2026-08-11 | Adenovirus Vector-Mediated In Vivo Knock-in Treatment of Neonatal Phenylketonuria Mice Using Terminally Cleaved Donor DNA.
Adenovirus vectors (AdVs) are widely used and have an advantage of large insert capacity compared with adeno-associated virus vectors. However, AdVs have scarcely been used in genome-editing knock-in strategies because of low efficiency. Novel AdVs possessing a very large, 3.7 kb donor DNA fragment and six or eight multiplex gRNA expression units were developed for CRISPR/Cas9-mediated knock-in to correct a phenylalanine hydroxylase (Pah) gene in a Pahenu2 phenylketonuria mouse model. These AdVs were co-infected to Hepa1-6 cells or liver cells in vivo together with an AdV expressing either native Cas9 or Cas9 nickase (Cas9n) for double-nicking cleavage. In vitro knock-in of the AdVs carrying 3.7 kb donor DNA and six gRNA units targeting the cell genome was observed in both cases using Cas9 and Cas9n, though their efficiencies were low. Therefore, we generated AdVs carrying an additional two gRNA units that cleave the donor DNA terminus in the AdV genome via native Cas9 or Cas9 nickase. The knock-in efficiency increased approximately twofold for both vectors and reached a maximum of 8% for native Cas9 without selection. Newborn phenylketonuria model mice were intravenously administered the knock-in AdV together with the native-Cas9 AdV. Although the knock-in efficiency by homologous recombination occurred in only approximately 1% of hepatocytes, blood phenylalanine levels were reduced by up to 30%. Also, unintended fragments produced by nonhomologous end-joining were observed between the cleavage site at the terminus of the donor DNA in the AdV genome and the target site in the cell genome. The knock-in efficiency of AdVs can be increased by cleaving the terminus of the donor DNA, although it would be desirable to avoid nonhomologous end-joining between double-strand break termini.
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2026-08-10 | Clinical practice considerations for restarting pegvaliase in adults with phenylketonuria
Objective To provide key considerations and best practices for restarting pegvaliase based on real-world experiences of healthcare professionals managing individuals with phenylketonuria (PKU). Methods An in-person advisory board with 11 experienced PKU practitioners identified strategies for restarting pegvaliase after a treatment pause. The advisors presented real-world restart cases detailing treatment history, reasons for discontinuation, restart approach, and outcomes, followed by discussions about best practices for clinical decision-making. Results Among 11 restart cases, reasons for treatment discontinuation included adverse events (AEs) (3 cases), limited blood phenylalanine (Phe) response (4 cases), clinical trial participation (1 case), and pregnancy (3 cases). Time off treatment ranged from 3 to 64 months. Eight cases restarted pegvaliase with an expedited titration schedule, while 2 cases resumed with a slowed approach and 1 case used the standard titration. At the time of the advisory board, 9 cases had achieved blood Phe ≤360 μmol/L, 1 case still had elevated blood Phe levels but had not yet completed titration and 1 case discontinued after 18 months on therapy. Key considerations for restarting pegvaliase after AE-related discontinuations include proactively addressing anxiety, optimizing premedications, ensuring access to on-demand medications and using a flexible, individualized titration approach. For individuals discontinuing primarily due to a limited blood Phe response, resuming at the previously tolerated dose and titrating adaptively as tolerated may be considered. For individuals desiring an expedited titration, more frequent blood Phe monitoring may be warranted to guide dietary adjustments and dosing. Life changes, individual preferences and social determinants of health should inform timing and resources for restart. For pregnancy-related discontinuations restart decisions should be made collaboratively with the individual and care team (geneticist, metabolic dietitian, obstetrician, and other relevant caregivers). Conclusion Pegvaliase can help achieve blood Phe control, which may mitigate neurocognitive effects and can result in greater dietary flexibility and improved quality of life. Restarting pegvaliase is feasible for most individuals, with many having a better experience during the restart process compared with the first treatment initiation. AEs were reported (eg, injection site reactions, rash, and arthralgia) but were milder compared to the previous treatment course in most cases and some individuals responded at lower doses or after shorter treatment duration. Restarting pegvaliase should be considered as part of a shared decision-making process for individuals seeking to further optimize outcomes.
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2026-08-07 | Sapropterin (BH4) challenge in phenylketonuria: Responder or non-responder?
Sapropterin dihydrochloride, the synthetic form of tetrahydrobiopterin (BH4), is an established pharmacological treatment for a subset of patients with phenylalanine hydroxylase (PAH) deficiency. Yet the clinically simple question of whether a patient is a "responder" or "non-responder" remains difficult to answer in a uniform way. Since the first description of BH4-responsive PAH deficiency, challenge protocols have varied substantially, ranging from short 4-8 h loading tests to 24-48 h protocols, 72 h to 7-day approaches, and treatment trials lasting several weeks or months. These protocols differ in dose, duration, sampling schedule, dietary conditions, baseline phenylalanine (Phe) concentration, use of Phe loading, and response definition. European, American, and Japanese guidance also differ in target ranges and in how responsiveness is confirmed. The widely used criterion of at least 30% reduction in blood Phe is practical for short biochemical testing, but it may be insufficient in patients who are already well controlled on diet, in slow responders, in infants or preschool children in whom BH4 bioavailability may be lower, and in patients whose main benefit is increased daily dietary Phe tolerance rather than further Phe reduction. Recent European guidance distinguishes potential sapropterin responsiveness from long-term responsiveness, defining the latter by increased natural protein intake and/or improved biochemical control within the recommended therapeutic target range. Japanese guidance adds an important Asian perspective by recommending BH4 administration tests for all hyperphenylalaninemia/phenylketonuria (PKU) cases rather than relying only on phenotype or blood Phe level, and recent Japanese data suggest that age and baseline Phe may influence blood biopterin peaks and the risk of false-negative tests. This narrative review summarizes the historical development, biological rationale, clinical evidence, geographical variation, genotype-specific considerations, and practical limitations of BH4/sapropterin responsiveness testing in PKU. We propose a pragmatic responder/non-responder framework based on three linked dimensions: blood Phe reduction, increase in daily dietary Phe tolerance, and maintenance of blood Phe within age-, pregnancy-, and guideline-specific therapeutic ranges. A responder is best defined as a patient who demonstrates either reproducible biochemical response or clinically meaningful therapeutic benefit, while a non-responder is a patient who demonstrates neither under adequate testing conditions. Evidence from extended Dutch testing further supports using 7-day protocols selectively when 48 h results are borderline or clinically discordant, while avoiding a simple 20% threshold because of false-positive risk.
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2026-07-28 | Use of a Slow-Release Phenylalanine-Free Microtablet Protein Substitute in Children and Adolescents with Phenylketonuria: An Observational Pilot Study.
Background/Objectives: In phenylketonuria (PKU), adherence to protein substitute (PS) is frequently suboptimal due to poor palatability, high volume, and sensory fatigue. A novel phenylalanine (Phe)-free L-amino acid microtablet PS has been developed to address these barriers. The microtablets incorporate a cellulose-based taste-masking coating and a sodium-alginate inner matrix intended to enable controlled amino acid release over approximately three hours. This study evaluated their short-term tolerability, acceptability, and adherence in children with PKU, with extended follow-up in a subgroup. Methods: A 7-day observational study was conducted in participants with PKU aged ≥3 years on a low-Phe diet in a single centre. The test product provided protein equivalent (PE) 54 g/100 g. Participants replaced at least one daily dose of their usual PS with the test product, providing 10 g or 20 g/day PE, with one child receiving 80 g/day. Daily gastrointestinal (GI) symptoms, PS intake, and adherence were recorded. Acceptability was assessed using structured ratings of palatability, ease of use, and overall preference. Longer-term tolerability and acceptability were then assessed in four adolescents who elected to continue the test product for an additional 28 days. Results: Ten participants completed the 7-day study. Adherence to the test product was high (80%), exceeding adherence to participants' usual PS (70%). At baseline, nine participants reported none/mild GI symptoms, most commonly flatulence. One participant reported moderate/severe symptoms including constipation, flatulence, diarrhoea, bloating, burping, and abdominal discomfort. By day 7, all participants reported no or mild symptoms, with complete resolution in the child with moderate/severe baseline symptoms. Acceptability ratings were comparable between the test product and usual PS, and nine children (90%) reported no difficulty taking the test product. During the 28-day extension, adherence remained high, GI tolerance was maintained, and improved preference was noted, particularly due to reduced aftertaste. Conclusions: This novel slow-release PS microtablet was well tolerated, acceptable, and associated with high adherence. Its taste-masked, low-volume format offered practical advantages that may support sustained dietary adherence. Longer-term controlled studies are necessary to confirm these findings.
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2026-07-23 | Modulation of phenylalanine assembly kinetics by gallic acid and its therapeutic implications in phenylketonuria (PKU).
Self-assembly of L-phenylalanine (Phe) results in the formation of assemblies that are structurally and functionally related to amyloid-like fibrils. Recently, Phe assembly has been correlated with the elevated Phe levels observed in phenylketonuria (PKU) patients. The presence of Phe aggregates in the post-mortem brain sections of PKU patients and in transgenic mouse models suggests their plausible role in disease pathology. To inhibit Phe assembly, we hypothesized that gallic acid (GA), a known antioxidant, could interfere with the assembly process owing to its generic anti-amyloid activity. Here, using multiple biophysical techniques, we demonstrate the characteristic features of Phe assembly and its disassembly in the presence of GA. Owing to its natural occurrence in plants and its therapeutic potential, GA may serve as a promising molecule for future preclinical testing in PKU mouse models.
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