small molecules

2026-09-28 | PAH genotypes and their impact on sapropterin responsiveness in Thai patients with phenylalanine hydroxylase deficiency

Phenylketonuria (PKU) and hyperphenylalaninemia (HPA) are inherited metabolic disorders caused by phenylalanine hydroxylase (PAH) deficiency, leading to phenylalanine (Phe) accumulation and subsequent neurological impairment. Sapropterin therapy effectively reduces Phe levels in responsive patients, yet its efficacy among Thai patients with PAH deficiency remains unclear. A cohort study incorporating prospective and retrospective data enrolled 13 Thai patients with PAH deficiency from three rare disease centers. Participants underwent a 48-hour sapropterin loading test (20 mg/kg/day) with serial Phe measurements. Responsiveness was defined as a > 30% reduction in Phe levels. Genotype and Allelic Phenotype Value (APV) analyses were performed. In the five responsive patients who continued long-term sapropterin therapy, natural protein intake and psychometric outcomes were assessed longitudinally over six months. Sapropterin responsiveness was observed in 61.5% of patients, higher than previously reported. No patients with classic PKU responded, compared with 80% of those with mild PKU and 100% with mild HPA. Concordance between APV-predicted and observed phenotypes was 77.8%. Thai-specific PAH variants, p.(Arg169Leu), p.(Asp229GlufsTer54), and p.(Tyr317Asn), and a novel variant, p.(Gln226Glu), were identified. The 48-hour protocol improved detection of responsiveness compared with shorter durations. Among the five responsive patients who continued sapropterin treatment, natural protein tolerance increased, whereas no clear change in short-term psychometric outcomes was observed. This study represents the first cohort evaluating sapropterin responsiveness in Thai patients with PAH deficiency. Although limited by the small sample size, our findings suggest that sapropterin responsiveness may be relatively common among patients with milder phenotypes and provide preliminary data on genotype–phenotype correlations in the Thai population.

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2026-09-23 | Lower blood phenylalanine concentrations may be of clinical benefit in adults with phenylketonuria.

Treatment in phenylketonuria (PKU) is aimed at lowering blood phenylalanine (Phe) levels, however the target blood Phe levels in adults with PKU are a matter of ongoing debate. To describe the experience of adults with PKU regarding mood and self-reported functioning during a period with higher and/or lower blood Phe levels than they were used to during an interventional trial with Sepiapterin. Eight adults with PKU were included in an open label interventional trial with Sepiapterin (Sephience®, Phase 3 APHENITY Extension Study NCT05166161). Per protocol, participants who were receiving Sapropterin (N = 4) had a washout week before starting with Sepiapterin. The experience of the participants concerning their functioning and feelings were described during the first weeks of the trial when their blood Phe levels changed to higher than usual (in N = 4 during a Sapropterin washout week) and to lower than usual (N = 8) during treatment with Sepiapterin. Participants reported worse functioning when their blood Phe levels were higher than usual and improved functioning when their blood Phe levels were lower than usual. Adults with PKU may experience worsened self-reported functioning at higher blood Phe levels and improved functioning when Phe is lower than their usual blood Phe levels. Therapies that reduce blood Phe levels without the need of a strict diet offer the possibility to objectively study the impact of lower blood Phe levels in adults with PKU and may eventually facilitate individualized target blood Phe levels.

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2026-09-03 | Global Trends in Phenylketonuria Treatment Research, 2000-2025: Bibliometric Analysis.

Phenylketonuria (PKU) is the most common inborn error of amino acid metabolism, and if untreated, leads to severe neurocognitive impairment. Over the past 2 decades, treatment strategies have evolved from strict dietary phenylalanine restriction to include pharmacological therapies such as tetrahydrobiopterin and, more recently, enzyme substitution with pegvaliase. Despite these advances, significant heterogeneity exists in global research priorities, collaboration patterns, and the translation of emerging therapies into clinical practice. A systematic overview of the field's development, thematic shifts, and remaining knowledge gaps is currently lacking. This study aimed to provide a systematic bibliometric analysis of global treatment research on PKU from 2000 to 2025. The aim was to quantify publication trends, collaboration patterns, thematic evolution, and research gaps, thereby informing future scientific and clinical directions. A search of the Web of Science Core Collection was performed on September 13, 2025. The search initially identified 1877 records. After screening, 1462 English-language articles and reviews were included. Publication trends were analyzed using Microsoft Excel (version 16.101), while VOSviewer 1.6.20 and CiteSpace 6.4R1 were used to visualize country- and institutional-level collaborations, journal networks, keyword co-occurrence, citation bursts, and thematic clusters. Statistical charts were generated with GraphPad Prism 10.2.1. Annual publication output demonstrated an overall upward trend, peaking in 2022 with 111 publications. The United States led in both publication volume (375 studies) and total citations (11,142 citations), maintaining strong collaborative ties with several European countries, particularly the Netherlands and the United Kingdom. China ranked seventh globally in publication volume, although its citation impact remains comparatively limited. Key institutions, including the University of Groningen and Birmingham Children's Hospital, as well as prominent scholars such as Francjan J van Spronsen and Anita MacDonald, have occupied central positions in the global PKU treatment research collaboration network over the study period. High-frequency and high-centrality keywords, such as "phenylalanine," "dietary treatment," and "tetrahydrobiopterin," highlighted continued emphasis on metabolic control and targeted therapies. Keyword burst analysis revealed a gradual shift from conventional dietary management toward enzyme replacement therapies, investigations of neurocognitive outcomes, and precision medicine-oriented approaches. Over the past 25 years, PKU treatment research has progressed from foundational dietary interventions to molecular mechanistic studies and individualized therapeutic strategies. Future research should prioritize longitudinal multiomics investigations, targeted metabolic correction technologies, gene-based therapeutic approaches, and enhanced international collaboration, particularly to strengthen diagnosis and management capacities in low- and middle-income regions. Such efforts will be critical to advancing global standards of PKU care.

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2026-08-31 | Natural polysaccharide-based molecularly imprinted polymers as sorbents for selective removal of amino acids as a potential approach for management of aminoacidopathies: Template and monomer selection study.

Phenylketonuria and histidinemia are metabolic conditions characterized by the buildup of certain amino acids resulting from enzymatic deficiencies. Approaches that diminish the intestinal uptake of these amino acids may assist in regulating their systemic concentrations. Molecularly imprinted polymers provide specialized recognition sites that facilitate the precise binding of particular molecules. Natural polymers like chitosan and alginate offer biocompatible frameworks that could improve the efficacy of these systems. Four molecularly imprinted polymers were created utilizing l-phenylalanine as the template and l-phenyllactic acid as a dummy template within chitosan and chitosan/alginate matrices. Characterization employing Fourier-transform infrared spectroscopy and scanning electron microscopy validated the successful imprinting and exhibited clearly defined microcavities, especially with the use of l-phenylalanine and chitosan/alginate polymer. This polymer exhibited an imprinting factor of 2.5 and a substantial binding capacity (15.1 mg/g), nearly double that of the chitosan-only variant (7.6 mg/g), underscoring the notable role of alginate. The polymer adhered to second-order adsorption kinetics, indicating chemical sorption, and Langmuir isotherms, supporting the formation of imprinted cavities. In terms of monomer selection, employing l-phenylalanine as a template demonstrated enhanced selectivity, evidenced by a specificity factor of <1 for alternative amino acids. Nonetheless, the l-phenyllactic acid-imprinted polymer exhibited a strong affinity for certain amino acids such as histidine, threonine, and tryptophan, compromising its selectivity. The evaluation of cytotoxicity validated positive biocompatibility. The results underscore the significant role of ALG in enhancing binding capacity, thereby augmenting the potential of MIPs as selective sorbents for amino acid-related metabolic disorders. This research lays the groundwork for the implementation of therapeutic MIP-based sorbents to enhance the well-being of individuals suffering from aminoacidopathies and other diet-related disorders.

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2026-08-27 | Mediterranean PAH Stratification in Phenylketonuria: Tracing Historical Maps to Point Toward Clinical Phenotype and Obesity Risk.

Apulia and neighbouring Southern Italian regions present a complex genetic stratification shaped by historical migrations. Phenylketonuria (PKU), caused by PAH mutations, serves as an excellent model for molecular epidemiology. This study maps the geographical distribution of PAH variants in a large Southern Italian cohort, evaluating their correlation with clinical phenotypes and long-term Body Mass Index (BMI). A retrospective cohort study was conducted on 344 patients followed for at least 10 years. Genetic characterization evolved from Sanger to Next-Generation Sequencing, and patients were stratified into micro-geographic clusters. The genetic landscape was dominated by three ancestral variants (c.1208C>T, c.1066-11G>A, c.898G>T), accounting for 58% of the allelic pool. Significant micro-geographic polarization was observed: c.1222C>T clustered in Central-Northern Apulia, mirroring historical Norman-Swabian migration, whereas c.441+5G>T enriched in Southern Apulia. At final follow-up, classic PKU adult patients exhibited a four-fold increased risk of obesity (p < 0.05), driven by severe null alleles that necessitate a lifelong dependency on engineered low-protein foods. PAH mutational stratification serves as a contemporary reflection of historical migratory maps. Incorporating regional and genotypic mapping provides a precision medicine framework to anticipate phenotype severity, optimize therapeutic management, and tailor long-term obesity risk monitoring.

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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.

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2026-08-27 | Effects of a Prolonged-Release Protein Substitute on 24-h Phenylalanine and Tyrosine Profiles in Phenylketonuria: A Randomized Crossover Study.

Achieving optimal metabolic control in phenylketonuria (PKU) remains challenging, particularly in adolescents and adults. Conventional free amino acid-based protein substitutes are absorbed rapidly, whereas prolonged-release formulations are designed to provide a more gradual amino acid release profile. Whether this translates into more favorable 24 h Phe and Tyr profiles and improved short-term biochemical parameters of metabolic control remains uncertain. This study evaluated whether a prolonged-release amino acid-based protein substitute (PR-AA) could improve 24 h metabolic control compared with standard protein substitutes. This randomized, controlled, open-label crossover study included adolescents and adults with PKU aged ≥16 years. Participants received either conventional protein substitutes (standard of care; SC) or PR-AA three times daily for two consecutive days, followed by crossover to the alternate treatment. Blood spot samples were collected at five time points over 24 h on the second day of each treatment period. The primary outcome was the 24 h blood Phe profile. Secondary outcomes included blood Tyr, Phe/Tyr ratio, and exploratory assessment of branched-chain amino acids (BCAAs). Twenty-one patients were enrolled, of whom 16 were included in the intention-to-treat analysis and 12 in the per-protocol analysis. PR-AA treatment resulted in significantly lower mean blood Phe levels over 24 h compared with SC (522 ± 194 vs. 561 ± 165 µmol/L; p = 0.0009), corresponding to a reduction of approximately 6-7%. The effect was consistently observed throughout the 24-h assessment period, particularly during the early morning, and became detectable after only two days of treatment. PR-AA was also associated with significantly higher blood Tyr levels (approximately +18-20%; p < 0.05) and a lower Phe/Tyr ratio (approximately -20%; p < 0.001) compared with SC. Exploratory analyses demonstrated modestly lower and more stable BCAA profiles with PR-AA. No significant carryover effects or treatment-related safety concerns were observed. Short-term treatment with a prolonged-release protein substitute was associated with modest but consistent improvements in short-term biochemical parameters of metabolic control in adolescents and adults with PKU, including lower blood Phe and higher Tyr levels, resulting in a reduced Phe/Tyr ratio. These findings suggest that optimizing amino acid delivery kinetics may represent a promising strategy to improve short-term metabolic control in PKU. Given the preliminary nature of these findings and the limited number of participants who completed the study protocol, larger studies with longer follow-up, including direct clinical and neurocognitive outcomes and formal variability metrics, are needed to determine the clinical relevance of these biochemical effects.

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2026-08-25 | TS Case Study: Dietary Management of Phenylketonuria (PKU)

Dietary management of PKU: Development of a phe-free medical food called “Glytactin,” a glycomacropeptide (GMP) for people with phenylketonuria.

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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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2026-07-28 | 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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cell therapies

2026-08-28 | Elucidating a potential role of the infant gut microbiome on L-tyrosine bioavailability in phenylketonuria using whole-body metabolic modelling

Phenylketonuria (PKU) is an inherited metabolic disorder caused by phenylalanine hydroxylase (PAH) deficiency, leading to elevated L-phenylalanine and severe neurological damage if untreated. While phenylalanine-based biomarkers are diagnostic and phenylalanine levels correlate with disease severity, the clinical manifestations of PKU are heterogeneous. To identify additional reliable, potentially novel biomarkers, we used germ-free sex-specific, organ-resolved infant whole-body metabolic models (infant-WBMs) to simulate PAH deficiency and predicted elevated L-phenylalanine and its derivatives, alongside reduced L-tyrosine fluxes, as the product of phenylalanine hydroxylation. To test the reliability of these predicted biomarkers to variations in microbiome composition, we combined the infant-WBMs with gut microbiome models from 48 female and 42 male healthy infants. Upon integrating microbiome data, we found that microbial metabolism significantly increased the predicted L-tyrosine availability, obscuring its utility as a universal biomarker. In ~ 23% of microbiome-PKU models, L-tyrosine fluxes remained low, indicating insufficient microbial compensation. These cases were enriched in Firmicutes and lacked specific Bifidobacterium and Escherichia strains linked to L-tyrosine biosynthesis via the pretyrosine pathway. Shadow price analysis identified microbial species critical for host L-tyrosine metabolism. However, some, such as Bifidobacterium dentium , also contributed to L-phenylalanine synthesis, potentially worsening the PKU phenotype. In contrast, L-phenylalanine, phenylpyruvate, and hydroxyphenylacetic acid remained reliably elevated across all models, validating their diagnostic relevance. Our study demonstrates that microbiome composition can modulate biomarker reliability in PKU, particularly for L-tyrosine. Integrating microbial metabolic models with whole-body physiology enables assessment of biomarker reliability and reveals subpopulations for whom secondary biomarkers or targeted probiotics may be beneficial. This approach offers a powerful framework for refining diagnostics and therapy monitoring in rare metabolic diseases and the development of possible targeted microbiome therapies. Video Abstract

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2026-08-18 | Synthetic biology strategies for engineering microbial therapeutics in metabolic diseases and cancer.

Synthetic biology has redefined the therapeutic role of microbes, transforming them from passive commensals or delivery vehicles into programmable living therapeutics capable of sensing, computing, and actuating within host tissues. This transformation is particularly relevant to metabolic disorders and oncological conditions in which pathophysiology is spatially heterogeneous, dynamically regulated and strongly modulated by host-microbe interactions. Engineered bacterial strains and other microbial platforms can be designed to degrade toxic metabolites, replace deficient enzymes or hormones, alter bile-acid and short-chain-fatty-acid profiles, modulate host immunity, and deliver antitumor payloads with spatial precision. In metabolic disorders, early live biotherapeutic programs have demonstrated that engineered Escherichia coli is capable of metabolically degrading phenylalanine in phenylketonuria, thereby providing a clinical proof of mechanism for gut-restricted metabolic interception. In oncology, tumor colonizing bacteria have been engineered to express cytokines, checkpoint inhibitors, lytic toxins, and diagnostic signals, and their application has been recently demonstrated in colorectal cancer detection and localized immunomodulation. Despite this progress, clinical translation remains limited by variable survival and functional activity in vivo, inconsistent engraftment, metabolic and genetic instability, biocontainment requirements, manufacturing complexity, and uncertain dose control, persist in biogenetic engineering. This review highlights chassis selection, circuit architectures, applications for metabolic diseases and cancer, metabolic bottlenecks, and future directions for precision microbial therapeutics.

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2026-07-21 | Minicells derived from Escherichia coli Nissle 1917 for efficient phenylalanine degradation.

Phenylketonuria is an inherited metabolic disorder characterized by impaired phenylalanine (Phe) catabolism and toxic Phe accumulation. Current therapies are limited by poor long-term adherence, high cost, and biosafety concerns associated with existing or emerging approaches. Here, a non-replicative yet metabolically active platform was developed using anucleate minicells derived from Escherichia coli Nissle 1917 (EcN). A dual phenylalanine degradation system was introduced to engineer these minicells into synthetic microreactors, designated PCB101. In vitro, PCB101 degraded 10 mM Phe within 1.5 h. In a murine hyperphenylalaninemia model, intravenous injection of PCB101 (109 cells/mL) reduced plasma Phe by 87.5%, and oral administration (1010 cells/mL) achieved a 66% reduction in a dose-dependent manner. These results demonstrate that EcN-derived minicells provide a controllable and biosafe chassis for efficient Phe degradation both in vitro and in vivo, supporting their potential applicability in biotherapeutic strategies requiring stable dosing and prevention of microbial proliferation. Engineered microbial systems are widely applied to perform defined metabolic functions, yet most designs rely on viable and replicating cells, intrinsically coupling functional output to population expansion. This growth-dependent paradigm complicates dose stability and raises biosafety concerns in practical applications. This study demonstrates that anucleate minicells derived from Escherichia coli Nissle 1917 retain translational and metabolic activity despite lacking chromosomal DNA and replicative capacity. By incorporating a dual phenylalanine degradation pathway, the engineered minicells efficiently reduced phenylalanine both in vitro and in vivo. This study illustrates that metabolic function can be preserved independently of cell division, providing a controllable microbial platform for applications requiring stable functional output without population expansion. This work expands the conceptual framework for non-replicative microbial systems in applied microbiology.

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2026-06-17 | Artificial Cells: Perspective on Reconstructing Biological Cells and "Out of the Box" Innovations.

The first artificial cell (Chang 1957, 1964) was in the form of artificial red blood cells (RBCs), one of the most important cells because all cells, tissues, organs, and indeed the human being need it for survival. I first reconstructed artificial RBCs in vitro. This is made of cellular dimensions with ultrathin membranes of polymer, crosslinked protein, protein-lipid, or lipid. The next step in this work is to develop this for actual use to replace RBCs in the body. This is a more complex task requiring modifying the configuration and dimensions. Instead of starting with all three functions, a bottom-up approach is used. First, one with only oxygen transport function shows promising results in clinical trials with patients. A future direction is the development of artificial cells that have all three red blood cell functions including antioxidant, oxygen, and CO2 transport. My research group has also been doing innovative research going outside the box of RBCs. This way, extensive variations in contents, membranes, dimensions, and configurations are possible. This allows for worldwide and innovative applications that encompass hemoperfusion, delivery systems, COVID-19 vaccines, cancer therapy, therapy for hereditary enzyme defects like phenylketonuria, cell/stem cell therapy, microbes, nanomedicine, biotherapeutics, gene therapy, regenerative medicine, agricultural applications, aquatic culture, fermentation, nanorobotics, and other areas.

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2026-06-10 | Pharmaco-microdynamics (PMD): Redefining dose, exposure, and control for living drug carriers.

Living drug delivery systems including probiotics, engineered microbial therapeutics, and live biotherapeutic products represent a rapidly emerging therapeutic modality whose behavior fundamentally diverges from the assumptions underlying classical pharmacokinetics and pharmacodynamics (PK/PD). Unlike chemically defined, non-replicating drugs, living therapeutics persist, replicate, adapt, and generate bioactive molecules in situ, such that therapeutic exposure is not externally imposed but biologically generated over time. As a result, administered dose functions only as an initiating condition, while realized exposure emerges from population dynamics, ecological establishment, spatial localization, and regulated functional output. These properties render concentration-based PK/PD frameworks insufficient for predicting efficacy, safety, and controllability of living drug carriers. We introduce pharmaco-microdynamics (PMD) as a quantitative delivery-science framework designed to define, measure, and control exposure for living therapeutics. PMD is operationalized through a set of formal metrics including the functional exposure integral (F-AUC), colonization efficiency (CE), residence-time-weighted activity (RTWA), effective functional concentration (EFC50), and the genetic stability index (GSI) that serve as living-system analogues of AUC, bioavailability, mean residence time, EC50, and product-identity specifications. PMD reconceptualizes exposure as a time-integrated biological process governed by four interdependent axes: population kinetics, functional output kinetics, spatial pharmacology, and evolutionary dynamics. By integrating principles from pharmacology, microbial ecology, synthetic biology, biomaterials science, and systems modeling, PMD provides an operational vocabulary for translating adaptive biological agents into predictable and engineerable delivery systems. We further delineate PMD from adjacent frameworks such as quantitative systems pharmacology (QSP) and ecological microbiome modeling, and critically discuss boundary conditions under which classical PK/PD remains applicable to non-replicating or transient microbial interventions. This review critically examines the limitations of classical PK/PD in modeling living drug carriers, formalizes the core principles of PMD, and illustrates them through three quantitative case studies: SYNB1618 for phenylketonuria, synchronized-lysis bacterial tumor therapies, and fecal microbiota transplantation for recurrent Clostridioides difficile infection. Regulatory and clinical implications are addressed, emphasizing the need to shift from dose- and concentration-centric evaluation toward functional biomarkers, persistence metrics, and model-informed assessment of biological activity. Collectively, pharmaco-microdynamics establishes a unifying conceptual and quantitative foundation for the rational development of living medicines.

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gene therapies

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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2026-07-15 | How Well Is Blood Phenylalanine Controlled in Maternal PKU in Europe? Results from 102 Pregnancies.

Background/Objectives: In phenylketonuria (PKU), high blood phenylalanine (Phe) levels during pregnancy negatively influence foetal organogenesis and growth, leading to maternal PKU syndrome. Pregnancies must be carefully planned in order to maintain blood Phe levels ≤ 360 µmol/L pre-conception and throughout pregnancy. Our aim was to study metabolic control in PKU pregnancies across Europe. Methods: Eleven centres managing PKU participated. Data on blood Phe levels (µmol/L), natural protein intake (g/day), protein substitute intake (g/day) and maternal weight (kg) during pregnancy were collected retrospectively from dietetic records between 2012 and 2018. Results: In total, 84 female patients with PKU, accounting for 102 pregnancies (mean age: 30.4 ± 4.8 years), participated. Of these, 7 had hyperphenylalaninemia (HPA), 26 had mild PKU, 55 had classical PKU and 14 were unclassified. Sapropterin was prescribed in two pregnancies. Only 27% (28/102) of pregnancies successfully achieved consistent blood Phe levels ≤ 360 µmol/L for at least 2 weeks pre-conception. During pregnancy, 88% of blood Phe levels were ≤360 µmol/L, with a mean Phe of 229 ± 65 µmol/L. The mean number of blood Phe samples was 60 (1.5 per week) per pregnancy. In pre-pregnancy, over a mean of 2.9 years, only 35% of blood Phe levels were ≤360 µmol/L and 61% were <600 µmol/L. Post-pregnancy, over a mean of 2.8 years, 43% of Phe levels were <600 µmol/L with mean Phe 462 ± 226 µmol/L and 724 ± 230 µmol/L, respectively. 25% (25/102) had no levels performed post-pregnancy (mean of 2.8 ± 1.6 years) compared to 7% (7/102) pre-pregnancy (mean of 2.9 ± 1.5 years). Mean prescribed Phe intake pre-/during/post-pregnancy was 810 ± 721 vs. 787 ± 552 vs. 1110 ± 722 mg/day. Natural protein intake was 17 ± 15 vs. 17 ± 11 vs. 23 ± 15 g/day. Protein equivalent from protein substitute intake was 57 ± 21 vs. 66 ± 16 vs. 50 ± 23 g/day and total protein remained stable, 73 ± 14 vs. 83 ± 14 vs. 71 ± 19 g/day (1.1 ± 0.3 vs. 1.1 ± 0.4 vs. 1.0 ± 0.4 g/kg/day). Conclusions: Although a high level of metabolic control was maintained during pregnancy, fewer than 30% of pregnancies achieved constant Phe levels ≤ 360 µmol/L prior to conception, with minimal monitoring post-pregnancy. The long-term impact on the offspring remains unknown and requires further investigation.

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2026-06-15 | In vivo adenine base editing rescues brain biochemistry and improves motor deficits in a common phenylketonuria variant.

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism caused by pathogenic variants in the phenylalanine hydroxylase (PAH) gene. Patients are unable to convert the amino acid phenylalanine (Phe) into tyrosine (Tyr), leading to neurotoxic Phe accumulation. Chronically elevated Phe results in intellectual disability, psychiatric disorders, motor impairments, and epilepsy in affected children. Although there are interventions that focus on reducing plasma Phe levels, no curative therapies exist for PKU. Utilizing an adenine base editor (ABE), we demonstrate efficient in vivo corrective editing of hepatocytes in humanized PKU mice homozygous for the common P281L (c.842C>T) variant of PAH. Delivery of the ABE via lipid nanoparticles (LNPs) at four weeks of age resulted in significant reductions in Phe levels in plasma and cortex, as well as increases in cerebral amino acid and neurotransmitter concentrations. Behavioral assessment post-treatment revealed improvements in abnormal motor phenotypes. These data provide increased support for the viability of ABE-based therapeutics as a durable treatment for patients with monogenic metabolic disorders.

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2026-05-15 | [Cognitive function, brain MRI characteristics and their association with blood phenylalanine levels in patients with phenylketonuria].

Objective: To investigate cognitive function and brain MRI characteristics in children aged 7~18 years with phenylketonuria (PKU) and their association with blood phenylalanine (Phe) control levels. Methods: This was a cross-sectional study. Clinical data including general characteristics, random blood Phe levels, Wechsler intelligence scale for children scores, and MRI scores were collected from 29 PKU patients aged 7-18 years treated at Guangzhou Women and Children's Medical Center, Guangzhou Medical University from July 2024 to July 2025. Patients were divided into two groups by random blood Phe>600 μmol/L and ≤600 μmol/L and by historical Phe target attainment rate into relatively well-controlled (≥50%) and poorly-controlled (<50%). Independent-samples t-test, Mann-Whitney U test and multiple linear regression were used to examine the effects of blood Phe control, clinical phenotype and other factors on full scale intelligence quotient (FSIQ) and MRI scores. Results: Among the 29 patients, 17 were males and 12 females, including 17 with classic PKU and 12 with mild PKU. Twenty-seven (93%) were diagnosed early via newborn screening. Physical development was generally normal in 27 patients except for 1 with short stature and 1 with obesity. The random blood Phe level was (634±262) μmol/L. The random blood Phe level in the relatively well-controlled group (19 cases) was significantly lower than that in the poorly-controlled group (10 cases) ((550±266) vs. (794±171) μmol/L, t=2.17, P=0.039). The FSIQ was 90±14. The overall abnormality rate on brain MRI was 86% (25/29), with predominant white matter lesions in the parieto-occipital lobe. The MRI score was 4 (2, 16) points and 10 cases (34%) had a score ≥10 points. Diffusion restriction was observed in 16 cases (55%), of whom 13 cases had random blood Phe>600 μmol/L. The >600 μmol/L group (18 cases) had lower FSIQ, verbal comprehension and working memory scores (all P<0.05), and higher MRI scores (6 (3, 20) vs. 2 (0, 4), Z=2.26, P=0.024) than the ≤600 μmol/L group (11 cases). FSIQ and the scores for verbal comprehension, perceptual reasoning, working memory, and processing speed were all significantly lower in the poorly-controlled group than in the relatively well-controlled group (all P<0.05). Multiple linear regression indicated that long-term Phe metabolic control status (β=0.63, P=0.003) was an independent predictor of FSIQ (F=3.18, P=0.022, adjusted R²=0.33), whereas age (β=0.44, P=0.015) and random blood Phe level (β=0.42, P=0.044) were independent predictors of MRI score (F=4.34, P=0.006, adjusted R²=0.37). Conclusions: In early-diagnosed PKU patients aged 7~18 years, FSIQ is within the average range, often accompanied by varying degrees of white matter abnormalities. Poor long-term metabolic control of blood Phe is closely associated with cognitive decline. Recent blood Phe level >600 μmol/L correlates with more extensive white matter lesions, frequently accompanied by acute brain injury with restricted diffusion.

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2026-02-25 | Exploring subthreshold functional network alterations in women with phenylketonuria by higher criticism.

OBJECTIVE: Phenylketonuria (PKU) is an inherited disorder of amino acid metabolism. Despite early dietary treatment, cognitive functioning of patients has been reported as being inferior to healthy controls. Objective of this study was to assess functional connectivity (FC) alterations in PKU in cognition-related brain networks by resting-state functional magnetic resonance imaging. We followed a hierarchical analysis approach partially based on higher criticism (HC) statistics as previously applied in a larger sister-project in fetal alcohol syndrome. RESULTS: After exclusions for excessive head movement, 11 female young adults with early-treated PKU (age: 27.2 ± 3.7 years) and 11 age-matched female healthy controls (age: 25.9 ± 3.8 years) were included in the analysis. We observed effects within attention networks and the default mode network, but not in fronto-parietal networks, at the HC-based intermediate analysis level. No between-network FC differences were found. In the most detailed analysis level, we could not identify single affected functional connections. Despite statistical power limitations in this small sample, these preliminary findings are in line with previously reported FC alterations in PKU and the cognitive profile in young adults with PKU, particularly the still uncertain notion that cognitive control deficits might become less pronounced when PKU patients reach adulthood.

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2026-09-23 | Construction of a self-amplifying mRNA-LNP complex for phenylalanine hydroxylase and its efficacy evaluation in gene therapy for phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessive disorder caused by deficient phenylalanine hydroxylase (PAH) activity, with current pharmacotherapies showing limited response rates and age-dependent restrictions. We compared two liver-targeted lipid nanoparticle formulations delivering PAH-encoding mRNA-a self-amplifying construct (saRNA) and a conventional non-replicating counterpart (nr-mRNA)-in Pah-knockout mice. Both constructs reduced circulating phenylalanine (Phe) after a single intravenous injection. The saRNA formulation achieved comparable efficacy at one-sixth the nr-mRNA dose and maintained physiological Phe levels for 14 days, whereas nr-mRNA produced only transient reductions. Repeated saRNA administration was well tolerated, with no detectable immunogenicity or hepatic toxicity. These results establish that saRNA-mediated PAH replacement provides sustained activity with substantial dose reduction, overcoming the short-lived effect of non-replicating mRNA. This platform may inform broader strategies for durable enzyme therapy in inherited metabolic diseases requiring liver-restored function.

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2025-12-15 | Rational design and applications of piperazine and cyclohexane ionizable lipids for PKU and SSADH deficiency.

A significant challenge of mRNA-based protein replacement therapies is the diminishing efficacy and escalating toxicity associated with repeated dosing of lipid nanoparticles (LNPs). Many existing lipid formulations were originally designed for vaccine and are not optimized for therapeutic applications. We developed two libraries of ionizable lipids-one based on piperazine and the other on a newly introduced cyclohexane structure-with variations in linker and tail groups to enhance molecular diversity. GC Biopharma's cyclohexane- and piperazine-based LNPs (GCP LNPs) supported stable and high-level expression without inducing liver toxicity under repeated dosing regimens. These LNPs effectively corrected disease markers in mouse models of phenylketonuria (PKU) and succinic semialdehyde dehydrogenase (SSADH) deficiency. Specially, we observed that the rigid structure and chemical stability of cyclohexane-based lipids contributed to sustained delivery performance. These findings offer a promising direction for the development of LNPs suitable for chronic mRNA-based therapies.

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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.

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2025-05-21 | Treatment of a metabolic liver disease in mice with a transient prime editing approach.

Prime editing is a versatile genome editing technology that circumvents the need for DNA double-strand break formation and homology-directed repair, making it particularly suitable for in vivo correction of pathogenic mutations. Here we developed liver-specific prime editing approaches with temporally restricted prime editor (PE) expression. We first established a dual-delivery approach where the prime editor guide RNA is continuously expressed from adeno-associated viral vectors and only the PE is transiently delivered as nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNP). This strategy achieved 26.2% editing with PEmax and 47.4% editing with PE7 at the Dnmt1 locus using a single 2 mg kg-1 dose of mRNA-LNP. When targeting the pathogenic Pahenu2 mutation in a phenylketonuria mouse model, gene correction rates reached 4.3% with PEmax and 20.7% with PE7 after three doses of 2 mg kg-1 mRNA-LNP, effectively reducing blood L-phenylalanine levels from over 1,500 µmol l-1 to below the therapeutic threshold of 360 µmol l-1. Encouraged by the high efficiency of PE7, we next explored a simplified approach where PE7 mRNA was co-delivered with synthetic prime editor guide RNAs encapsulated in LNP. This strategy yielded 35.9% editing after two doses of RNA-LNP at the Dnmt1 locus and 8.0% editing after three doses of RNA-LNP at the Pahenu2 locus, again reducing L-phenylalanine levels below 360 µmol l-1. These findings highlight the therapeutic potential of mRNA-LNP-based prime editing for treating phenylketonuria and other genetic liver diseases, offering a scalable and efficient platform for future clinical translation.

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2024-06-21 | Splice-Switching Antisense Oligonucleotides Correct Phenylalanine Hydroxylase Exon 11 Skipping Defects and Rescue Enzyme Activity in Phenylketonuria.

The PAH gene encodes the hepatic enzyme phenylalanine hydroxylase (PAH), and its deficiency, known as phenylketonuria (PKU), leads to neurotoxic high levels of phenylalanine. PAH exon 11 is weakly defined, and several missense and intronic variants identified in patients affect the splicing process. Recently, we identified a novel intron 11 splicing regulatory element where U1snRNP binds, participating in exon 11 definition. In this work, we describe the implementation of an antisense strategy targeting intron 11 sequences to correct the effect of PAH mis-splicing variants. We used an in vitro assay with minigenes and identified splice-switching antisense oligonucleotides (SSOs) that correct the exon skipping defect of PAH variants c.1199+17G>A, c.1199+20G>C, c.1144T>C, and c.1066-3C>T. To examine the functional rescue induced by the SSOs, we generated a hepatoma cell model with variant c.1199+17G>A using CRISPR/Cas9. The edited cell line reproduces the exon 11 skipping pattern observed from minigenes, leading to reduced PAH protein levels and activity. SSO transfection results in an increase in exon 11 inclusion and corrects PAH deficiency. Our results provide proof of concept of the potential therapeutic use of a single SSO for different exonic and intronic splicing variants causing PAH exon 11 skipping in PKU.

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