting a low Phe diet generally prevented susceptibility to AGS within 12 h. Although return to a standard diet raised Phe levels to hyperphenylalaninemic within 12 h in animals treated with a low Phe diet for 2 weeks, more than 7 weeks were needed for a complete resumption of AGS. Transient decrease in Phe levels within hyperphenylalaninemic range may be a necessary condition for PKU-related seizures to occur. A low Phe diet prevents susceptibility to seizures, which can resume with the significant delay after termination of dietary treatment.

Gene therapy for phenylketonuria mouse model using pseudotyped adeno‐associated virus vector

Phenylketonuria (PKU) is an inherited metabolic disorder caused by a deficiency of phenylalanine hydroxylase. The accumulation of phenylalanine leads to severe mental and psychomotor retardation. Phenylalanine restriction diet can prevent irreversible damage if instituted from birth. Recently, we reported the cognitive outcome of biochemical and phenotypic reversal of PKU mouse model, Pahenu2, by the AAV 2-mediated gene delivery of a human PAH transgene (Pediatr Res 56:–284, 2004). However, the therapeutic effectiveness had been observed only in male PKU mice. No normalization in coat color or plasma phenylalanine level was observed in gene therapy on female PKU mice. In this study, we generated pseudotyped AAV2/8 vector encoding the human PAH gene. The rAAV2/8-hPAH vector was infused into female PKU mice via the hepatic portal vein. Six weeks after injection, the coat color change to black was clearly observed in female PKU as in male. Plasma phenylalanine level in female PKU mice decreased to 120_μM/L. The PAH enzyme activities of treated mouse liver increased to 65–70% of wild-type in female PKU mice, to 90–95% in male PKU mice. These corrections were sustained over 6 month. Furthermore, we observed rAAV2/8-hPAH treated female PKU mice can overcome maternal PKU syndrome. These results indicate that recombinant AAV 2/8 mediated gene therapy can be a useful therapeutic candidate for patients with PKU.

207. Long-Term Therapy for PKU in Mice by rAAV Pseudotype 8 Gene Transfer to Liver

Phenylketonuria (PKU) is caused by an autosomal recessive deficiency of the hepatic phenylalanine hydroxylase (PAH) leading to high serum phenylalanine and irreversible damage of the brain. We recently reported on the use of recombinant AAV2 to deliver the PAH gene to the liver of PKU mice (C57Bl/6-Pah-enu2), which is an exceptionally faithful model for the human disease (Ref: Ding, Georgiev and Thony, Gene Ther, advanced online publication Dec 2005). In this study we used an AAV2/8 hybrid vector expressing the murine PAH-cDNA under the control of the composite CMV enhancer-chicken beta-actin promoter and which was pseudotyped with capsids from serotype 8 and delivered to the liver of PKU mice via single injections of portal vein with 5x10e12 vg per mouse or tail vein with 2x10e13 vg per mouse. Untreated PKU mice exhibit normal expression of mutant Pah-mRNA and PAH protein, but no detectable enzyme activity. They consequently had Phe levels between 1500 to 2500 micromol/l (25 to 42 mg/dl), and we (and others) have noticed that Phe concentrations in PKU mice were slightly higher for females (1866 micromol/l) than males (1695 micromol/l) rendering females putatively more resistive to PKU treatment. Two weeks post hepatic or systemic delivery of AAV2/8 a complete phenotypic correction was observed in PKU mice with blood Phe levels decreasing from high to normal values (<120 micromol/l or 2mg/dl). The therapeutic levels of plasma Phe concentrations (360 micromol/l or 6 mg/dl), as determined approximately every four weeks, were observed for a period of at least 7-10 months. Here we report on the follow-up of this gene therapy approach during a period of over a year where animals were successively euthanized for analyzing their hepatic viral load and PAH expression, and only two females were kept in each study until the end of the experiment. For tail vein injection, one female started to rise Phe concentrations after 35 weeks, and at 56 weeks the two females had Phe blood levels of 1203 and 1453 micromol/l. For portal vein infusion, mice remained at therapeutic levels at least until week 61, and three weeks later the remaining two females had Phe values of 284 and 517 micromol/l. Thus far we have collected data on viral titers after 8 and 46 weeks for portal vein injection, and found a decrease from 3400 to below 1000 viral genome copies per diploid hepatocytes genome. PAH expression 8 weeks after portal vein injection was found to be moderate with a 32% increase of Pah-mRNA and a 2.2-fold increase of PAH protein, and PAH enzyme activity comparable to wild-type mice. Expression analysis of PAH and parameter important for the enzymatic activity at different treatment intervals are under way. In conclusion, a 4-fold lower concentration of the AAV2/8 vector directly injected into the portal vein as compared to tail vein resulted in a more efficient long-term correction of serum Phe levels. Furthermore, understanding the mechanism for the re-rise of blood Phe levels might help to improve a gene therapy protocol to eventually perform complete and persistent correction of PKU by hepatic gene transfer with AAV vectors.

404. Therapeutic Correction of PKU in a Mouse Model by Ectopic Expression of PAH and Its BH4- Cofactor Genes in Skeletal Muscle by a Recombinant Triple-Cistronic AAV2-Based Pseudotype 1 Vector

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism with a deficiency of the hepatic phenylalanine hydroxylase (PAH) leading to toxic accumulation of circulating phenylalanine (Phe) in blood, resulting in growth failure, microcephaly, seizures, and mental retardation. PAH catalyzes the hydroxylation of Phe to tyrosine and needs oxygen and tetrahydrobiopterin (BH4) as cofactor. BH4 biosynthesis requires the consecutive action of the enzymes GTPCH and PTPS, with dihydroneopterin triphosphate as the first intermediate. Here we aimed at expressing the PAH system in skeletal muscle to degrade serum Phe in PKU patients, as this tissue is abundant, easy accessible, and persistent due to its post-mitotic nuclei. However, BH4 is abundant in liver but scarce in skeletal muscle, as the cofactor- synthesizing enzyme GTPCH is absent in muscle tissue, and PTPS is expressed at low levels. We first demonstrated that transgenic PKU mice that had no liver PAH and expressed coordinately PAH along with GTPCH in skeletal muscle tissue accumulated dihydroneopterin triphosphate and remained hyperphenylalaninemic unless synthetic BH4-cofactor was supplied by intraperitoneal injections. Thus, PTPS activity is definitely limiting in skeletal muscle to synthesize sufficient BH4 and to support Phe hydroxylation. A recombinant triple-cistronic AAV2- based pseudotype 1 vector expressing PAH along with the two cDNA-genes for BH4 biosynthesis, GTPCH and PTPS, was then generated. Upon single injections of at least 3.5x10e12 recombinant triple-cistronic AAV2/1 vector particles into each of the gastrocnemius muscles of the hind legs of the PKU mouse model Pah-enu2 resulted in long-term clearance of blood Phe, including complete phenotypic reversion. A similar therapeutic effect was achieved when a combination of two AAV2/1 vectors expressing individually PAH and GTPCH-PTPS were co-injected into the same hind leg muscles. As a control, an AAV2/1-vector expressing only PAH with GTPCH was not therapeutic. This non-invasive application is the basis to develop an efficient therapy for PKU using a triple-cistronic gene transfer into skeletal muscle.

411. The Metabolic Basis of Sexual Dimorphism in PKU Mice after PAH Gene Treatment

Phenylketonuria (PKU) is a metabolic disorder secondary to a hepatic deficiency of phenylalanine hydroxylase (PAH) that predisposes affected individuals to the development of severe mental retardation. PAH is the enzyme that catalyzes the conversion of phenylalanine to tyrosine, and its deficiency leads to hyperphenylalaninemia as well as hypopigmentation in patients. We have previously reported a transgene delivery system based on phiBT1 bacteriophage integrase that result in targeted insertion of transgenes into the mammalian genome, and the use of it to transfer a CAG-driven mouse PAH cDNA expression cassett in the hepatocytes of PKU mice.

227. Sustained Correction of Hyperphenylalaninemia in Female Pahenu2 Mouse by a Self-Complementary Adeno-Associated Virus Vector

The classic form of phenylketonuria (PKU) is caused by deficiency of phenylalanine hydroxylase (PAH), a critical liver enzyme converting phenylalanine (Phe) to tyrosine. If untreated, elevated serum Phe levels predispose afflicted children to severe brain damage. The present therapy for PKU mandates strict restriction of protein intake, preferably for the lifetime. Therefore long-term curing means such as gene therapy are awaited. Among preclinical studies with a mouse model of PKU (Pahenu2 strain carrying a missense mutation in the PAH gene), vectors derived from adeno- associated virus (AAV) have given the most promising results. We and other investigators have successfully corrected hyperphenylalaninemia by portal delivery of recombinant AAV vectors. Similar to other AAV-mediated liver transduction models, however, female animals required much higher vector dose than males, and tended to lose the therapeutic effect over time. Aiming at better gene transfer to the liver, we investigated the efficacy of the self-complementary AAV system in Pahenu2. A self-complementary AAV vector (scAAV8/LP1-mPAH) was constructed by replacing the human factor IX gene of scAAV8/LP1-hFIX (a gift from Dr. JT Gray, St. Jude Research Hospital) with the murine PAH gene. For comparison, a conventional single strand AAV8 vector (AAV8/CAG-mPAH) was constructed by transferring the PAH expression cassette from AAV5/CAG-mPAH (Mochizuki S et al, Gene Therapy 11:1081, 2004). AAV type 8 capsid was chosen for packaging because of its stronger liver tropism than AAV2 and AAV5. In most cases, vectors were given to the peritoneal cavity of Pahenu2 mice of 4-5 weeks of age, because AAV8 vectors accumulate mainly in the liver regardless of the route of administration. In parallel to our previous experiment with AAV5, female PKU mice were refractory to AAV8/CAG-mPAH. While 1x1012 particles of the vector reduced blood Phe to the therapeutic level in males, no Phe reduction was obtained by injecting tenfold more AAV8/CAG-mPAH (1x1013 particles) to females. Portal injection of the same vector dose (1x1013 particles of AAV8/CAG-mPAH) was effective initially, but hyperphenylalaninemia revisited after 16 weeks of the treatment. In contrast, the self-complementary AAV vector brought about a dramatic effect. Within 2 weeks after receiving 3x1012 or 1x1013 particles of scAAV8/LP1-mPAH, blood Phe was completely normalized in the female Pahenu2 mice. The therapeutic effect has been stable up to 16 weeks (3x1012) and 30 weeks (1x1013), and the follow-up study is ongoing. Injection of 1x1012 particles of scAAV8/ LP1-mPAH had no effect, suggesting that the threshold dose lies somewhere between 1x1012 and 3x1012 particles when the vector is administered intraperitoneally. These results indicated that the efficacy of scAAV8/LP1-mPAH was about tenfold higher than AAV8/CAG-mPAH. Dissection of the mechanisms for this superior performance of scAAV8/LP1-mPAH should help further vector improvement for PKU treatment.

Administration-route and gender-independent long-term therapeutic correction of phenylketonuria (PKU) in a mouse model by recombinant adeno-associated virus 8 pseudotyped vector-mediated gene transfer

Phenylketonuria (PKU) is an inborn error of metabolism caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH) which leads to high blood phenylalanine (Phe) levels and consequent damage of the developing brain with severe mental retardation if left untreated in early infancy. The current dietary Phe restriction treatment has certain clinical limitations. To explore a long-term nondietary restriction treatment, a somatic gene transfer approach in a PKU mouse model (C57Bl/6-Pahenu2) was employed to examine its preclinical feasibility. A recombinant adeno-associated virus (rAAV) vector containing the murine Pah-cDNA was generated, pseudotyped with capsids from AAV serotype 8, and delivered into the liver of PKU mice via single intraportal or tail vein injections. The blood Phe concentrations decreased to normal levels (< or =100 microM or 1.7 mg/dl) 2 weeks after vector application, independent of the sex of the PKU animals and the route of application. In particular, the therapeutic long-term correction in females was also dramatic, which had previously been shown to be difficult to achieve. Therapeutic ranges of Phe were accompanied by the phenotypic reversion from brown to black hair. In treated mice, PAH enzyme activity in whole liver extracts reversed to normal and neither hepatic toxicity nor immunogenicity was observed. In contrast, a lentiviral vector expressing the murine Pah-cDNA, delivered via intraportal vein injection into PKU mice, did not result in therapeutic levels of blood Phe. This study demonstrates the complete correction of hyperphenylalaninemia in both males and females with a rAAV serotype 8 vector. More importantly, the feasibility of a single intravenous injection may pave the way to develop a clinical gene therapy procedure for PKU patients.

Reversal of gene expression profile in the phenylketonuria mouse model after adeno-associated virus vector-mediated gene therapy

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by phenylalanine hydroxylase (PAH) deficiency. Accumulation of phenylalanine leads to severe mental and psychomotor retardation, and hypopigmentation of skin and hair. We have demonstrated the cognitive outcome of biochemical and phenotypic reversal by the adeno-associated virus vector-mediated gene delivery of a human PAH transgene. In this study, we identified the expression of genes related to pathologic abnormalities of the PKU-affected brain, in which the symptoms of PKU are mainly manifest, and transcriptional changes in effective gene therapy treatment using oligonucleotide array. Therapeutic effectiveness was verified by change in enzyme activity (15 ± 5.84%), phenylalanine plasma level (261 ± 108 μM), and coat color. Our data indicated that 12 genes were significantly up-regulated in PKU. Four are involved in defense and inflammatory responses of neutrophils (NE, MPO, NGP, and CRAMP), three other overexpressed genes are related to extracellular matrix organization and degradation (COL1A1, COL1A2, and MMP13); the remainder were a nociceptor in sensory neurons ( MrgA1), a structural gene of P lysozyme (Lzp-s), an immunoglobulin α heavy chain constant region gene (Igh-2), an osteocalcin-related protein precursor (Bglap-rs1), and a membrane-spanning 4 domain, subfamily A, member 3 (Ms4a3). Data demonstrated that elevated genes in the PKU-affected brain could be normalized by human PAH gene delivery. Although we could not precisely link transcript level changes and neurologic pathogenesis, this study provides a more comprehensive understanding of the PKU-affected brain at the molecular level, possibly resulting in better therapeutic approaches.

Development of Pegylated Forms of Recombinant Rhodosporidium toruloides Phenylalanine Ammonia-Lyase for the Treatment of Classical Phenylketonuria

Phenylketonuria (PKU) is a metabolic disorder due primarily to mutations in the PAH gene that impair both phenylalanine hydroxylase activity and disposal of l-phenylalanine from the normal diet. Excess phenylalanine is toxic to cognitive development and a low-phenylalanine diet prevents mental retardation, but it is a difficult therapeutic option. Previous studies with recombinant phenylalanine ammonia-lyase, PAL, demonstrated pharmacologic and physiologic proofs of principle for PAL as an alternative therapy for PKU but its immunogenicity was problematic. From a series of formulations of linear and branched polyethylene glycols chemically conjugated to PAL, we have created a parenteral therapeutic agent for PKU treatment. All the pegylated molecules were fully characterized in vitro and the most promising formulations were then tested in vivo in the PKU mouse model. The linear 20-kDa PEG-PAL combination abolished in vivo immunogenicity after repeated challenge while retaining full catabolic activity against phenylalanine, suggesting potential as a novel PKU therapeutic.

Expression of calpastatin, minopontin, NIPSNAP1, rabaptin-5 and neuronatin in the phenylketonuria (PKU) mouse brain: Possible role on cognitive defect seen in PKU

Phenylketonuria (PKU) is an inborn error of amino acid metabolism. Phenylalanine hydroxylase (PAH) deficiency results in accumulation of phenylalanine (Phe) in the brain and leads to pathophysiological abnormalities including cognitive defect, if Phe diet is not restricted. Neuronatin and 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) reportedly have role in memory. Therefore, gene expression was examined in the brain of mouse model for PKU. Microarray expression analysis revealed reduced expression of calpastatin, NIPSNAP 1, rabaptin-5 and minopontin genes and overexpression of neuronatin gene in the PKU mouse brain. Altered expression of these genes was further confirmed by one-step real time RT-PCR analysis. Western blot analysis of the mouse brain showed reduced levels of calpastatin and rabaptin-5 and higher amount of neuronatin in PKU compared to the wild type. These observations in the PKU mouse brain suggest that altered expression of these genes resulting in abnormal proteome. These changes in the PKU mouse brain are likely to contribute cognitive impairment seen in the PKU mouse, if documented also in patients with PKU.

Altered expression of myocilin in the brain of a mouse model for phenylketonuria (PKU)

Phenylketonuria (PKU) is an inborn error of amino acid metabolism. Phenylalanine hydroxylase ( PAH) mutations resulting reduced enzyme levels lead to accumulation of phenylalanine (Phe) in brain, if Phe diet is not restricted. Patients with PKU show neurophysiological abnormalities including demyelination and cognitive defect. How PAH defect causes events seen in PKU is not obvious. Therefore, expression analysis was performed in the brain of a mouse model for PKU. Microarray expression profile of the brain showed lower expression of myocilin ( Myoc) in the PKU mouse. Reduced expression of Myoc was further confirmed by one-step real-time RT-PCR. Western blotting analysis of the brain using equal quantities of protein showed a thin band in PKU compared to a prominent band in the wild type brain. In addition, expression of genes associated with transcription was found to be altered in the PKU mouse brain as observed by microarray analysis. These data suggest that PAH defect alters other genes expression likely to contribute neurophysiological abnormalities seen in the mouse, if documented also in patients with PKU.