FINAL PROGRAM AND ABSTRACTS

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1st EUROPEANPHENYLKETONURIA GROUP

(EPG) SYMPOSIUM

“ADVANCES ANDCHALLENGES IN PKU”

Barcelona, Spain- J

anua

ry16

-17,

2009

GENERAL INFORMATION

VENUEThe Symposium will take place at the:

Hotel Hilton Diagonal Mar Passeig del Taulat 262-264

Barcelona, Spain

LANGUAGEThe official language of this Symposium will be English.

TRAVEL INFORMATIONBarcelona was founded on the Mediterranean coast

between two rivers more than two thousand years ago.It is located in the North East of the Iberian Peninsula,

just a short distance from France. Romans, Arabs andChristians all influenced Barcelona throughout the

centuries. Traces of this diverse history with contrastingcultures can be found all over the city. From the Gothic

Quarter built on Roman ruins to its Art Noveau Exampledistrict dominated by Gaudí's exuberant architecture:

Barcelona surely is a city of contrasts.

1st European Phenylketonuria Group (EPG) Symposium - Advances and Challenges in PKU

Serono Symposia International FoundationSymposium on:

1ST EUROPEAN PHENYLKETONURIA GROUP (EPG) SYMPOSIUM “ADVANCES AND CHALLENGES IN PKU”Barcelona, Spain - January 16-17, 2009

AIM OF THE SYMPOSIUM The aim of the Symposium is to create an interdisciplinary forum for discussing present and future developments in the fieldof phenylketonuria (PKU). PKU was formerly one of the most frequent definable causes of mental retardation. Thanks tonewborn screening and early dietary therapy, mental retardation is now rarely seen in PKU. As a consequence of thisremarkable and dramatic development, PKU has become the flagship for treatable genetic disease. Despite this wonderfuladvance, however, there remains a number of very troubling problems in long term outcome. Included among these isdisagreement on how long the diet should be continued. Thus, new approaches such as enzyme replacement therapy, genetherapy, large neutral amino acids (LNAA), and tetrahydrobiopterin are of interest. This Symposium is the first in the series ofCME events and is organized by the European PKU Group (EPG). Discussed will be past, present and future efforts on PKUtreatment and four workshops will be included. The end results of these synergistic and open communications will benefit thepatients and their families.

LEARNING OBJECTIVESAfter participating in this symposium, the participants will have an updated knowledge on:• Treatment with large neutral amino acids • Treatment with tetrahydrobiopterin • Enzyme replacement and gene therapy • Compliance with the management of PKU • Nutritional care of PKU • Maternal PKU • Follow-up and outcome of PKU

TARGET AUDIENCEClinicians (pediatric and adult metabolic specialists), dietitians, clinical biochemists, experts in genetics, basic scientists, peoplewithin the area of public health.

ACCREDITATIONSerono Symposia International Foundation (www.seronosymposia.org) has submitted this program “1st EuropeanPhenylketonuria Group (EPG) Symposium - Advances and Challenges in PKU” (January 16-17, 2009 - Barcelona, Spain)for accreditation by the European Accreditation Council for Continuing Medical Education (EACCME), by the Royal College ofPhysicians, UK, and by the Italian Ministry of Health (ref. number: 13776 - 9000514).The Royal College of Physicians, UK, has accredited this program “1st European Phenylketonuria Group (EPG) Symposium- Advances and Challenges in PKU” (January 16-17, 2009 - Barcelona, Spain) with 7 (seven) credit hours.

All Serono Symposia International Foundation programs are organized solely to promote the exchange and dissemination of scientific and medicalinformation. No forms of promotional activities are permitted. There may be presentations discussing investigational uses of various products. Theseviews are the responsibility of the named speakers, and do not represent an endorsement or recommendation on the part of Serono SymposiaInternational Foundation.

This program is supported by an unrestricted educational grant from Serono Symposia International Foundation.

SCIENTIFIC COMMITTEE

Amaya Bélanger QuintanaUnidad de Enfermedades Metabólicas Servicio de Pediatría Hospital Ramón y Caja Madrid, Spain

Nenad BlauDivision of Clinical Chemistry and Biochemistry University Children's Hospital Zürich, Switzerland

Mübeccel DemirkolDepartment of Nutrition and Metabolism Istanbul Faculty of Medicine Children's Hospital Istanbul, Turkey

François Feillet Centre de Référence des Maladies Héréditaires du Métabolisme Hôpital d’Enfants, CHU Brabois Vandoeuvre les Nancy, France

Marcello Giovannini Department of Pediatrics San Paolo Hospital University of Milan Milan, Italy

Anita MacDonaldDietetic Department The Children's Hospital Birmingham, UK

Friedrich K. TrefzChildren’s Hospital Kreis Kliniken Reutlingen GmbH Klinikum Am Steinberg School of Medicine University of Tuebingen Reutlingen, Germany

Francjan J. van SpronsenBeatrix Childrens Hospital University Medical Center of Groningen Groningen, The Netherlands

CHAIRMAN

Nenad Blau Division of Clinical Chemistry and Biochemistry University Children's Hospital Zürich, Switzerland

ORGANIZING COMMITTEE

Nenad Blau Division of Clinical Chemistry and Biochemistry University Children's Hospital Zürich, Switzerland

Anita MacDonaldDietetic Department The Children's Hospital Birmingham, UK

Francjan J. van SpronsenBeatrix Childrens Hospital University Medical Center Groningen Groningen, The Netherlands

SCIENTIFIC SECRETARIAT

Serono Symposia International FoundationSalita di San Nicola da Tolentino, 1/b - 00187 Rome, ItalySenior Project Manager: Annamaria Nesticò Tel.: +39-06-420413 591 - Fax: +39-06-420413 677 E-mail: info@seronosymposia.org

Serono Symposia International Foundationis a Swiss Foundation with headquarters in14, rue du Rhône, 1204 Genève, Switzerland

ORGANIZING SECRETARIAT

Meridiano Congress International Via Mentana, 2/B - 00185 Rome - ItalyPoint Person: Simona Germoni Phone: +39-06-88595 209 - Fax: +39-06-88595 234 E-mail: s.germoni@meridiano.it

LIST OF SPEAKERS AND CHAIRMEN

Carlo AgostoniDepartment of Pediatrics University of Milan Milan, Italy

Kirsten Kiaer AhringPKU Department Kennedy Centre Glostrup, Denmark

Amaya Bélanger QuintanaUnidad de Enfermedades Metabólicas Servicio de Pediatría Hospital Ramón y Caja Madrid, Spain

Nenad BlauDivision of Clinical Chemistry and Biochemistry University Children's Hospital Zürich, Switzerland

Olaf A. Bodamer Division of Biochemical and Paediatric Genetics Austrian Newborn Screening programme University Children's Hospital Vienna, Austria

Peter BurgardDepartment of General Pediatrics / METABNET StudyCoordination Centre for Pediatric and Adolescent MedicineUniversity of Heidelberg Heidelberg, Germany

Jaime Campistol PlanaHospital Sant Joan de DeuNeuropediatricsBarcelona, Spain

Olivier DunantPresident Serono Symposia International FoundationGeneva, Switzerland

François FeilletCentre de Référence des Maladies Héréditaires du MétabolismeHôpital d’Enfants, CHU Brabois Vandoeuvre les Nancy, France

Ivar FøllingSection of EndocrinologyUniversity of Oslo Lørenskog, Norway

LIST OF SPEAKERS AND CHAIRMEN

Cary HardingMolecular and Medical Genetics Department Oregon Health & Science University Portland, OR, USA

Richard KochDepartment of Pediatrics University of Southern California School of Medicine Los Angeles, CA, USA

Philip J. LaipisBiochemistry and Molecular Biology University of Florida College of Medicine Gainesville, FL, USA

Harvey L. LevyChildren's Hospital Boston Medicine Genetics Department Boston, MA, USA

Anita MacDonaldDietetic Department The Children's Hospital Birmingham, UK

Denise M. Ney Nutritional Sciences University of Wisconsin-Madison Madison, WI, USA

Rani H. SinghHuman Genetics Department Emory University Decatur, GA, USA

Beat ThönyDepartment of Pediatrics University of Zürich Zürich, Switzerland

Friedrich K. TrefzChildren’s Hospital Kreis Kliniken Reutlingen GmbH Klinikum Am Steinberg School of Medicine University of Tuebingen Reutlingen, Germany

Francjan J. van SpronsenBeatrix Childrens Hospital University Medical Center of Groningen Groningen, The Netherlands

SCIENTIFICPROGRAM

11.00 Welcome Lunch and Registration

12.25 Welcome Host Chairman: Jaime Campistol Plana, Spain

12.30 Serono Symposia International Foundation (SSIF) Opening Olivier Dunant, Switzerland

12.35 Introduction Chairman: Nenad Blau, Switzerland

SESSION I

LARGE NEUTRAL AMINO ACIDS

12.40 Introduction:Francjan J. van Spronsen, The Netherlands

12.50 L1: Serine and LNAA transportOlaf A. Bodamer, Austria

13.10 L2: Dietary glycomacropeptide and LNAA transportDenise M. Ney, USA

13.30 L3: LNAA in daily practiceKirsten Kiaer Ahring, Denmark

13.50 Discussion

14.00 Coffee break

SESSION II

TETRAHYDROBIOPTERIN

14.25 Introduction:Nenad Blau, Switzerland

14.35 L4: Treatment modalities with BH4 in PKU patientsAmaya Bélanger Quintana, Spain

14.55 L5: BH4 or BH4 + PKU diet?Rani H. Singh, USA

15.15 L6: Long-term follow-up of PKU patients on BH4Friedrich K. Trefz, Germany

15.35 Discussion

15.45 Coffee break and transfer to parallel workshop rooms

FRIDAY - JANUARY 16, 2009

16.15 PARALLEL WORKSHOPS

WORKSHOP 1: Compliance with the management of PKU

Chairmen: Peter Burgard, Germany - Anita MacDonald, UK

• What is the definition of good compliance in PKU?• What is compliance with diet (compared with national

recommendations)?• Children• Teenagers• Adults• How good is the evidence?• Is there any evidence to suggest that trends are changing?• What data should we be collecting nationally and internationally

about compliance?

WORKSHOP 2: Nutritional care of PKU

Chairmen: François Feillet, France - Carlo Agostoni, Italy

• Identify main nutritional issues?• What is the magnitude of each issue and why?• What are the trends from country to country?• How well is the nutritional state of patients monitored? • Define minimal routine nutritional status data (and its frequency)

that should be collected?

SCIENTIFIC PROGRAM

WORKSHOP 3: Maternal PKU

Chairmen: Friedrich K. Trefz, Germany - Harvey L. Levy, USA

• How well is maternal PKU managed on an International basis?• What is the proportion of women managed pre-conception and during

pregnancy? • What are the gaps in the knowledge about maternal PKU care?• How should maternal PKU women be monitored throughout pregnancy:

define minimal and optimal standards of care? • Long term follow up of their children - what do we know and don’t

know?

WORKSHOP 4: Follow-up and outcome of PKU

Chairmen: Francjan J. van Spronsen, The Netherlands Amaya Bélanger Quintana, Spain

• What is known about the long term outcome of adult patients?• How robust is the data? What are the gaps in knowledge? What

minimal follow-up data should be collected internationally?• What do we know about the psychosocial outcome of patients- school

achievement, work, relationships etc?• How frequently should patients be monitored (clinics and blood testing)?• Children, teenagers, adults • What should be monitored? • Blood phenylalanine, psycho-metric tests?, Other

18.15 End of the day

SESSION III

ENZYME REPLACEMENT AND GENE THERAPY

08.30 Introduction:Cary Harding, USA

08.40 L7: PKU: Gene therapy, enzyme therapy, molecular chaperones?Philip J. Laipis, USA

09.00 L8: Long-term correction of PKU by a viral gene transfer: liver vs.muscleBeat Thöny, Switzerland

09.20 L9: Therapeutic liver repopulation for PKUCary Harding, USA

09.40 Discussion

09.50 Coffee break

10.20 Workshop summaries and general discussionModerators Workshops 1-4

ASBJØRN FØLLING LECTURE “GIANTS IN PKU”

11.50 Introduction:Ivar Følling, Norway

12.00 L10: The Giant Achievers in Phenylketonuria ResearchRichard Koch, USA

12.25 Presentation of the Award Nenad Blau, Switzerland - Ivar Følling, Norway

12.30 Closing remarks and end of the Symposium

SATURDAY - JANUARY 17, 2009

DISCLOSURE OF FACULTY RELATIONSHIPS

Serono Symposia International Foundation adheres to guidelines of the European Accreditation Council for Continuing Medical Education(EACCME) and all other professional organizations, as applicable, which state that programs awarding continuing education credits mustbe balanced, independent, objective, and scientifically rigorous. Investigative and other uses for pharmaceutical agents, medical devices,and other products (other than those uses indicated in approved product labeling/package insert for the product) may be presented in theprogram (which may reflect clinical experience, the professional literature or other clinical sources known to the presenter). We ask allpresenters to provide participants with information about relationships with pharmaceutical or medical equipment companies that mayhave relevance to their lectures. This policy is not intended to exclude faculty who have relationships with such companies; it is onlyintended to inform participants of any potential conflicts so participants may form their own judgments, based on full disclosure of thefacts. Further, all opinions and recommendations presented during the program and all program-related materials neither imply anendorsement, nor a recommendation, on the part of Serono Symposia International Foundation. All presentations solely represent theindependent views of the presenters/authors.

The following faculty provided information regarding significant commercial relationships and/or discussions of investigational or non-EMEA/FDA approved (off-label) uses of drugs:

Carlo Agostoni Declared no potential conflict of interest.

Kirsten Kiaer Ahring Declared no potential conflict of interest.

Amaya Bélanger Quintana Declared that her presentation will contain discussion of off-labelled or otherwise non-approved use ofproducts. The product is currently approved in the US and in the process of being approved in Europe. Ithas been used as a “compassive use” medication, approved by the Spanish legislation, with theinformed consent of patients or their legal guardians.

Nenad Blau Declared to be a member of Merck Serono SA Advisory Board.

Olaf A. Bodamer Declared no potential conflict of interest.

Peter Burgard Declared no potential conflict of interest.

Jaime Campistol Plana Declared no potential conflict of interest.

François Feillet Declared to be a member of Merck Serono SA Advisory Board.

Ivar Følling Declared no potential conflict of interest.

Cary Harding Declared no potential conflict of interest.

Philip J. Laipis Declared receipt of a research grant and contract from BioMarin Pharmaceuticals Inc. and from theUniversity of Florida.

Harvey L. Levy Declared receipt of honoraria or consultation fees from BioMarin Pharmaceuticals, Inc. and to be amember of company advisory board, board of directors or other similar group of the same company.

Anita MacDonald Declared no potential conflict of interest.

Denise M. Ney Declared receipt of grants and contracts from NIH at the end of July 2008

Rani H. Singh Declared no potential conflict of interest.

Beat Thöny Declared no potential conflict of interest.

Francjan J. Van Spronsen Declared receipt of honoraria or consultation fees from European Working Group on Tyrosinemia Type 1

The following faculty has provided no information regarding significant relationship with commercial supporters and/or discussion ofinvestigational or non-EMEA/FDA approved (off-label) uses of drugs as of December 18, 2008.

Richard Koch

Friedrich K. Trefz

This program is supported by an unrestricted educational grant from Serono Symposia International Foundation. All Serono Symposia InternationalFoundation program are organized solely to promote the exchange and dissemination of scientific and medical information. No form of promotionalactivity is permitted. There may be presentations discussing investigational uses of various products. All views are the responsibility of the namedspeakers, and do not represent an endorsement, nor recommendation on the part of Serono Symposia International Foundation.

ABSTRACTS(L1 - L10)

ABSTRACTS

L1SERINE AND LNAA TRANSPORT

Olaf A. BodamerDivision of Biochemical and Paediatric Genetics, Austrian Newborn Screening programme, University Children's Hospital,Vienna, Austria

Large Neutral Amino Acids (LNAA) and serine transport across the blood brain barrier and the intestinal mucosa are facilitated throughspecific transporter systems that reside in the luminal and abluminal sections of the respective membranes. LNAA including phenylalanine,tyrosine, branch chain amino acids, threonine, tryptophan, histidine, methionine and glutamine are transported via the L1 transporter andthe Na+ dependent LNAA transporter whereas serine is transported via the ASC transporter complex. LNAA compete with phenylalanineat the level of the L1 and Na+ dependent transporter although with different KM. Based on this concept it has been proposed to usedifferent LNAAs as additional dietary supplements in patients with PKU to reduce phenylalanine uptake to the brain or to reduce intestinaluptake in order to reduce phenylalanine blood levels thereby increasing phenylalanine tolerance. In fact, studies both in knock-out mousemodels and patients with PKU have demonstrated the validity of this approach, although long-term effects have not been investigated.

L2DIETARY GLYCOMACROPEPTIDE AND LARGE NEUTRAL AMINO ACIDTRANSPORT

Denise M. NeyNutritional Sciences and Waisman Center, University of Wisconsin-Madison,Madison, WI, USA

The primary treatment for PKU is life-long adherence to a low-phenylalanine (phe) diet that provides the majority of protein from an aminoacid (AA) formula. Dietary compliance is poor and new approaches are needed to improve the PKU diet. Glycomacropeptide (GMP) is anintact, natural protein produced during cheese making that is uniquely suited to the PKU diet because it contains minimal phe (2.5-5 mgphe/g protein) and has high concentrations of the large neutral amino acids (LNAA) threonine and isoleucine. GMP must be supplementedwith histidine, leucine, tryptophan and tyrosine to provide a complete source of protein. We developed low-phe foods and beverages madewith GMP to provide a palatable source of protein for the PKU diet (Mol Genet Metab 92:176, 2007). Our objective is to evaluate thesafety and efficacy of dietary GMP in the PKU mouse (Pah enu2) and in individuals with PKU.

PKU and wild type mice fed the GMP diets showed gains in body weight that were not different compared to the AA or casein dietsdemonstrating that GMP provides a nutritionally adequate source of protein for growing mice (J Nutr 138:316, 2008). With similar pheintake, PKU mice fed the GMP diet showed significant decreases in the concentrations of phe in plasma (11% decrease) and in brain (20%decrease) compared to the AA diet. The concentration of phe in brain was inversely correlated with the concentrations of isoleucine,threonine and valine in plasma (p<0.0001, R 2=0.74) suggesting competitive inhibition of phe transport into the brain.

A metabolic study was conducted in 11 subjects with PKU (7 male, 4 female; ages 11.5-31 years) to evaluate the safety, acceptability andefficacy of GMP compared to the usual AA diet. The design included 3 stages: establish phe tolerance (2-3 months), AA diet (6 days) andGMP diet (4 days). Subjects consumed their usual AA diet for 4 days at home and then were admitted to the hospital where they continuedthe AA diet for 2 days followed by 4 days of the GMP diet. Foods and beverages made with GMP replaced all of the AA formula and aconstant intake of phe, protein and energy was provided during the AA and GMP dietary treatments. The GMP diet was well-accepted andthere were no adverse reactions to GMP. The chemistry panel and liver function tests did not change except for a decrease in BUN withthe GMP diet. Although there was no significant difference in postprandial plasma phe level, GMP resulted in less daily variation in plasmaphe level, postprandial vs fasting states, compared with the AA diet.In addition, an adult with PKU who successfully followed the GMP diet for 10 weeks at home showed a significant 13-14% reduction inblood phe level (J Inher Metab Dis, Vol 32, 2009).

In summary, food products made with GMP provide a palatable source of low-phe, intact protein enriched in LNAA for individuals withPKU that may improve blood phe concentrations and reduce transport of phe into brain.

ABSTRACTS

L3USE OF LARGE NEUTRAL AMINO ACIDS (LNAA) IN DAILY PRACTICE

Kirsten Kiær AhringKennedy Centre, Glostrup, Denmark

LNAA has been used as alternative treatment for PKU in Denmark since 1985. Screening was implemented in Denmark in 1967 and it wasnormal procedure to stop treatment with low protein (LP) diet and amino acid (AA) supplementation from age of 18. However, it wasnoticed that the patients didn’t feel well after discontinuation of diet, but had no desire to stay on diet and compliance was poor.Supplementation of Tryptophan combined with a semi-free diet had a positive effect on HVA/5-HIAA ratio in PKU (1). The first LNAA tabletcontained only the two amino acids tyrosine and tryptophan and was used for young adults from the age of 15 years and up. Later, theproduct was further developed and today it contains all the essential amino acids. More than 100 patients in Denmark are using LNAA.

References:- J. B. Nielsen, H C. Lou, F Güttler: Effects of Diet Discontinuation and Dietary Tryptophan Supplementation on Neurotransmitter Metabolism in

Phenylketonuria. Brain Dysfunction 1988; 1:51-56

L4TREATMENT MODALITIES WITH BH4 IN PKU PATIENTS

Amaya Bélanger QuintanaUnidad de Enfermedades Metabólicas, Servicio de Pediatría, Hospital Ramón y Caja, Madrid, Spain

The investigation and use of BH4 in PKU patients in Europe has been determined by the lack of registration of the medication and its highcost. These factors made BH4 unavailable in many centers. In our center in Madrid, we have been using BH4 in PKU patients for the last 6years within clinical investigations, which have allowed us to perform over 70 short-term and 10 long-term BH4 overload tests in PKUpatients of all clinical phenotypes. We have 23 PKU patients treated on a regular basis with BH4, ages ranging from the neonatal periodto adulthood. This treatment may consist only on BH4 or on a combination of BH4 and diet. The evolution of these patients, and the factorswe have found most useful, in the determination of treatment and management of these patients, are discussed.

ABSTRACTS

L5BH4 OR BH4 + PKU DIET?

Rani H. SinghDepartment of Human Genetics, Emory University School of Medicine, Decatur, GA, USA

OBJECTIVE: While improvements in plasma phenylalanine (Phe) concentrations have been the primary outcome measure oftetrahydrobiopterin (BH4) responsiveness thus far, the implications for diet and nutrition status are lacking. The objective of thispresentation is to report the impact of BH4 on Phe tolerance, long-term dietary patterns, medical food continuation and nutritional status.

METHODS: At the Emory Genetics Clinic, 7 of 12 children with well-controlled phenylketonuria (PKU) responded to a dose of 20mg/kg/day of BH4 (sapropterin dihydrochloride) with a ≥ 30% decrease in plasma Phe concentrations after 8 days (p = 0.014). Six of theresponders were enrolled in a 12-month follow-up study to evaluate further the impact of BH4 on Phe tolerance and nutritional status.Maximum dietary Phe tolerance was determined by progressively increasing milk or egg powder over a six-week period while maintainingplasma Phe concentrations between 120 and 360 μmol/L. Subsequently, protein from medical food was decreased by 25% each weekprovided that plasma Phe concentrations and nutrition status markers remained within the therapeutic range and the average proteinintake met or exceeded US Dietary Reference Intakes (DRIs).

RESULTS: Six weeks: Dietary Phe tolerance increased to a mean±SD of 1380±395 mg/d (baseline 575mg/d±215) (p = 0.001).Twelve months: Mean plasma Phe concentrations persisted within the therapeutic range of 120-360 μmol/L throughout the 12-monthfollow-up period, while the mean dietary Phe tolerance was 1545±348 mg/d. Four of the six patients were able to completely eliminatemedical food from their diet, while the remaining two took medical food below baseline intakes. While mean total protein intake did notsignificantly decrease and continued to exceed DRIs for each patient, vitamin and mineral supplementation was required for those whodiscontinued formula to meet micronutrient DRIs. There was no significant change in mean energy intake and weight percentiles, butconcentrations of prealbumin, hemoglobin and hematocrit and cholesterol improved significantly (P <0.01).

CONCLUSIONS: These results demonstrate the need to systematically reduce medical food, to maintain nutrient adequacy of the diet,while maintaining plasma Phe levels within therapeutic range and to personalize diet recommendations. Vitamin and mineralsupplementation may be necessary, particularly if medical food has been discontinued.

L6LONG TERM FOLLOW UP OF PKU PATIENTS ON BH4

Friedrich K. TrefzKlinik fuer Kinder und Jugendmedizin, Kreiskliniken Reutlingen GmbH

In the last 10 years we treated nine patients with phenylketonuria by tetrahydrobiopterin (Schircks, Switzerland) and from 2007 six patientsalso with sapropterin (Biomarin, CA,USA) in the PKU 008 study. Patients were seen at regular intervals at least 2 times per year performingclinical status and routine blood chemistry. Blood phenylalanine was measured in the first year of life at weekly intervals, from the secondyear 2 times per month and in adults once per month according to the German recommendations for treating PKU using capillary blooddrawing performed by parents/patient on dried filer paper. Blood phe was measured by tandem mass spectrometry. No side effects wereobserved possibly related to BH4. There was an improvement of weight gain and growth under BH4 when treatment was changed fromphe restricted diet alone to a more relaxed diet under BH4 treatment. In two patients there was no effect of sapropterin on blood mediumphe concentration before and after the start of BH4 treatment, and even BH4 loading showed a response >30% indicating an effect bychance when tested at day 1 and day 8 after the challenge. There was a clear genotype-phenotype correlation in most of the patientsshowing that BH4-responsiveness is helpful for selecting PKU patients for a BH4 response loading test. However, this test should beperformed using preloading baseline blood phe measurements for a better evaluation for BH4 responsiveness. Future strategies may alsoconsider drug monitoring to confirm good compliance of drug intake in those patients with unsatisfactory blood phe control despite provenBH4 responsiveness.

ABSTRACTS

L7PKU: GENE THERAPY, ENZYME THERAPY, MOLECULAR CHAPERONES?

P.J. Laipis 1, W. Zeile 1, J. Embury 1, S. Bell 3, P. Fitzpatrick 3, R. Zori 2, Don Musson 3, C. O’Neill 3 and L. Tsuruda 3;Departments of Biochemistry and Molecular Biology 1 and Pediatrics 2, College of Medicine, University of Florida, Gainesville, FL, USAand BioMarin Pharmaceutical Inc. 3, Novato, CA, USA

Phenylketonuria (PKU) is the most frequent disorder of amino acid metabolism (~1 in 104 births) in U.S. populations of European origin.PKU patients accumulate phenylalanine (Phe) to abnormally high concentrations, usually due to reduced or absent phenylalaninehydroxylase (PAH) enzymatic activity. PAH converts Phe to tyrosine, using the cofactor tetrahydrobiopterin and molecular oxygen. Theaccumulation of Phe in PKU patients is neurotoxic and leads to a range of complications for PKU patients including abnormal mentaldevelopment, mental retardation, behavioral and neurocognitive abnormalities, depression and sleeping disorders. Although deleteriouseffects can be minimized by a Phe-restricted diet instituted at birth, the diet is demanding and most adult PKU patients are poorlycompliant leading to cognitive and behavioral deficits. Additional therapies would be valuable, especially for treatment of Maternal PKUSyndrome. Possibilities include gene therapy (introduction of a functional PAH gene), enzyme therapy (reduction of Phe levels using analternative enzymatic activity) and molecular chaperones (stabilization or activation of a mutant PAH protein). Each approach hasadvantages and disadvantages, which will be discussed. However, the most promising alternatives to diet in the immediate term aremolecular chaperones (already in clinical use) and enzyme substitution therapy using phenylalanine ammonium lyase (PAL), the majorsubject of this presentation.

Recombinant PAL is derived from Anabaena variabilis, expressed in E. coli and modified with a linear form of methoxypolyethylene glycol(PEG- 20 kDa; rAvPAL PEG). Point mutations were introduced into the native rAvPAL sequence to enhance enzyme stability and decreaseaggregation. rAvPAL-PEG was injected subcutaneously into BTBR PAHenu2 mice, an animal model of PKU that exhibits similar clinicalcharacteristics to PKU patients including hyperphenylalanemia, (baseline plasma Phe concentrations of 1.0 to 2.0 mM), behavioralchanges, and hypopigmentation. Changes in plasma Phe concentrations were followed as the primary pharmacodynamic endpoint toevaluate the effectiveness of rAvPAL-PEG. Male PKU mice receiving weekly subcutaneous injections of rAvPAL PEG initially rapidly reducedplasma Phe levels to less than 200 μM. An interim attenuated pharmacodynamic response was usually seen between weeks 3 7 withplasma Phe concentrations increasing from less than 200 μM towards baseline during the week interval between rAVPAL-PEG doses.However, by weeks 7 to 8, plasma Phe concentrations remained at concentrations below 200 µM for the entire period. Phe levels rose tobaseline after 8-12 days, but immediately returned to below 200 μM if rAvPAL-PEG injections resumed within 4 weeks; a longer hiatusof 78 days resulted in a similar interim attenuated response, which again stabilized to below 200 μM. Lower weekly doses of rAvPAL-PEG(20 mg/kg) also resulted in Phe levels below 0.5 mM for periods of 4-5 days. Interestingly, 5 mg/kg doses were not effective. Treated micehad increased body weight and pigmentation with no injection site reactions. Weekly administration of 80 mg/kg rAvPAL PEG resulted inlong-term correction of plasma Phe levels (> 6 months). Single dose PK/PD indicates that increased drug levels, in general, correlated withdecreased plasma Phe levels. We previously reported that histological abnormalities and large increases in the number of microglial cellswere seen in dopaminergic neuron-containing brain regions of PKU mice; this microglial cell infiltrate could be reduced to normal levelsby treatment with a gene therapy vector expressing PAH. A similar result was seen in the brains of PKU mice treated with rAvPAL-PEG.Microglial cell populations increased once rAvPAL-PEG treatment was halted.

Female mice also responded well to treatment with rAvPAL-PEG, although somewhat higher doses were required, consistent with thehigher plasma Phe levels seen in female BTBR PAH enu2 mice. Treated female mice became pregnant and delivered live pups. Interestingly,it appears that in mice, good survival to adulthood will require a modest increase in maternal Phe levels, perhaps to provide sufficient Phefor the large mass of developing pups when compared to maternal body weight.

Based on these and additional studies, a Phase I clinical trial was begun in mid 2008 and is currently progressing through dose escalation.In summary, these results suggest that rAvPAL-PEG could be a viable alternative to diet for non BH4 responding PKU patients.

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L8LONG-TERM CORRECTION OF PKU BY A VIRAL GENE TRANSFER: LIVER VS.MUSCLE

Beat ThönyDivision of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zürich, Zürich, Switzerland

PKU is a frequent inherited disorder of amino acid metabolism caused by deficiency of hepatic phenylalanine hydroxylase (PAH) resultingin accumulation of phenylalanine (Phe) and its metabolites in blood and other tissues. Treatment consists of Phe intake restriction, whichprevents severe neurological damage in PKU patients, although mild neuropsychological findings, such as poor school performance, a slightreduction in intelligence quotient, and the presence of tremor may arise, especially when careful dietary compliance is not achieved.Therefore, investigations of alternative or complementary therapeutic approaches are highly encouraged, including gene therapy. We havepreviously shown life-long therapeutic correction of PKU in a PKU mouse model by using a recombinant AAV2 pseudotype-8-mediatedtransfer of the murine PAH gene to liver after portal vein or tail vein administration 1. Recently, we demonstrated effective long-termcorrection for PKU also following intramuscular delivery of a recombinant triple-cistronic AAV2 serotype 1 expressing ectopically, in muscletissue, PAH along with two essential genes for tetrahydrobiopterin biosynthesis 2. This non-invasive approaches are the basis to developstrategies for an efficient and safe gene therapy procedure for PKU.

References:1. Ding, Z., Georgiev, P. & Thöny, B. 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. Gene Ther 13, 587-93 (2006).2. Ding, Z. et al. Correction of murine PKU following AAV-mediated intramuscular expression of a complete phenylalanine hydroxylating system. Mol Ther

16, 673-81 (2008).

ABSTRACTS

L9THERAPEUTIC LIVER REPOPULATION FOR PHENYLKETONURIA

Cary HardingMolecular and Medical Genetics Department. Oregon Health & Science University, Portland, OR, USA

Cell-directed therapies including gene therapy and stem cell transplantation are promising novel treatment approaches for inborn errorsof metabolism including phenylketonuria (PKU). This presentation will update the status of preclinical studies in the Pahenu2 mouse, amodel of human PKU, to develop effective cell-mediated therapeutic liver repopulation. Experiments with hepatocyte transplantation havedefined the threshold for therapeutic success but also demonstrate that a selective growth advantage for donor hepatocytes is necessaryto achieve physiologically relevant levels of liver repopulation. In an erythropoietic stem cell-mediated approach, bone marrow-derivedwild type cells are capable of fusing with PAH-deficient hepatocytes and leading to partial correction of hyperphenylalaninemia. However,random chromosomal segregation during subsequent division of the marrow-hepatocyte fusion product likely prevents completephenotypic correction as there is no selective growth advantage for PAH-expressing cells in the liver and ultimately only a small populationof PAH positive cells persist. These experiments are instructive with regards to phenylalanine metabolism and liver physiology but alsoclearly illustrate the considerable barriers to therapeutic liver repopulation as potential therapy for PKU.

L10THE GIANT ACHIEVERS IN PHENYLKETONURIA RESEARCH

Richard KochDepartment of Pediatrics, University of Southern California, School of Medicine, Los Angeles, CA, USA

Introduction: Asbjorn Folling, a Norwegian physician with biochemical training, first described phenylketonuria (PKU) in 1934. Despite hisexcellent presentation some 75 years ago, treatment for the disorder was not developed until 1951 by Horst Bickel, who isolatedphenylpyruvic acid from the urine of two children with the disorder. His knowledge of biochemistry, lead him to treat a mentally disabledchild with the disorder with a diet restricted in phenylalanine. Here again, it was due to his medical and biochemical skills, that hediscovered the use of a phenylalanine restricted diet as a treatment for phenylketonuria and the subsequent publication of his articlerecommending this treatment. Then an unusual event occurred, which caught the attention of Robert Guthrie, a physician with abacteriologic background. One of his close relatives had a child with phenylketonuria and he became interested in the idea that a diagnosisof the disorder shortly after birth might be amenable to early treatment with a diet restricted in phenylalanine. It happened that he wasworking at the University of Buffalo in New York and he met Robert Warner, who was the medical director of the PKU program there.Within a year, Dr. Guthrie developed the first newborn screening test to detect elevated blood phenylalanine levels shortly after birth. Thismade it possible to institute a phenylalanine restricted diet before brain damage had occurred in a child. There is universal agreement thatthe work of Drs. Folling, Bickel and Guthrie would be considered the giants of the first 35 years of the PKU story.

With the development of newborn screening facilitating early diagnosis, the first generation of treated patients with phenylketonuria wereable to go to school, participate in society, marry and have children. Then, to the consternation of many, the offspring of untreated womenwith PKU were born with microcephaly, Congenital Heart Disease and other problems. In 1980, Harvey Levy, a pediatrician at HarvardMedical School published the first comprehensive assessment of the Maternal PKU Syndrome and showed that women with significantlyelevated blood phenylalanine were most likely to have severely retarded offspring and those with lower blood phenylalanine levels. Thisresulted in efforts to improve control of maternal blood phenylalanine levels during pregnancy. Subsequently Flemming Guttler, a physicianin Denmark, categorized all of the Danish cases according to their blood phenylalanine levels and suggested that there were four types ofPKU: classic with blood phenylalanine levels greater than 1200 micromoles/L; moderate with levels between 900-1200 micromoles/L; mildwith levels between 600-900 and hyperphenylalaninemic with levels between 240-600 phenylalanine levels. The latter group did not needtreatment. Charles Scriver, a physician in Canada, then developed a sophisticated data base, which was facilitated by the discovery, by Dr.Savio Woo, of the mutation abnormalities of the phenylalanine hydroxylase gene. This put a firm scientific foundation for the continuedstudy of phenylketonuria.

Thus Dr. Levy, Guttler, Woo and Scriver brought a scientific basis to all of the efforts to develop adequate programs for life-long therapy tothose affected by severe mutations. As a result recommendations were established for treatment. During childhood, the recommendedrange of blood phenylalanine was 120-360 micromoles/L and 360-900 micromoles/L thereafter.

Dr. Isabel Smith in the British Isles and Professor Magdalena Ugarte in Spain provided additional data supporting these observations bydetailed studies of children with PKU. The discovery of the mutation abnormalities in the gene substantially improved our knowledge ofclinical care, as did knowledge gained from research with the PKU mouse by Ian McDonald and his associates. Finally Dr. Shigeo Kure’sdiscovery of the therapeutic value of biopterin, the cofactor for phenylalanine hydroxylase may be as significant an advance as that of HorstBickel some 50 years ago. Finally I do need to give credit to Virginia Schuett for her development of the PU Newsletter that is issued threetimes a year. She started this publication 19 years ago and it has become an international guide for families with PKU.

In summary, I have undoubtedly, left many other persons from my “Giants” in PKU, but I have one more to nominate and that is my wife,Jean, who has been my co-worker for the years since I retired from the Childrens Hospital of Los Angeles staff where I was a pediatricintern in 1951. I would never have been able to continue my work in the use of biopterin in the treatment of persons with PKU or inmaternal PKU. I have now treated maternal PKU pregnancies, with biopterin alone or in combination with dietary restriction, and I amexcited about the excellent results that I have observed. Of course this therapy will require more study and evaluation.

One other thought I would like to leave with you is that schizophrenia has occurred in three of my PKU adult patients, who were off dietarycontrol. Recovery has occurred in one patient, improvement in the second and the effect in the third is uncertain. Therein lies a whole newfuture discovery of perhaps another important use of biopterin in combination with dietary control. There is still much more to learn!

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