RARE DISEASE
Hyperphenylalaninemia due to tetrahydrobiopterin deficiency
Hyperphenylalaninemia due to tetrahydrobiopterin deficiency
Hyperphenylalaninemia due to tetrahydrobiopterin deficiency
Synonyms: Hyperphenylalaninemia due to BH4 deficiency
Synonyms: Hyperphenylalaninemia due to BH4 deficiency
Synonyms: Hyperphenylalaninemia due to BH4 deficiency
Drug discovery
8
drugs
With orphan designations
Overview
Hyperphenylalaninemia due to tetrahydrobiopterin (BH4) deficiency is a rare autosomal recessive disorder caused by defects in BH4 biosynthesis or recycling, impairing phenylalanine metabolism and neurotransmitter synthesis. BH4 serves as a cofactor for phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase, leading to elevated phenylalanine levels and dopamine/serotonin deficiency. Clinical features include progressive neurological deterioration, dystonia, seizures, and developmental delays. Diagnosis combines newborn screening for hyperphenylalaninemia with urinary pterin analysis and genetic testing [1][3][7].
Therapies
BH4 supplementation (2–20 mg/kg/day) or phenylalanine-restricted diet to normalize phenylalanine levels [3][7].
Neurotransmitter replacement (L-dopa/carbidopa and 5-hydroxytryptophan) to address dopamine/serotonin deficiency [3][7].
Folinic acid for dihydropteridine reductase (DHPR) deficiency to prevent cerebral folate depletion [7][9].
Categories: rare genetic diseases, rare inborn errors of metabolism, rare neurological diseases
Research Papers
506 drug discovery papers about Hyperphenylalaninemia due to tetrahydrobiopterin deficiency, with 6 first-in-class and 14 next-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
506 drug discovery papers about Hyperphenylalaninemia due to tetrahydrobiopterin deficiency, with 6 first-in-class and 14 next-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
categories:
Small molecules
small molecules
2026-09-11 | A phase 1/2 dose-escalation study of sepiapterin in patients with 6-pyruvoyl-tetrahydropterin synthase deficiency with hyperphenylalaninemia.
Oral sepiapterin (PTC923), a precursor of tetrahydrobiopterin (BH4), was evaluated in patients with 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency in a phase 1/2, multicenter, open-label, randomized, intra-individual dose-escalation study. Patients stopped sapropterin (used to control hyperphenylalaninemia) for 2-4-days before being randomized 1:1 into 2 cohorts receiving sepiapterin at 2 doses (Cohort 1: 2.5 and 10 mg/kg/day; Cohort 2: 5 and 20 mg/kg/day) each for 7 days, separated by a 2-4-day washout period. Eight participants (5 male, 3 female, aged 2.2-20.0 years) completed the study (4 in each cohort). Overall, 7/8 participants (87.5%) experienced a total of 35 treatment-emergent adverse events; none were severe, serious, or led to discontinuation. Mean phenylalanine levels increased (up to 800-1200 μmol/L) after discontinuation of sepiapterin or sapropterin, and normalized (<130 μmol/L) with sepiapterin at all tested doses by Day 2. Blood BH4 concentrations increased with increasing single doses of sepiapterin, reaching a peak at approximately 3-4 hours, which was 2-3 hours after the sepiapterin peak. These results show that in patients with PTPS deficiency, sepiapterin is rapidly converted to BH4 and normalizes blood phenylalanine concentrations, with no dose-limiting toxicity or dose-related adverse events.
2026-08-19 | Targeting Mutant Phenylalanine Hydroxylase With Pyrimidine-Triazole Conjugates: A Primary Framework for Candidate Chaperone-Based Strategies in Phenylketonuria.
Phenylketonuria (PKU) is a rare metabolic disorder caused by pathogenic mutations in the phenylalanine hydroxylase (PAH) gene, which impair the conversion of phenylalanine to tyrosine, leading to subsequent neurotoxicity. Dietary management, sapropterin dihydrochloride, and pegvaliase are the current therapies; however, their limited efficacy and adverse effects highlight the pressing need for pharmacological treatments that restore PAH activity. Therefore, in this work, we designed and synthesized pyrimidine-triazole derivatives (Pyr-TZ) as candidate pharmacological chaperones targeting the catalytic domain of PAH. Molecular docking disclosed that Pyr-TZ-2O and Pyr-TZ-2S exhibited higher binding energies (-8.5 to -10.3 kcal/mol) for the wild-type (WT) and mutant (R252Q) PAH compared to sapropterin (-7.0 kcal/mol). Molecular Mechanics with Generalized Born and Surface Area solvation (MM-GBSA) was used here as comparative estimates suggested enhanced binding stability of Pyr-TZ compounds, primarily driven by van der Waals and lipophilic interactions. Drug-likeness and ADMET profiling indicated favorable pharmacokinetic properties. Biological validation in R252Q mutant cells demonstrated significant upregulation of PAH and key tetrahydrobiopterin (BH4) pathway genes, including quinonoid dihydropteridine reductase (QDPR), sepiapterin reductase (SPR), and 6-pyruvoyl-tetrahydropterin synthase (PTS), following Pyr-TZ-2O treatment. Consistent with these findings, sandwich ELISA revealed a dose-dependent increase in PAH protein abundance post-Pyr-TZ-2O treatment. Conclusively, Pyr-TZ-2O can be considered a potential pharmacological chaperone capable of stabilizing mutant PAH and enhancing cofactor regeneration, offering a rational therapeutic approach for PKU.
2026-07-24 | DNAJC12 Stabilizes Phenylalanine Hydroxylase and Facilitates Its Substrate-Dependent Activation.
Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH4)-dependent enzyme that converts L-phenylalanine (L-Phe) to L-tyrosine. PAH dysfunction leads to the accumulation of L-Phe in the blood (hyperphenylalaninemia; HPA), which may reach neurotoxic levels, resulting in phenylketonuria (PKU). PKU is associated with pathogenic variants in PAH, mostly causing misfolding and instability, leading to decreased levels of PAH protein and activity. Recently, variants in the J-domain protein DNAJC12 have also been associated with HPA in patients, demonstrating the importance of protein homeostasis regulation for proper PAH function. DNAJC12 and PAH have previously been reported to interact, but the molecular and structural mechanisms behind complex formation have remained unclear. In this work, we show that DNAJC12 binds to PAH but presents higher affinity for its L-Phe activated form, which resembles the conformation of unliganded tyrosine hydroxylase, a structurally and functionally related enzyme that also binds to DNAJC12. At saturation, four DNAJC12 monomers bind and stabilize the PAH tetramer, protecting it from aggregation and lowering the L-Phe concentration necessary for substrate-induced activation, without affecting the interaction of the enzyme with its cofactor BH4. Importantly, DNAJC12 also stabilizes and delays the aggregation of the PKU-associated variant PAH-p.R261Q. This study provides the first detailed characterization of the molecular determinants driving PAH:DNAJC12 complex formation and reveals how this interaction modulates enzyme stability and activity, and stimulates Hsc70 ATPase activity. These findings provide mechanistic insight into the pathogenic basis of DNAJC12 deficiency and identify the PAH:DNAJC12 complex as a promising therapeutic target for HPA.
2026-07-15 | Blood Phenylalanine Control in Paediatric and Adult Centres in the UK: Data from 2012-2018.
Background: Metabolic control in phenylketonuria (PKU) is known to deteriorate with age, but national-level data describing blood phenylalanine (Phe) control across the United Kingdom (UK) are limited. Objective: To characterise blood Phe control in individuals with PKU attending UK metabolic centres. Methods: Sixteen UK centres (nine paediatric, six adult, one mixed) retrospectively extracted blood Phe results collected between 2012 and 2018. Demographic, phenotypic and monitoring-related variables were analysed. Written consent for data collection was obtained from all patients or their caregivers. Results: Data were available for 871 individuals (55% female), of whom 744 (85%) were classified as follows: classical PKU, 75%, mild PKU, 22% and hyperphenylalaninaemia, 3%. Mean blood Phe concentrations were significantly higher in adults than children (491 ± 308 vs. 303 ± 199 µmol/L; p < 0.001), and the proportion of samples within target range declined steadily with age, from 78% in children < 2 years to 36% in adults ≥ 41 years. Individuals with classical PKU had higher mean Phe concentrations and lower target attainment than those with HPA (386 vs. 300 µmol/L; 61% vs. 78%; p < 0.001), while mild PKU and HPA showed comparable control. Females generally demonstrated better metabolic control than males. More frequent dried blood spot sampling for blood Phe was strongly associated with improved metabolic control: weekly (254 ± 175 µmol/L; 82% within target), fortnightly (319 ± 207 µmol/L; 70%), monthly (397 ± 231 µmol/L; 61%), and less than monthly (624 ± 349 µmol/L; 44%). Nearly half of the blood Phe samples (47%) with recorded timing were taken in a non-fasting state. Conclusions: Achieving lifelong metabolic stability on a Phe-restricted diet alone remains challenging. These national data highlight the need for broader therapeutic options to support individuals with PKU across the lifespan.
2026-06-26 | Molecular Genetic and Biochemical Characterization of Hyperphenylalaninemia Based on Expanded Neonatal Screening Data from 2023 to 2024 in the Russian Federation.
Since January 2023, the Russian Federation has implemented expanded neonatal screening for 36 hereditary disorders, which has changed the diagnostic algorithm for hyperphenylalaninemia/phenylketonuria (HPA/PKU) by introducing tandem mass spectrometry (MS/MS) on the second day of life, followed by confirmatory biochemical and molecular testing in newborns at risk. We analyzed 1247 newborns aged 5-15 days with elevated phenylalanine levels (≥120 µmol/L) and a phenylalanine to tyrosine ratio of at least 1 detected during the first stage of screening using MS/MS. At the reference center, newborns underwent repeat biochemical testing and stepwise molecular analysis of HPA-associated genes. Two pathogenic variants in HPA-associated genes were identified in 538 newborns, including 534 newborns with biallelic pathogenic variants in PAH and 4 with BH4-deficient forms (PTS, QDPR). The incidence of molecularly confirmed HPA was 1:4518 newborns (95% CI: 1:4152-1:4925). The PAH variant spectrum was dominated by p.Arg408Trp (c.1222C>T) (33.4%). Genotype-based analysis indicated that 73 newborns (13.7%) were likely responsive to cofactor therapy, whereas 222 (41.6%) were potentially responsive. These findings define the molecular epidemiology of HPA in Russia and support early genetic stratification for diagnosis and treatment.
proteins
2026-09-04 | Postprandial metabolic responses to a glycomacropeptide-based protein substitute in different meal contexts - Implications for phenylketonuria (PKU).
Casein glycomacropeptide supplemented with amino acids (CGMP-AA) is used in the treatment of phenylketonuria (PKU). However, its acute metabolic effects when consumed with other foods remain poorly understood. This study investigated the impact CGMP-AA consumed alone or with two low-protein meals providing carbohydrates with either fibre or fat. In this randomised crossover trial, fifteen healthy adults (27.0 ± 7.9 y; 7 females) consumed: A) CGMP-AA alone; B) CGMP-AA with low-protein bread and unpeeled apple; or C) CGMP-AA with low-protein bread and olive oil. Blood samples were collected over 120 min to assess glucose, insulin, C-peptide, amino acids, and appetite-related hormones (glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and peptide YY (PYY)). Co-ingestion with low-protein foods had limited effects on total essential and large neutral amino acid concentrations over 120 min, but increased Tyr incremental area under the curve (iAUC(0-120min)) and peak concentration (Cmax) compared with CGMP-AA alone. Meal B elicited the highest glucose, insulin, and C-peptide iAUC(0-120min) and Cmax, followed by meal C, whereas meal A induced minimal glycaemic and insulinaemic excursions. Meal C produced the greatest GIP response, whereas GLP-1 and PYY did not differ between test meals. In healthy adults, CGMP-AA elicited distinct acute postprandial responses depending on whether it was consumed alone or with low-protein foods. CGMP-AA consumed with food is not metabolically equivalent to CGMP-AA consumed alone, and carbohydrate-with-fibre and carbohydrate-with-fat meal contexts affect distinct aspects of the postprandial response. These findings should be confirmed in individuals with PKU before being translated into dietary recommendations.
2025-12-31 | Gut Microbiota and Metabolic Modulation by Slow-Release Protein Substitutes in Phenylketonuria: Findings from the PREMP Study.
Background/Objectives: Phenylketonuria (PKU) is an inherited metabolic disorder requiring early and lifelong dietary management through a low-phenylalanine (Phe) diet supplemented with Phe-free protein substitutes (PS). Recently developed slow-release PS formulations aim to mimic natural protein absorption, enhancing metabolic stability and tolerability. The PREMP study (effect of Protein RElease on the Microbiota composition and function in Phenylketonuric subjects) assessed the effects of a slow-release, Phe-free PS on gut microbiota composition and metabolic parameters in patients with PKU. Methods: Patients aged ≥6 years with PKU were enrolled from two Italian centers (Milan and Naples). Participants replaced ≥50% of their usual protein equivalent (P.Eq.) intake from Phe-free PS with a slow-release PS for 4 months. Clinical, biochemical, and nutritional assessments were performed at baseline and post-intervention. Gut microbiota composition was analyzed by 16S rRNA gene sequencing, and fecal fatty acids were quantified by gas chromatography-mass spectrometry. Results: Thirteen patients (median age 17 years) completed the intervention, replacing on average 78% of their usual P.Eq. intake with the slow-release formulation. Plasma phenylalanine and tyrosine levels remained stable, while fasting insulin (p = 0.0185) and HOMA-IR (p = 0.0099) significantly decreased, indicating improved insulin sensitivity. Anthropometric and dietary parameters showed no significant changes. Gut microbiota diversity remained stable, with modest increases in microbial richness and beneficial genera such as Bacteroides, Bifidobacterium, and Gemmiger, while Hafnia, Anaerostipes and Romboutsia decreased. Fecal butyrate and other fatty acids showed slight, non-significant increases. Conclusions: The slow-release PS was safe, well-tolerated, and improved insulin sensitivity without affecting amino acid or nutritional status. Microbial changes suggest potential benefits for gut health, warranting confirmation in larger, long-term studies.
2025-12-26 | Metabolic microenvironment-forming porous hydrogels for the protection of phenylalanine ammonia-lyase in the intestine.
Biological therapeutics drive biomedical innovation, yet their oral delivery is hindered by gastrointestinal instability. Phenylalanine ammonia-lyase (PAL), a promising enzyme for phenylketonuria management, exemplifies this challenge. Here, we present ultraporous template-based hydrogels (TGels) that protect and deliver PAL in the small intestine. PAL-TGels are obtained via UV crosslinking of poly(ethylene glycol) diacrylamide in the presence of sacrificial CaCO3 templates, followed by absorption-based loading of PAL and a protease inhibitor. The interconnected porous matrix retains PAL while allowing phenylalanine and trans-cinnamic acid diffusion for continuous catalysis. In vitro studies confirmed robust protein retention and preserved enzymatic activity in simulated intestinal fluid, without impairing global trypsin function, while large animal tests validated matrix properties and transit. This fabrication-efficient platform enables localized intestinal enzyme protection, offering a promising route toward safe and accessible oral PAL therapy for phenylketonuria.
2024-11-24 | The effects of casein glycomacropeptide on general health status in children with PKU: A randomized crossover trial.
In PKU, it is suggested that casein glycomacropeptide based protein substitute (GMP) may have physiological advantage when satiety, oxidative stress, renal function and inflammation are considered. Its prebiotic properties may also help gastrointestinal (GI) tolerance. In children with PKU, a randomized/crossover trial comparing phenylalanine-free amino acids (AA) vs GMP as the single source of protein substitute for 12-weeks in each arm was conducted. There was a 4-week wash out period with AA in-between. At baseline and end of each intervention, blood and fecal samples were taken to monitor gut health, oxidative stress, renal function, inflammatory markers and plasma amino acids. Satiety and Pediatric Quality of Life (PedsQL) GI symptoms questionnaires were completed. Usual weekly blood spots for phenylalanine and tyrosine were done. Twelve patients (8 males; aged 4-9y) with PKU participated. GMP improved the following GI symptoms: stomach pain (p = 0.003), heartburn and reflux (p = 0.041) wind and bloating (p = 0.018). With GMP, there was also a trend for less constipation (p = 0.068), discomfort with eating (p = 0.065) and nausea and vomiting (p = 0.087). There were no changes on stool gut health markers (IgA, short chain fatty acids and fecal calprotectin). There were no statistically significant differences for renal, oxidative stress, inflammatory and gut health markers or measures of satiety except for adiponectin (p = 0.028) and total antioxidant capacity (p = 0.049), although the latter was possibly without clinical significance. Mean dried blood spot phenylalanine (Phe) was 114 μmol/L higher with GMP vs AA (p < 0.001). There was no difference in tyrosine levels. In conclusion, GI symptoms statistically significantly improved with GMP versus AA. The Phe content of GMP may present challenges when it is used as the only protein substitute in children with classical PKU with low Phe tolerance.
2024-10-16 | Protein requirements in adults with phenylketonuria and bioavailability of glycomacropeptide compared to an l-amino acid-based product.
Phenylketonuria (PKU) is caused by phenylalanine hydroxylase deficiency. Treatment is primarily a low-Phe diet combined with l-amino acid-based products (l-AA). Protein requirements in adults with PKU have not been directly determined. A formula with glycomacropeptide (GMP) and low phenylalanine is available, yet untested for optimal protein synthesis. To determine the protein requirements in adults with PKU and the bioavailability of GMP-AA in the same patients using the indicator amino acid oxidation (IAAO) technique. Each participant was allocated to 7 separate l-AA intakes (range: 0.1-1.8 g/kg/day) in Experiment 1. In Experiment 2, the same patients participated in 4 GMP-AA intakes (range: 0.1-0.9 g/kg/day). The IAAO method with l-[1-13C]-lysine as the indicator amino acid and its oxidation to 13CO2 was used as the primary indicator of protein synthesis. Protein requirements were identified with a breakpoint, and bioavailability was determined by comparing 13CO2 slope from GMP-AA versus l-AA. Six adults with PKU (4 M: 2F) completed a total of 54 study days over the 2 experiments. The estimated average requirement (EAR) for protein was determined to be 1.11 g/kg/day (R2 = 0.20). The bioavailability of protein from GMP-AA was determined to be 99.98%, which was high and near to 100% comparable to l-AA; although, the results apply only to the tested GMP-AA blend. To our knowledge, this is the first study to directly define a quantitative protein requirement and indicates that current PKU protein recommendations for adults with PKU may be underestimated. The bioavailability of protein in the GMP-AA blend was high and optimal for protein synthesis in adults with PKU.
gene therapies
2025-09-13 | Advancing Gene Therapy for Phenylketonuria: From Precision Editing to Clinical Translation.
Phenylketonuria (PKU) is an inherited disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene that result in the amino acid phenylalanine (Phe) building up in the blood. Current therapies suggest low-Phe dietary management and (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) therapy, which are limited in efficacy and require lifelong treatment. Recent advances in gene therapy, including gene editing and viral-mediated gene delivery, produce therapeutic effects. Advancements in gene editing technologies, notably adenine base editors (ABEs) and CRISPR-based systems, in conjunction with enhanced delivery methods such as lipid nanoparticles (LNPs) and recombinant viruses, have demonstrated substantial promise in preclinical studies. This review details the pathophysiology of PKU treatment, and progress in preclinical and clinical gene therapy strategies. Emphasis is on adenine base editing using LNPs, recombinant adeno-associated virus (rAAV)-mediated gene transfer, and the translational challenges associated with these technologies. We also discuss future directions for therapeutic reach and ensuring long-term safety and efficacy.
2024-10-09 | Genetically humanized phenylketonuria mouse model as a testing tool for human genome editing in fertilized eggs.
Targeted genome editing has made significant advancements; however, safety and ethical issues have not been fully elucidated, resulting in strict control of the technique. We tested genome editing tools on gametes from a genetically humanized mouse model using a phenylketonuria (PKU) mouse model to gain insights into genome editing in human embryos. The human PKU mouse model PahhR111X mice was generated. The junctional region between exon 3 and intron 3 of Pah was replaced with a 120 bp corresponding human PAH sequence, including the pathogenic common variant c.331C > T in PahhR111X mice. PahhR111X mice successfully recapitulated the PKU phenotype and showed cognitive dysfunction and depressive-like behavior, which are observed in human patients with PKU. Genome editing was applied to fertilized eggs of PahhR111X mice utilizing sgRNA that targets the human sequence. Mice with the corrected allele exhibited normal serum phenylalanine levels. Through genome editing, we validated the utility of sgRNA. The genetically humanized mouse model suggested that germ-line genome editing of the pathogenic variant may be feasible for monogenic disorders by revealing the recovery of the phenotype; however, there are remaining issues with the tool, including its efficiency and accuracy. This genome editing protocol using a genetically humanized mouse model will provide insights for improving current issues and contribute to the establishment of heritable human genome editing protocols.
2024-01-16 | State-of-the-art 2023 on gene therapy for phenylketonuria.
Phenylketonuria (PKU) or hyperphenylalaninemia is considered a paradigm for an inherited (metabolic) liver defect and is, based on murine models that replicate all human pathology, an exemplar model for experimental studies on liver gene therapy. Variants in the PAH gene that lead to hyperphenylalaninemia are never fatal (although devastating if untreated), newborn screening has been available for two generations, and dietary treatment has been considered for a long time as therapeutic and satisfactory. However, significant shortcomings of contemporary dietary treatment of PKU remain. A long list of various gene therapeutic experimental approaches using the classical model for human PKU, the homozygous enu2/2 mouse, witnesses the value of this model to develop treatment for a genetic liver defect. The list of experiments for proof of principle includes recombinant viral (AdV, AAV, and LV) and non-viral (naked DNA or LNP-mRNA) vector delivery methods, combined with gene addition, genome, gene or base editing, and gene insertion or replacement. In addition, a list of current and planned clinical trials for PKU gene therapy is included. This review summarizes, compares, and evaluates the various approaches for the sake of scientific understanding and efficacy testing that may eventually pave the way for safe and efficient human application.
2023-11-03 | [Development of analytics in newborn screening-from the Guthrie card to genetics].
For more than five decades, all newborns in Germany have been offered a screening examination for the early detection of congenital treatable diseases. Since its inception, about 35 million children have been screened in this way.Originally, screening exams only included early detection of phenylketonuria, which, without timely treatment, would lead to mental retardation that could no longer be corrected. The bacteriological Guthrie test allowed the detection of elevated concentrations of phenylalanine. The methods used today are the result of decades of development. They have been expanded to include tests to determine enzyme activities, immunoassays for the early detection of important hormonal disorders such as congenital hypothyroidism, and high-pressure liquid chromatography for the diagnosis of pathologic hemoglobins. The very sophisticated tandem mass spectrometry enables the simultaneous detection of amino acid and fatty acid compounds. Steroids can also be identified. The specificity can be further increased by combining tandem mass spectrometry with chromatographic pre-separation. In recent years, chemical-analytical analyses have been supplemented by genetic diagnostic methods such as quantitative or qualitative polymerase chain reaction (PCR).The current state of laboratory technology is by no means final. Both classical analytics and especially genetic methods are facing further rapid development. Although the expansion of screening is also a consequence of technical development, the inclusion of further congenital diseases is fundamentally dependent on the given therapy. But it is precisely here that many innovations are currently being investigated. Gene therapy is at the forefront of interest. Seit mehr als 5 Jahrzehnten wird allen Neugeborenen in Deutschland eine Vorsorgeuntersuchung zur Früherkennung angeborener behandelbarer Krankheiten angeboten. Seit Beginn sind so etwa 35 Mio. Kinder untersucht worden.Anfangs ging es nur um die Früherkennung der Phenylketonurie, die ohne frühzeitige Behandlung zu nicht mehr korrigierbarer geistiger Behinderung führt. Der bakteriologische Guthrie-Test erlaubte den Nachweis erhöhter Konzentrationen von Phenylalanin. Die heute eingesetzten Methoden sind das Ergebnis einer über Jahrzehnte verlaufenden Entwicklung. Hinzugekommen sind Tests zur Bestimmung von Enzymaktivitäten, Immunoassays zur Früherkennung wichtiger hormoneller Störungen wie der angeborenen Schilddrüsenunterfunktion sowie Hochdruck-Flüssigkeits-Chromatografie zur Identifizierung pathologischer Hämoglobine. Die sehr anspruchsvolle Tandem-Massenspektrometrie ermöglicht die gleichzeitige Erfassung von Aminosäuren und Derivaten organischer Säuren und Fettsäuren. Auch Steroide können damit identifiziert werden. Die Spezifität lässt sich durch Kombination mit chromatografischer Vortrennung noch erhöhen. In den letzten Jahren wurden die chemisch-analytischen Untersuchungen ergänzt durch gendiagnostische Verfahren, wie beispielsweise quantitative oder qualitative Polymerasekettenreaktion (PCR).Der Stand der Labortechnik ist keineswegs endgültig. Sowohl die klassische Analytik als auch besonders die genetischen Verfahren stehen vor einer weiteren rasanten Entwicklung. Während die Ausweitung des Screenings auch eine Folge der technischen Entwicklung ist, hängt die Einbeziehung weiterer angeborener Erkrankungen grundsätzlich von einer jeweiligen Therapie ab. Aber gerade hier werden gegenwärtig viele Neuerungen erprobt. Im Vordergrund des Interesses steht dabei die Gentherapie.
2022-04-26 | In vivo prime editing of a metabolic liver disease in mice.
Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain (PE2ΔRnH) and an intein-split construct (PE2 p.1153) for adeno-associated virus-mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2ΔRnH via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pahenu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 1014 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.
cell therapies
2025-11-05 | Synthetic closed-loop gene circuit for phenylalanine regulation.
Genetic programming of cells offers significant potential for developing next-generation cell-based therapies that detect and respond to signals within the patient's body to treat chronic diseases. Closed-loop systems designed to self-regulate in response to abnormal biomarker levels are particularly attractive for biomedical applications. In this study, we engineered PRO (phenylalanine regulation orchestration), a cell-autonomous genetic system capable of sensing and degrading elevated levels of phenylalanine, an essential amino acid that accumulates to pathological levels in patients with phenylketonuria (PKU). Central to this system is a transcriptional switch relying on the regulatory domain of human phenylalanine hydroxylase, which dimerizes in the presence of phenylalanine to reconstitute a split transcription factor, thereby inducing gene expression from a synthetic promoter. This sensing module was optimized through random mutagenesis to ensure responsiveness to high concentrations of phenylalanine, enabling robust and dose-dependent activation of protein expression. Engineered human PRO cells containing the sensor driving the expression of phenylalanine-degrading enzymes effectively detected and degraded excess phenylalanine. Microencapsulated PRO cells efficiently reduced phenylalanine levels to the normal physiological range when cultured in human whole blood. Finally, as proof of concept, we showed that alginate-encapsulated PRO cells intraperitoneally implanted in a PKU mouse model significantly lowered blood phenylalanine levels compared to controls implanted with non-engineered cells. Our results suggest that synthetic self-regulating systems are promising candidates for the treatment of metabolic diseases characterized by accumulation of toxic metabolites.
2024-10-25 | Therapeutic liver cell transplantation to treat murine PKU.
For gene therapy of the liver, in vivo applications based on adeno-associated virus are the most advanced vectors despite limitations, including low efficacy and episomal loss, potential integration and safety issues, and high production costs. Alternative vectors and/or delivery routes are of high interest. The regenerative ability of the liver bears the potential for ex vivo therapy using liver cell transplantation for disease correction if provided with a selective advantage to expand and replace the existing cell mass. Here we present such treatment of a mouse model of human phenylketonuria (PKU). Primary hepatocytes from wild-type mice were gene modified in vitro (with a lentiviral vector) that carries a gene editing system (CRISPR) to inhibit Cypor. Cypor inactivation confers paracetamol (or acetaminophen) resistance to hepatocytes and thus a growth advantage to eliminate the pre-existing liver cells upon grafting (via the spleen) and exposure to repeated treatment with paracetamol. Grafting Cypor-inactivated wild-type hepatocytes into inbred young adult enu2 (PKU) mice, followed by selective expansion by paracetamol dosing, resulted in replacing up to 5% of cell mass, normalization of blood phenylalanine, and permanent correction of PKU. Hepatocyte transplantation offers thus an armamentarium of novel therapy options for genetic liver defects.
2024-04-18 | Complete correction of murine phenylketonuria by selection-enhanced hepatocyte transplantation.
Hepatocyte transplantation for genetic liver diseases has several potential advantages over gene therapy. However, the low efficiency of cell engraftment has limited its clinical implementation. This problem could be overcome by selectively expanding transplanted donor cells until they replace enough of the liver mass to achieve therapeutic benefit. We previously described a gene therapy method to selectively expand hepatocytes deficient in cytochrome p450 reductase (Cypor) using acetaminophen (APAP). Because Cypor is required for the transformation of APAP to a hepatotoxic metabolite, Cypor-deficient cells are protected from toxicity and are able to expand following APAP-induced liver injury. Here, we apply this selection system to correct a mouse model of phenylketonuria by cell transplantation. Hepatocytes from a wild-type donor animal were edited in vitro to create Cypor deficiency and then transplanted into phenylketonuric animals. Following selection with APAP, blood phenylalanine concentrations were fully normalized and remained stable following APAP withdrawal. Cypor-deficient hepatocytes expanded from < 1% to ~14% in corrected animals, and they showed no abnormalities in blood chemistries, liver histology, or drug metabolism. We conclude that APAP-mediated selection of transplanted hepatocytes is a potential therapeutic for phenylketonuria with long-term efficacy and a favorable safety profile.
2023-11-14 | The treatment of biochemical genetic diseases: From substrate reduction to nucleic acid therapies.
Newborn screening (NBS) began a revolution in the management of biochemical genetic diseases, greatly increasing the number of patients for whom dietary therapy would be beneficial in preventing complications in phenylketonuria as well as in a few similar disorders. The advent of next generation sequencing and expansion of NBS have markedly increased the number of biochemical genetic diseases as well as the number of patients identified each year. With the avalanche of new and proposed therapies, a second wave of options for the treatment of biochemical genetic disorders has emerged. These therapies range from simple substrate reduction to enzyme replacement, and now ex vivo gene therapy with autologous cell transplantation. In some instances, it may be optimal to introduce nucleic acid therapy during the prenatal period to avoid fetopathy. However, as with any new therapy, complications may occur. It is important for physicians and other caregivers, along with ethicists, to determine what new therapies might be beneficial to the patient, and which therapies have to be avoided for those individuals who have less severe problems and for which standard treatments are available. The purpose of this review is to discuss the "Standard" treatment plans that have been in place for many years and to identify the newest and upcoming therapies, to assist the physician and other healthcare workers in making the right decisions regarding the initiation of both the "Standard" and new therapies. We have utilized several diseases to illustrate the applications of these different modalities and discussed for which disorders they may be suitable. The future is bright, but optimal care of the patient, including and especially the newborn infant, requires a deep knowledge of the disease process and careful consideration of the necessary treatment plan, not just based on the different genetic defects but also with regards to different variants within a gene itself.
2022-02-28 | A quantitative model for metabolic intervention using gut microbes.
As medicine shifts toward precision-based and personalized therapeutics, utilizing more complex biomolecules to treat increasingly difficult and rare conditions, microorganisms provide an avenue for realizing the production and processing necessary for novel drug pipelines. More so, probiotic microbes can be co-opted to deliver therapeutics by oral administration as living drugs, able to survive and safely transit the digestive tract. As living therapeutics are in their nascency, traditional pharmacokinetic-pharmacodynamic (PK-PD) models for evaluating drug candidates are not appropriate for this novel platform. Using a living therapeutic in late-stage clinical development for phenylketonuria (PKU) as a case study, we adapt traditional oral drug delivery models to properly evaluate and inform the engineering of living therapeutics. We develop the adapted for living therapeutics compartmental absorption and transit (ALT-CAT) model to provide metrics for drug efficacy across nine age groups of PKU patients and evaluate model parameters that are influenced by patient physiology, microbe selection and therapeutic production, and dosing formulations. In particular, the ALT-CAT model describes the mathematical framework to model the behavior of orally delivered engineered bacteria that act as living therapeutics by adapting similar methods that have been developed and widely-used for small molecular drug delivery and absorption.
other
2024-05-07 | Characterizing the mechanism of action for mRNA therapeutics for the treatment of propionic acidemia, methylmalonic acidemia, and phenylketonuria.
Messenger RNA (mRNA) therapeutics delivered via lipid nanoparticles hold the potential to treat metabolic diseases caused by protein deficiency, including propionic acidemia (PA), methylmalonic acidemia (MMA), and phenylketonuria (PKU). Herein we report results from multiple independent preclinical studies of mRNA-3927 (an investigational treatment for PA), mRNA-3705 (an investigational treatment for MMA), and mRNA-3210 (an investigational treatment for PKU) in murine models of each disease. All 3 mRNA therapeutics exhibited pharmacokinetic/pharmacodynamic (PK/PD) responses in their respective murine model by driving mRNA, protein, and/or protein activity responses, as well as by decreasing levels of the relevant biomarker(s) when compared to control-treated animals. These preclinical data were then used to develop translational PK/PD models, which were scaled allometrically to humans to predict starting doses for first-in-human clinical studies for each disease. The predicted first-in-human doses for mRNA-3927, mRNA-3705, and mRNA-3210 were determined to be 0.3, 0.1, and 0.4 mg/kg, respectively.
2023-04-18 | Myelin basic protein recovery during PKU mice lifespan and the potential role of microRNAs on its regulation.
Untreated phenylketonuria (PKU) patients and PKU animal models show hypomyelination in the central nervous system and white matter damages, which are accompanied by myelin basic protein (MBP) impairment. Despite many assumptions, the primary explanation of the mentioned cerebral outcomes remains elusive. In this study, MBP protein and mRNA expression on brains of wild type (WT) and phenylketonuric (ENU2) mice were analyzed throughout mice lifespan (14-60-180-270-360-540 post-natal days, PND). The results confirmed the low MBP expression at first PND times, while revealed an unprecedented progressive MBP protein expression recovery in aged ENU2 mice. Unexpectedly, unaltered MBP mRNA expression between WT and ENU2 was always observed. Additionally, for the same time intervals, a significant decrease of the phenylalanine concentration in the peripheral blood and brain of ENU2 mice was detected, to date, for the first time. In this scenario, a translational hindrance of MBP during initial and late cerebral development in ENU2 mice was hypothesized, leading to the execution of a microRNA microarray analysis on 60 PND brains, which was followed by a proteomic assay on 60 and 360 PND brains in order to validate in silico miRNA-target predictions. Taken together, miR-218-1-3p, miR-1231-3p and miR-217-5p were considered as the most impactful microRNAs, since a downregulation of their potential targets (MAG, CNTNAP2 and ANLN, respectively) can indirectly lead to a low MBP protein expression. These miRNAs, in addition, follow an opposite expression trend compared to MBP during adulthood, and their target proteins revealed a complete normalization in aged ENU2 mice. In conclusion, these results provide a new perspective on the PKU pathophysiology understanding and on a possible treatment, emphasizing the potential modulating role of differentially expressed microRNAs in MBP expression on PKU brains during PKU mouse lifespan.
2022-09-08 | Present and future of lipid nanoparticle-mRNA technology in phenylketonuria disease treatment.
Phenylketonuria (PKU) is a metabolic rare disease characterized by a failure of the body to clear out the high levels of Phenylalanine (Phe), leading to devastating neurological defects and growth retardation. PKU was discovered in 1934 by AsbjrØrn FØlling, and even though there have been continuous efforts from the scientific community to find therapeutic approaches to modulate the high levels of phenylalanine found in the body of the PKU patients, an efficient therapy still needs to be developed. Current standard of care includes low phenylalanine diets, but the strict restrictions for patients and families makes it very difficult to adequately being implemented. FDA has approved two drugs to help reduce Phe levels in PKU patients: an enzyme substitution therapy, Palynziq® (PEGylated recombinant phenylalanine ammonia lyase), and Kuvan®, a supplemental tetrahydrobiopterin (BH4) cofactor that enhances residual enzyme activity. Both treatments are restricted to certain PKU patients' population, and, therefore, there are still high unmet needs for most of the patients. The present review will focus on current advancements in lipid nanoparticles (LNP)-mRNA technologies and their potential in treating the root cause of PKU, a therapeutic approach that will be analyzed in the context of other promising therapeutic approaches that are been developed for PKU.
2022-07-21 | Genetic etiology and clinical challenges of phenylketonuria.
This review discusses the epidemiology, pathophysiology, genetic etiology, and management of phenylketonuria (PKU). PKU, an autosomal recessive disease, is an inborn error of phenylalanine (Phe) metabolism caused by pathogenic variants in the phenylalanine hydroxylase (PAH) gene. The prevalence of PKU varies widely among ethnicities and geographic regions, affecting approximately 1 in 24,000 individuals worldwide. Deficiency in the PAH enzyme or, in rare cases, the cofactor tetrahydrobiopterin results in high blood Phe concentrations, causing brain dysfunction. Untreated PKU, also known as PAH deficiency, results in severe and irreversible intellectual disability, epilepsy, behavioral disorders, and clinical features such as acquired microcephaly, seizures, psychological signs, and generalized hypopigmentation of skin (including hair and eyes). Severe phenotypes are classic PKU, and less severe forms of PAH deficiency are moderate PKU, mild PKU, mild hyperphenylalaninaemia (HPA), or benign HPA. Early diagnosis and intervention must start shortly after birth to prevent major cognitive and neurological effects. Dietary treatment, including natural protein restriction and Phe-free supplements, must be used to maintain blood Phe concentrations of 120-360 μmol/L throughout the life span. Additional treatments include the casein glycomacropeptide (GMP), which contains very limited aromatic amino acids and may improve immunological function, and large neutral amino acid (LNAA) supplementation to prevent plasma Phe transport into the brain. The synthetic BH4 analog, sapropterin hydrochloride (i.e., Kuvan®, BioMarin), is another potential treatment that activates residual PAH, thus decreasing Phe concentrations in the blood of PKU patients. Moreover, daily subcutaneous injection of pegylated Phe ammonia-lyase (i.e., pegvaliase; PALYNZIQ®, BioMarin) has promised gene therapy in recent clinical trials, and mRNA approaches are also being studied.
2022-07-14 | Antisense Oligonucleotide Rescue of Deep-Intronic Variants Activating Pseudoexons in the 6-Pyruvoyl-Tetrahydropterin Synthase Gene
We report two new 6-pyruvoyl-tetrahydropterin synthase splicing variants identified through genomic sequencing and transcript analysis in a patient with tetrahydrobiopterin deficiency, presenting with hyperphenylalaninemia and monoamine neurotransmitter deficiency. Variant c.243 + 3A>G causes exon 4 skipping. The deep-intronic c.164–672C>T variant creates a potential 5′ splice site that leads to the inclusion of four overlapping pseudoexons, corresponding to exonizations of an antisense short interspersed nuclear element AluSq repeat sequence. Two of the identified pseudoexons have been reported previously, activated by different deep-intronic variants, and were also detected at residual levels in control cells. Interestingly, the predominant pseudoexon is nearly identical to a disease causing activated pseudoexon in the F8 gene, with the same 3′ and 5′ splice sites. Splice switching antisense oligonucleotides (SSOs) were designed to hybridize with splice sites and/or predicted binding sites for regulatory splice factors. Different SSOs corrected the aberrant pseudoexon inclusion, both in minigenes and in fibroblasts from patients carrying the new variant c.164–672C>T or the previously described c.164–716A>T. With SSO treatment PTPS protein was recovered, illustrating the therapeutic potential of the approach, for patients with different pseudoexon activating variants in the region. In addition, the natural presence of pseudoexons in the wild type context suggests the possibility of applying the antisense strategy in patients with hypomorphic PTS variants with the purpose of upregulating their expression to increase overall protein and activity.
small molecules
2026-09-11 | A phase 1/2 dose-escalation study of sepiapterin in patients with 6-pyruvoyl-tetrahydropterin synthase deficiency with hyperphenylalaninemia.
Oral sepiapterin (PTC923), a precursor of tetrahydrobiopterin (BH4), was evaluated in patients with 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency in a phase 1/2, multicenter, open-label, randomized, intra-individual dose-escalation study. Patients stopped sapropterin (used to control hyperphenylalaninemia) for 2-4-days before being randomized 1:1 into 2 cohorts receiving sepiapterin at 2 doses (Cohort 1: 2.5 and 10 mg/kg/day; Cohort 2: 5 and 20 mg/kg/day) each for 7 days, separated by a 2-4-day washout period. Eight participants (5 male, 3 female, aged 2.2-20.0 years) completed the study (4 in each cohort). Overall, 7/8 participants (87.5%) experienced a total of 35 treatment-emergent adverse events; none were severe, serious, or led to discontinuation. Mean phenylalanine levels increased (up to 800-1200 μmol/L) after discontinuation of sepiapterin or sapropterin, and normalized (<130 μmol/L) with sepiapterin at all tested doses by Day 2. Blood BH4 concentrations increased with increasing single doses of sepiapterin, reaching a peak at approximately 3-4 hours, which was 2-3 hours after the sepiapterin peak. These results show that in patients with PTPS deficiency, sepiapterin is rapidly converted to BH4 and normalizes blood phenylalanine concentrations, with no dose-limiting toxicity or dose-related adverse events.
2026-08-19 | Targeting Mutant Phenylalanine Hydroxylase With Pyrimidine-Triazole Conjugates: A Primary Framework for Candidate Chaperone-Based Strategies in Phenylketonuria.
Phenylketonuria (PKU) is a rare metabolic disorder caused by pathogenic mutations in the phenylalanine hydroxylase (PAH) gene, which impair the conversion of phenylalanine to tyrosine, leading to subsequent neurotoxicity. Dietary management, sapropterin dihydrochloride, and pegvaliase are the current therapies; however, their limited efficacy and adverse effects highlight the pressing need for pharmacological treatments that restore PAH activity. Therefore, in this work, we designed and synthesized pyrimidine-triazole derivatives (Pyr-TZ) as candidate pharmacological chaperones targeting the catalytic domain of PAH. Molecular docking disclosed that Pyr-TZ-2O and Pyr-TZ-2S exhibited higher binding energies (-8.5 to -10.3 kcal/mol) for the wild-type (WT) and mutant (R252Q) PAH compared to sapropterin (-7.0 kcal/mol). Molecular Mechanics with Generalized Born and Surface Area solvation (MM-GBSA) was used here as comparative estimates suggested enhanced binding stability of Pyr-TZ compounds, primarily driven by van der Waals and lipophilic interactions. Drug-likeness and ADMET profiling indicated favorable pharmacokinetic properties. Biological validation in R252Q mutant cells demonstrated significant upregulation of PAH and key tetrahydrobiopterin (BH4) pathway genes, including quinonoid dihydropteridine reductase (QDPR), sepiapterin reductase (SPR), and 6-pyruvoyl-tetrahydropterin synthase (PTS), following Pyr-TZ-2O treatment. Consistent with these findings, sandwich ELISA revealed a dose-dependent increase in PAH protein abundance post-Pyr-TZ-2O treatment. Conclusively, Pyr-TZ-2O can be considered a potential pharmacological chaperone capable of stabilizing mutant PAH and enhancing cofactor regeneration, offering a rational therapeutic approach for PKU.
2026-07-24 | DNAJC12 Stabilizes Phenylalanine Hydroxylase and Facilitates Its Substrate-Dependent Activation.
Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH4)-dependent enzyme that converts L-phenylalanine (L-Phe) to L-tyrosine. PAH dysfunction leads to the accumulation of L-Phe in the blood (hyperphenylalaninemia; HPA), which may reach neurotoxic levels, resulting in phenylketonuria (PKU). PKU is associated with pathogenic variants in PAH, mostly causing misfolding and instability, leading to decreased levels of PAH protein and activity. Recently, variants in the J-domain protein DNAJC12 have also been associated with HPA in patients, demonstrating the importance of protein homeostasis regulation for proper PAH function. DNAJC12 and PAH have previously been reported to interact, but the molecular and structural mechanisms behind complex formation have remained unclear. In this work, we show that DNAJC12 binds to PAH but presents higher affinity for its L-Phe activated form, which resembles the conformation of unliganded tyrosine hydroxylase, a structurally and functionally related enzyme that also binds to DNAJC12. At saturation, four DNAJC12 monomers bind and stabilize the PAH tetramer, protecting it from aggregation and lowering the L-Phe concentration necessary for substrate-induced activation, without affecting the interaction of the enzyme with its cofactor BH4. Importantly, DNAJC12 also stabilizes and delays the aggregation of the PKU-associated variant PAH-p.R261Q. This study provides the first detailed characterization of the molecular determinants driving PAH:DNAJC12 complex formation and reveals how this interaction modulates enzyme stability and activity, and stimulates Hsc70 ATPase activity. These findings provide mechanistic insight into the pathogenic basis of DNAJC12 deficiency and identify the PAH:DNAJC12 complex as a promising therapeutic target for HPA.
2026-07-15 | Blood Phenylalanine Control in Paediatric and Adult Centres in the UK: Data from 2012-2018.
Background: Metabolic control in phenylketonuria (PKU) is known to deteriorate with age, but national-level data describing blood phenylalanine (Phe) control across the United Kingdom (UK) are limited. Objective: To characterise blood Phe control in individuals with PKU attending UK metabolic centres. Methods: Sixteen UK centres (nine paediatric, six adult, one mixed) retrospectively extracted blood Phe results collected between 2012 and 2018. Demographic, phenotypic and monitoring-related variables were analysed. Written consent for data collection was obtained from all patients or their caregivers. Results: Data were available for 871 individuals (55% female), of whom 744 (85%) were classified as follows: classical PKU, 75%, mild PKU, 22% and hyperphenylalaninaemia, 3%. Mean blood Phe concentrations were significantly higher in adults than children (491 ± 308 vs. 303 ± 199 µmol/L; p < 0.001), and the proportion of samples within target range declined steadily with age, from 78% in children < 2 years to 36% in adults ≥ 41 years. Individuals with classical PKU had higher mean Phe concentrations and lower target attainment than those with HPA (386 vs. 300 µmol/L; 61% vs. 78%; p < 0.001), while mild PKU and HPA showed comparable control. Females generally demonstrated better metabolic control than males. More frequent dried blood spot sampling for blood Phe was strongly associated with improved metabolic control: weekly (254 ± 175 µmol/L; 82% within target), fortnightly (319 ± 207 µmol/L; 70%), monthly (397 ± 231 µmol/L; 61%), and less than monthly (624 ± 349 µmol/L; 44%). Nearly half of the blood Phe samples (47%) with recorded timing were taken in a non-fasting state. Conclusions: Achieving lifelong metabolic stability on a Phe-restricted diet alone remains challenging. These national data highlight the need for broader therapeutic options to support individuals with PKU across the lifespan.
2026-06-26 | Molecular Genetic and Biochemical Characterization of Hyperphenylalaninemia Based on Expanded Neonatal Screening Data from 2023 to 2024 in the Russian Federation.
Since January 2023, the Russian Federation has implemented expanded neonatal screening for 36 hereditary disorders, which has changed the diagnostic algorithm for hyperphenylalaninemia/phenylketonuria (HPA/PKU) by introducing tandem mass spectrometry (MS/MS) on the second day of life, followed by confirmatory biochemical and molecular testing in newborns at risk. We analyzed 1247 newborns aged 5-15 days with elevated phenylalanine levels (≥120 µmol/L) and a phenylalanine to tyrosine ratio of at least 1 detected during the first stage of screening using MS/MS. At the reference center, newborns underwent repeat biochemical testing and stepwise molecular analysis of HPA-associated genes. Two pathogenic variants in HPA-associated genes were identified in 538 newborns, including 534 newborns with biallelic pathogenic variants in PAH and 4 with BH4-deficient forms (PTS, QDPR). The incidence of molecularly confirmed HPA was 1:4518 newborns (95% CI: 1:4152-1:4925). The PAH variant spectrum was dominated by p.Arg408Trp (c.1222C>T) (33.4%). Genotype-based analysis indicated that 73 newborns (13.7%) were likely responsive to cofactor therapy, whereas 222 (41.6%) were potentially responsive. These findings define the molecular epidemiology of HPA in Russia and support early genetic stratification for diagnosis and treatment.
proteins
2026-09-04 | Postprandial metabolic responses to a glycomacropeptide-based protein substitute in different meal contexts - Implications for phenylketonuria (PKU).
Casein glycomacropeptide supplemented with amino acids (CGMP-AA) is used in the treatment of phenylketonuria (PKU). However, its acute metabolic effects when consumed with other foods remain poorly understood. This study investigated the impact CGMP-AA consumed alone or with two low-protein meals providing carbohydrates with either fibre or fat. In this randomised crossover trial, fifteen healthy adults (27.0 ± 7.9 y; 7 females) consumed: A) CGMP-AA alone; B) CGMP-AA with low-protein bread and unpeeled apple; or C) CGMP-AA with low-protein bread and olive oil. Blood samples were collected over 120 min to assess glucose, insulin, C-peptide, amino acids, and appetite-related hormones (glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and peptide YY (PYY)). Co-ingestion with low-protein foods had limited effects on total essential and large neutral amino acid concentrations over 120 min, but increased Tyr incremental area under the curve (iAUC(0-120min)) and peak concentration (Cmax) compared with CGMP-AA alone. Meal B elicited the highest glucose, insulin, and C-peptide iAUC(0-120min) and Cmax, followed by meal C, whereas meal A induced minimal glycaemic and insulinaemic excursions. Meal C produced the greatest GIP response, whereas GLP-1 and PYY did not differ between test meals. In healthy adults, CGMP-AA elicited distinct acute postprandial responses depending on whether it was consumed alone or with low-protein foods. CGMP-AA consumed with food is not metabolically equivalent to CGMP-AA consumed alone, and carbohydrate-with-fibre and carbohydrate-with-fat meal contexts affect distinct aspects of the postprandial response. These findings should be confirmed in individuals with PKU before being translated into dietary recommendations.
2025-12-31 | Gut Microbiota and Metabolic Modulation by Slow-Release Protein Substitutes in Phenylketonuria: Findings from the PREMP Study.
Background/Objectives: Phenylketonuria (PKU) is an inherited metabolic disorder requiring early and lifelong dietary management through a low-phenylalanine (Phe) diet supplemented with Phe-free protein substitutes (PS). Recently developed slow-release PS formulations aim to mimic natural protein absorption, enhancing metabolic stability and tolerability. The PREMP study (effect of Protein RElease on the Microbiota composition and function in Phenylketonuric subjects) assessed the effects of a slow-release, Phe-free PS on gut microbiota composition and metabolic parameters in patients with PKU. Methods: Patients aged ≥6 years with PKU were enrolled from two Italian centers (Milan and Naples). Participants replaced ≥50% of their usual protein equivalent (P.Eq.) intake from Phe-free PS with a slow-release PS for 4 months. Clinical, biochemical, and nutritional assessments were performed at baseline and post-intervention. Gut microbiota composition was analyzed by 16S rRNA gene sequencing, and fecal fatty acids were quantified by gas chromatography-mass spectrometry. Results: Thirteen patients (median age 17 years) completed the intervention, replacing on average 78% of their usual P.Eq. intake with the slow-release formulation. Plasma phenylalanine and tyrosine levels remained stable, while fasting insulin (p = 0.0185) and HOMA-IR (p = 0.0099) significantly decreased, indicating improved insulin sensitivity. Anthropometric and dietary parameters showed no significant changes. Gut microbiota diversity remained stable, with modest increases in microbial richness and beneficial genera such as Bacteroides, Bifidobacterium, and Gemmiger, while Hafnia, Anaerostipes and Romboutsia decreased. Fecal butyrate and other fatty acids showed slight, non-significant increases. Conclusions: The slow-release PS was safe, well-tolerated, and improved insulin sensitivity without affecting amino acid or nutritional status. Microbial changes suggest potential benefits for gut health, warranting confirmation in larger, long-term studies.
2025-12-26 | Metabolic microenvironment-forming porous hydrogels for the protection of phenylalanine ammonia-lyase in the intestine.
Biological therapeutics drive biomedical innovation, yet their oral delivery is hindered by gastrointestinal instability. Phenylalanine ammonia-lyase (PAL), a promising enzyme for phenylketonuria management, exemplifies this challenge. Here, we present ultraporous template-based hydrogels (TGels) that protect and deliver PAL in the small intestine. PAL-TGels are obtained via UV crosslinking of poly(ethylene glycol) diacrylamide in the presence of sacrificial CaCO3 templates, followed by absorption-based loading of PAL and a protease inhibitor. The interconnected porous matrix retains PAL while allowing phenylalanine and trans-cinnamic acid diffusion for continuous catalysis. In vitro studies confirmed robust protein retention and preserved enzymatic activity in simulated intestinal fluid, without impairing global trypsin function, while large animal tests validated matrix properties and transit. This fabrication-efficient platform enables localized intestinal enzyme protection, offering a promising route toward safe and accessible oral PAL therapy for phenylketonuria.
2024-11-24 | The effects of casein glycomacropeptide on general health status in children with PKU: A randomized crossover trial.
In PKU, it is suggested that casein glycomacropeptide based protein substitute (GMP) may have physiological advantage when satiety, oxidative stress, renal function and inflammation are considered. Its prebiotic properties may also help gastrointestinal (GI) tolerance. In children with PKU, a randomized/crossover trial comparing phenylalanine-free amino acids (AA) vs GMP as the single source of protein substitute for 12-weeks in each arm was conducted. There was a 4-week wash out period with AA in-between. At baseline and end of each intervention, blood and fecal samples were taken to monitor gut health, oxidative stress, renal function, inflammatory markers and plasma amino acids. Satiety and Pediatric Quality of Life (PedsQL) GI symptoms questionnaires were completed. Usual weekly blood spots for phenylalanine and tyrosine were done. Twelve patients (8 males; aged 4-9y) with PKU participated. GMP improved the following GI symptoms: stomach pain (p = 0.003), heartburn and reflux (p = 0.041) wind and bloating (p = 0.018). With GMP, there was also a trend for less constipation (p = 0.068), discomfort with eating (p = 0.065) and nausea and vomiting (p = 0.087). There were no changes on stool gut health markers (IgA, short chain fatty acids and fecal calprotectin). There were no statistically significant differences for renal, oxidative stress, inflammatory and gut health markers or measures of satiety except for adiponectin (p = 0.028) and total antioxidant capacity (p = 0.049), although the latter was possibly without clinical significance. Mean dried blood spot phenylalanine (Phe) was 114 μmol/L higher with GMP vs AA (p < 0.001). There was no difference in tyrosine levels. In conclusion, GI symptoms statistically significantly improved with GMP versus AA. The Phe content of GMP may present challenges when it is used as the only protein substitute in children with classical PKU with low Phe tolerance.
2024-10-16 | Protein requirements in adults with phenylketonuria and bioavailability of glycomacropeptide compared to an l-amino acid-based product.
Phenylketonuria (PKU) is caused by phenylalanine hydroxylase deficiency. Treatment is primarily a low-Phe diet combined with l-amino acid-based products (l-AA). Protein requirements in adults with PKU have not been directly determined. A formula with glycomacropeptide (GMP) and low phenylalanine is available, yet untested for optimal protein synthesis. To determine the protein requirements in adults with PKU and the bioavailability of GMP-AA in the same patients using the indicator amino acid oxidation (IAAO) technique. Each participant was allocated to 7 separate l-AA intakes (range: 0.1-1.8 g/kg/day) in Experiment 1. In Experiment 2, the same patients participated in 4 GMP-AA intakes (range: 0.1-0.9 g/kg/day). The IAAO method with l-[1-13C]-lysine as the indicator amino acid and its oxidation to 13CO2 was used as the primary indicator of protein synthesis. Protein requirements were identified with a breakpoint, and bioavailability was determined by comparing 13CO2 slope from GMP-AA versus l-AA. Six adults with PKU (4 M: 2F) completed a total of 54 study days over the 2 experiments. The estimated average requirement (EAR) for protein was determined to be 1.11 g/kg/day (R2 = 0.20). The bioavailability of protein from GMP-AA was determined to be 99.98%, which was high and near to 100% comparable to l-AA; although, the results apply only to the tested GMP-AA blend. To our knowledge, this is the first study to directly define a quantitative protein requirement and indicates that current PKU protein recommendations for adults with PKU may be underestimated. The bioavailability of protein in the GMP-AA blend was high and optimal for protein synthesis in adults with PKU.
gene therapies
2025-09-13 | Advancing Gene Therapy for Phenylketonuria: From Precision Editing to Clinical Translation.
Phenylketonuria (PKU) is an inherited disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene that result in the amino acid phenylalanine (Phe) building up in the blood. Current therapies suggest low-Phe dietary management and (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) therapy, which are limited in efficacy and require lifelong treatment. Recent advances in gene therapy, including gene editing and viral-mediated gene delivery, produce therapeutic effects. Advancements in gene editing technologies, notably adenine base editors (ABEs) and CRISPR-based systems, in conjunction with enhanced delivery methods such as lipid nanoparticles (LNPs) and recombinant viruses, have demonstrated substantial promise in preclinical studies. This review details the pathophysiology of PKU treatment, and progress in preclinical and clinical gene therapy strategies. Emphasis is on adenine base editing using LNPs, recombinant adeno-associated virus (rAAV)-mediated gene transfer, and the translational challenges associated with these technologies. We also discuss future directions for therapeutic reach and ensuring long-term safety and efficacy.
2024-10-09 | Genetically humanized phenylketonuria mouse model as a testing tool for human genome editing in fertilized eggs.
Targeted genome editing has made significant advancements; however, safety and ethical issues have not been fully elucidated, resulting in strict control of the technique. We tested genome editing tools on gametes from a genetically humanized mouse model using a phenylketonuria (PKU) mouse model to gain insights into genome editing in human embryos. The human PKU mouse model PahhR111X mice was generated. The junctional region between exon 3 and intron 3 of Pah was replaced with a 120 bp corresponding human PAH sequence, including the pathogenic common variant c.331C > T in PahhR111X mice. PahhR111X mice successfully recapitulated the PKU phenotype and showed cognitive dysfunction and depressive-like behavior, which are observed in human patients with PKU. Genome editing was applied to fertilized eggs of PahhR111X mice utilizing sgRNA that targets the human sequence. Mice with the corrected allele exhibited normal serum phenylalanine levels. Through genome editing, we validated the utility of sgRNA. The genetically humanized mouse model suggested that germ-line genome editing of the pathogenic variant may be feasible for monogenic disorders by revealing the recovery of the phenotype; however, there are remaining issues with the tool, including its efficiency and accuracy. This genome editing protocol using a genetically humanized mouse model will provide insights for improving current issues and contribute to the establishment of heritable human genome editing protocols.
2024-01-16 | State-of-the-art 2023 on gene therapy for phenylketonuria.
Phenylketonuria (PKU) or hyperphenylalaninemia is considered a paradigm for an inherited (metabolic) liver defect and is, based on murine models that replicate all human pathology, an exemplar model for experimental studies on liver gene therapy. Variants in the PAH gene that lead to hyperphenylalaninemia are never fatal (although devastating if untreated), newborn screening has been available for two generations, and dietary treatment has been considered for a long time as therapeutic and satisfactory. However, significant shortcomings of contemporary dietary treatment of PKU remain. A long list of various gene therapeutic experimental approaches using the classical model for human PKU, the homozygous enu2/2 mouse, witnesses the value of this model to develop treatment for a genetic liver defect. The list of experiments for proof of principle includes recombinant viral (AdV, AAV, and LV) and non-viral (naked DNA or LNP-mRNA) vector delivery methods, combined with gene addition, genome, gene or base editing, and gene insertion or replacement. In addition, a list of current and planned clinical trials for PKU gene therapy is included. This review summarizes, compares, and evaluates the various approaches for the sake of scientific understanding and efficacy testing that may eventually pave the way for safe and efficient human application.
2023-11-03 | [Development of analytics in newborn screening-from the Guthrie card to genetics].
For more than five decades, all newborns in Germany have been offered a screening examination for the early detection of congenital treatable diseases. Since its inception, about 35 million children have been screened in this way.Originally, screening exams only included early detection of phenylketonuria, which, without timely treatment, would lead to mental retardation that could no longer be corrected. The bacteriological Guthrie test allowed the detection of elevated concentrations of phenylalanine. The methods used today are the result of decades of development. They have been expanded to include tests to determine enzyme activities, immunoassays for the early detection of important hormonal disorders such as congenital hypothyroidism, and high-pressure liquid chromatography for the diagnosis of pathologic hemoglobins. The very sophisticated tandem mass spectrometry enables the simultaneous detection of amino acid and fatty acid compounds. Steroids can also be identified. The specificity can be further increased by combining tandem mass spectrometry with chromatographic pre-separation. In recent years, chemical-analytical analyses have been supplemented by genetic diagnostic methods such as quantitative or qualitative polymerase chain reaction (PCR).The current state of laboratory technology is by no means final. Both classical analytics and especially genetic methods are facing further rapid development. Although the expansion of screening is also a consequence of technical development, the inclusion of further congenital diseases is fundamentally dependent on the given therapy. But it is precisely here that many innovations are currently being investigated. Gene therapy is at the forefront of interest. Seit mehr als 5 Jahrzehnten wird allen Neugeborenen in Deutschland eine Vorsorgeuntersuchung zur Früherkennung angeborener behandelbarer Krankheiten angeboten. Seit Beginn sind so etwa 35 Mio. Kinder untersucht worden.Anfangs ging es nur um die Früherkennung der Phenylketonurie, die ohne frühzeitige Behandlung zu nicht mehr korrigierbarer geistiger Behinderung führt. Der bakteriologische Guthrie-Test erlaubte den Nachweis erhöhter Konzentrationen von Phenylalanin. Die heute eingesetzten Methoden sind das Ergebnis einer über Jahrzehnte verlaufenden Entwicklung. Hinzugekommen sind Tests zur Bestimmung von Enzymaktivitäten, Immunoassays zur Früherkennung wichtiger hormoneller Störungen wie der angeborenen Schilddrüsenunterfunktion sowie Hochdruck-Flüssigkeits-Chromatografie zur Identifizierung pathologischer Hämoglobine. Die sehr anspruchsvolle Tandem-Massenspektrometrie ermöglicht die gleichzeitige Erfassung von Aminosäuren und Derivaten organischer Säuren und Fettsäuren. Auch Steroide können damit identifiziert werden. Die Spezifität lässt sich durch Kombination mit chromatografischer Vortrennung noch erhöhen. In den letzten Jahren wurden die chemisch-analytischen Untersuchungen ergänzt durch gendiagnostische Verfahren, wie beispielsweise quantitative oder qualitative Polymerasekettenreaktion (PCR).Der Stand der Labortechnik ist keineswegs endgültig. Sowohl die klassische Analytik als auch besonders die genetischen Verfahren stehen vor einer weiteren rasanten Entwicklung. Während die Ausweitung des Screenings auch eine Folge der technischen Entwicklung ist, hängt die Einbeziehung weiterer angeborener Erkrankungen grundsätzlich von einer jeweiligen Therapie ab. Aber gerade hier werden gegenwärtig viele Neuerungen erprobt. Im Vordergrund des Interesses steht dabei die Gentherapie.
2022-04-26 | In vivo prime editing of a metabolic liver disease in mice.
Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain (PE2ΔRnH) and an intein-split construct (PE2 p.1153) for adeno-associated virus-mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2ΔRnH via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pahenu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 1014 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.
cell therapies
2025-11-05 | Synthetic closed-loop gene circuit for phenylalanine regulation.
Genetic programming of cells offers significant potential for developing next-generation cell-based therapies that detect and respond to signals within the patient's body to treat chronic diseases. Closed-loop systems designed to self-regulate in response to abnormal biomarker levels are particularly attractive for biomedical applications. In this study, we engineered PRO (phenylalanine regulation orchestration), a cell-autonomous genetic system capable of sensing and degrading elevated levels of phenylalanine, an essential amino acid that accumulates to pathological levels in patients with phenylketonuria (PKU). Central to this system is a transcriptional switch relying on the regulatory domain of human phenylalanine hydroxylase, which dimerizes in the presence of phenylalanine to reconstitute a split transcription factor, thereby inducing gene expression from a synthetic promoter. This sensing module was optimized through random mutagenesis to ensure responsiveness to high concentrations of phenylalanine, enabling robust and dose-dependent activation of protein expression. Engineered human PRO cells containing the sensor driving the expression of phenylalanine-degrading enzymes effectively detected and degraded excess phenylalanine. Microencapsulated PRO cells efficiently reduced phenylalanine levels to the normal physiological range when cultured in human whole blood. Finally, as proof of concept, we showed that alginate-encapsulated PRO cells intraperitoneally implanted in a PKU mouse model significantly lowered blood phenylalanine levels compared to controls implanted with non-engineered cells. Our results suggest that synthetic self-regulating systems are promising candidates for the treatment of metabolic diseases characterized by accumulation of toxic metabolites.
2024-10-25 | Therapeutic liver cell transplantation to treat murine PKU.
For gene therapy of the liver, in vivo applications based on adeno-associated virus are the most advanced vectors despite limitations, including low efficacy and episomal loss, potential integration and safety issues, and high production costs. Alternative vectors and/or delivery routes are of high interest. The regenerative ability of the liver bears the potential for ex vivo therapy using liver cell transplantation for disease correction if provided with a selective advantage to expand and replace the existing cell mass. Here we present such treatment of a mouse model of human phenylketonuria (PKU). Primary hepatocytes from wild-type mice were gene modified in vitro (with a lentiviral vector) that carries a gene editing system (CRISPR) to inhibit Cypor. Cypor inactivation confers paracetamol (or acetaminophen) resistance to hepatocytes and thus a growth advantage to eliminate the pre-existing liver cells upon grafting (via the spleen) and exposure to repeated treatment with paracetamol. Grafting Cypor-inactivated wild-type hepatocytes into inbred young adult enu2 (PKU) mice, followed by selective expansion by paracetamol dosing, resulted in replacing up to 5% of cell mass, normalization of blood phenylalanine, and permanent correction of PKU. Hepatocyte transplantation offers thus an armamentarium of novel therapy options for genetic liver defects.
2024-04-18 | Complete correction of murine phenylketonuria by selection-enhanced hepatocyte transplantation.
Hepatocyte transplantation for genetic liver diseases has several potential advantages over gene therapy. However, the low efficiency of cell engraftment has limited its clinical implementation. This problem could be overcome by selectively expanding transplanted donor cells until they replace enough of the liver mass to achieve therapeutic benefit. We previously described a gene therapy method to selectively expand hepatocytes deficient in cytochrome p450 reductase (Cypor) using acetaminophen (APAP). Because Cypor is required for the transformation of APAP to a hepatotoxic metabolite, Cypor-deficient cells are protected from toxicity and are able to expand following APAP-induced liver injury. Here, we apply this selection system to correct a mouse model of phenylketonuria by cell transplantation. Hepatocytes from a wild-type donor animal were edited in vitro to create Cypor deficiency and then transplanted into phenylketonuric animals. Following selection with APAP, blood phenylalanine concentrations were fully normalized and remained stable following APAP withdrawal. Cypor-deficient hepatocytes expanded from < 1% to ~14% in corrected animals, and they showed no abnormalities in blood chemistries, liver histology, or drug metabolism. We conclude that APAP-mediated selection of transplanted hepatocytes is a potential therapeutic for phenylketonuria with long-term efficacy and a favorable safety profile.
2023-11-14 | The treatment of biochemical genetic diseases: From substrate reduction to nucleic acid therapies.
Newborn screening (NBS) began a revolution in the management of biochemical genetic diseases, greatly increasing the number of patients for whom dietary therapy would be beneficial in preventing complications in phenylketonuria as well as in a few similar disorders. The advent of next generation sequencing and expansion of NBS have markedly increased the number of biochemical genetic diseases as well as the number of patients identified each year. With the avalanche of new and proposed therapies, a second wave of options for the treatment of biochemical genetic disorders has emerged. These therapies range from simple substrate reduction to enzyme replacement, and now ex vivo gene therapy with autologous cell transplantation. In some instances, it may be optimal to introduce nucleic acid therapy during the prenatal period to avoid fetopathy. However, as with any new therapy, complications may occur. It is important for physicians and other caregivers, along with ethicists, to determine what new therapies might be beneficial to the patient, and which therapies have to be avoided for those individuals who have less severe problems and for which standard treatments are available. The purpose of this review is to discuss the "Standard" treatment plans that have been in place for many years and to identify the newest and upcoming therapies, to assist the physician and other healthcare workers in making the right decisions regarding the initiation of both the "Standard" and new therapies. We have utilized several diseases to illustrate the applications of these different modalities and discussed for which disorders they may be suitable. The future is bright, but optimal care of the patient, including and especially the newborn infant, requires a deep knowledge of the disease process and careful consideration of the necessary treatment plan, not just based on the different genetic defects but also with regards to different variants within a gene itself.
2022-02-28 | A quantitative model for metabolic intervention using gut microbes.
As medicine shifts toward precision-based and personalized therapeutics, utilizing more complex biomolecules to treat increasingly difficult and rare conditions, microorganisms provide an avenue for realizing the production and processing necessary for novel drug pipelines. More so, probiotic microbes can be co-opted to deliver therapeutics by oral administration as living drugs, able to survive and safely transit the digestive tract. As living therapeutics are in their nascency, traditional pharmacokinetic-pharmacodynamic (PK-PD) models for evaluating drug candidates are not appropriate for this novel platform. Using a living therapeutic in late-stage clinical development for phenylketonuria (PKU) as a case study, we adapt traditional oral drug delivery models to properly evaluate and inform the engineering of living therapeutics. We develop the adapted for living therapeutics compartmental absorption and transit (ALT-CAT) model to provide metrics for drug efficacy across nine age groups of PKU patients and evaluate model parameters that are influenced by patient physiology, microbe selection and therapeutic production, and dosing formulations. In particular, the ALT-CAT model describes the mathematical framework to model the behavior of orally delivered engineered bacteria that act as living therapeutics by adapting similar methods that have been developed and widely-used for small molecular drug delivery and absorption.
other
2024-05-07 | Characterizing the mechanism of action for mRNA therapeutics for the treatment of propionic acidemia, methylmalonic acidemia, and phenylketonuria.
Messenger RNA (mRNA) therapeutics delivered via lipid nanoparticles hold the potential to treat metabolic diseases caused by protein deficiency, including propionic acidemia (PA), methylmalonic acidemia (MMA), and phenylketonuria (PKU). Herein we report results from multiple independent preclinical studies of mRNA-3927 (an investigational treatment for PA), mRNA-3705 (an investigational treatment for MMA), and mRNA-3210 (an investigational treatment for PKU) in murine models of each disease. All 3 mRNA therapeutics exhibited pharmacokinetic/pharmacodynamic (PK/PD) responses in their respective murine model by driving mRNA, protein, and/or protein activity responses, as well as by decreasing levels of the relevant biomarker(s) when compared to control-treated animals. These preclinical data were then used to develop translational PK/PD models, which were scaled allometrically to humans to predict starting doses for first-in-human clinical studies for each disease. The predicted first-in-human doses for mRNA-3927, mRNA-3705, and mRNA-3210 were determined to be 0.3, 0.1, and 0.4 mg/kg, respectively.
2023-04-18 | Myelin basic protein recovery during PKU mice lifespan and the potential role of microRNAs on its regulation.
Untreated phenylketonuria (PKU) patients and PKU animal models show hypomyelination in the central nervous system and white matter damages, which are accompanied by myelin basic protein (MBP) impairment. Despite many assumptions, the primary explanation of the mentioned cerebral outcomes remains elusive. In this study, MBP protein and mRNA expression on brains of wild type (WT) and phenylketonuric (ENU2) mice were analyzed throughout mice lifespan (14-60-180-270-360-540 post-natal days, PND). The results confirmed the low MBP expression at first PND times, while revealed an unprecedented progressive MBP protein expression recovery in aged ENU2 mice. Unexpectedly, unaltered MBP mRNA expression between WT and ENU2 was always observed. Additionally, for the same time intervals, a significant decrease of the phenylalanine concentration in the peripheral blood and brain of ENU2 mice was detected, to date, for the first time. In this scenario, a translational hindrance of MBP during initial and late cerebral development in ENU2 mice was hypothesized, leading to the execution of a microRNA microarray analysis on 60 PND brains, which was followed by a proteomic assay on 60 and 360 PND brains in order to validate in silico miRNA-target predictions. Taken together, miR-218-1-3p, miR-1231-3p and miR-217-5p were considered as the most impactful microRNAs, since a downregulation of their potential targets (MAG, CNTNAP2 and ANLN, respectively) can indirectly lead to a low MBP protein expression. These miRNAs, in addition, follow an opposite expression trend compared to MBP during adulthood, and their target proteins revealed a complete normalization in aged ENU2 mice. In conclusion, these results provide a new perspective on the PKU pathophysiology understanding and on a possible treatment, emphasizing the potential modulating role of differentially expressed microRNAs in MBP expression on PKU brains during PKU mouse lifespan.
2022-09-08 | Present and future of lipid nanoparticle-mRNA technology in phenylketonuria disease treatment.
Phenylketonuria (PKU) is a metabolic rare disease characterized by a failure of the body to clear out the high levels of Phenylalanine (Phe), leading to devastating neurological defects and growth retardation. PKU was discovered in 1934 by AsbjrØrn FØlling, and even though there have been continuous efforts from the scientific community to find therapeutic approaches to modulate the high levels of phenylalanine found in the body of the PKU patients, an efficient therapy still needs to be developed. Current standard of care includes low phenylalanine diets, but the strict restrictions for patients and families makes it very difficult to adequately being implemented. FDA has approved two drugs to help reduce Phe levels in PKU patients: an enzyme substitution therapy, Palynziq® (PEGylated recombinant phenylalanine ammonia lyase), and Kuvan®, a supplemental tetrahydrobiopterin (BH4) cofactor that enhances residual enzyme activity. Both treatments are restricted to certain PKU patients' population, and, therefore, there are still high unmet needs for most of the patients. The present review will focus on current advancements in lipid nanoparticles (LNP)-mRNA technologies and their potential in treating the root cause of PKU, a therapeutic approach that will be analyzed in the context of other promising therapeutic approaches that are been developed for PKU.
2022-07-21 | Genetic etiology and clinical challenges of phenylketonuria.
This review discusses the epidemiology, pathophysiology, genetic etiology, and management of phenylketonuria (PKU). PKU, an autosomal recessive disease, is an inborn error of phenylalanine (Phe) metabolism caused by pathogenic variants in the phenylalanine hydroxylase (PAH) gene. The prevalence of PKU varies widely among ethnicities and geographic regions, affecting approximately 1 in 24,000 individuals worldwide. Deficiency in the PAH enzyme or, in rare cases, the cofactor tetrahydrobiopterin results in high blood Phe concentrations, causing brain dysfunction. Untreated PKU, also known as PAH deficiency, results in severe and irreversible intellectual disability, epilepsy, behavioral disorders, and clinical features such as acquired microcephaly, seizures, psychological signs, and generalized hypopigmentation of skin (including hair and eyes). Severe phenotypes are classic PKU, and less severe forms of PAH deficiency are moderate PKU, mild PKU, mild hyperphenylalaninaemia (HPA), or benign HPA. Early diagnosis and intervention must start shortly after birth to prevent major cognitive and neurological effects. Dietary treatment, including natural protein restriction and Phe-free supplements, must be used to maintain blood Phe concentrations of 120-360 μmol/L throughout the life span. Additional treatments include the casein glycomacropeptide (GMP), which contains very limited aromatic amino acids and may improve immunological function, and large neutral amino acid (LNAA) supplementation to prevent plasma Phe transport into the brain. The synthetic BH4 analog, sapropterin hydrochloride (i.e., Kuvan®, BioMarin), is another potential treatment that activates residual PAH, thus decreasing Phe concentrations in the blood of PKU patients. Moreover, daily subcutaneous injection of pegylated Phe ammonia-lyase (i.e., pegvaliase; PALYNZIQ®, BioMarin) has promised gene therapy in recent clinical trials, and mRNA approaches are also being studied.
2022-07-14 | Antisense Oligonucleotide Rescue of Deep-Intronic Variants Activating Pseudoexons in the 6-Pyruvoyl-Tetrahydropterin Synthase Gene
We report two new 6-pyruvoyl-tetrahydropterin synthase splicing variants identified through genomic sequencing and transcript analysis in a patient with tetrahydrobiopterin deficiency, presenting with hyperphenylalaninemia and monoamine neurotransmitter deficiency. Variant c.243 + 3A>G causes exon 4 skipping. The deep-intronic c.164–672C>T variant creates a potential 5′ splice site that leads to the inclusion of four overlapping pseudoexons, corresponding to exonizations of an antisense short interspersed nuclear element AluSq repeat sequence. Two of the identified pseudoexons have been reported previously, activated by different deep-intronic variants, and were also detected at residual levels in control cells. Interestingly, the predominant pseudoexon is nearly identical to a disease causing activated pseudoexon in the F8 gene, with the same 3′ and 5′ splice sites. Splice switching antisense oligonucleotides (SSOs) were designed to hybridize with splice sites and/or predicted binding sites for regulatory splice factors. Different SSOs corrected the aberrant pseudoexon inclusion, both in minigenes and in fibroblasts from patients carrying the new variant c.164–672C>T or the previously described c.164–716A>T. With SSO treatment PTPS protein was recovered, illustrating the therapeutic potential of the approach, for patients with different pseudoexon activating variants in the region. In addition, the natural presence of pseudoexons in the wild type context suggests the possibility of applying the antisense strategy in patients with hypomorphic PTS variants with the purpose of upregulating their expression to increase overall protein and activity.
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Drug Discovery Landscape
8 orphan drug designations for Hyperphenylalaninemia due to tetrahydrobiopterin deficiency, including 1 approved therapy.
8 orphan drug designations for Hyperphenylalaninemia due to tetrahydrobiopterin deficiency, including 1 approved therapy.
Drug | Therapy type | Orphan designation | Clinical trial status | Sponsor |
|---|---|---|---|---|
5-hydroxytryptophan | small molecules | FDA | — | Kalevala Therapeutics, Inc. |
2,4-Diamino-5-[[5-(1H-pyrazol-5-yl)-2-thienyl]methyl]-1H-pyrimidin-6-one | small molecules | EMA | — | Pluvia AS |
Escherichia coli, strain Nissle 1917, expressing High affinity phenylalanine transporter, Phenylalanine ammonia lyase and L-amino acid deaminase | cell therapies | EMA | — | Orphix Consulting GmbH |
(S)-2-amino-6-(2-hydroxypropanoyl)-7,8-dihydropteridin-4(3H)-one [Sephience] | small molecules | EMA | Approved | PTC Therapeutics International Limited |
sepiapterin | small molecules | FDA | PTC Therapeutics, Inc. (PTC) | |
Particles comprised of methacrylic acid based co-polymer, cross-linked with a bi-functional cross-linker, purified to bind L-phenylalanine and L-phenylalanine containing peptides | small molecules | EMA | — | MipSalus ApS |
5,6,7,8-Tetrahydrobiopterin | small molecules | EMA | Orphanetics Pharma Entwicklungs GmbH | |
L-5-hydroxytryptophan | small molecules | FDA | — | Watson Laboratories, Inc. |
“Terminated” – programs with discontinued clinical trials, or with no updates reported for 5 years after completion or 12 years after the last trial started.
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