Efficient in vivo prime editing corrects the most frequent phenylketonuria variant, associated with high unmet medical need.

The c.1222C>T (p.Arg408Trp) variant in the phenylalanine hydroxylase gene (PAH) is the most frequent cause of phenylketonuria (PKU), the most common inborn error of metabolism. This autosomal-recessive disorder is characterized by accumulation of blood phenylalanine (Phe) to neurotoxic levels. Using real-world data, we observed that despite dietary and medical interventions, most PKU individuals harboring at least one c.1222C>T variant experience chronic, severe Phe elevations and do not comply with Phe monitoring guidelines. Motivated by these findings, we generated an edited c.1222C>T hepatocyte cell line and humanized c.1222C>T mouse models, with which we demonstrated efficient in vitro and in vivo correction of the variant with prime editing. Delivery via adeno-associated viral (AAV) vectors reproducibly achieved complete normalization of blood Phe levels in PKU mice, with up to 52% whole-liver corrective PAH editing. These studies validate a strategy involving prime editing as a potential treatment for a large proportion of individuals with PKU.

Long-term comparative effectiveness of pegvaliase versus medical nutrition therapy with and without sapropterin in adults with phenylketonuria.

Phenylketonuria is characterized by intellectual disability and behavioral, psychiatric, and movement disorders resulting from phenylalanine (Phe) accumulation. Standard-of-care treatment involves a Phe-restricted diet plus medical nutrition therapy (MNT), with or without sapropterin dihydrochloride, to reduce blood Phe levels. Pegvaliase is an injectable enzyme substitution treatment approved for adult patients with blood Phe >600 µmol/L despite ongoing management. A previous comparative effectiveness analysis using data from the Phase 3 PRISM trials of pegvaliase (NCT01819727 and NCT01889862) and the Phenylketonuria Demographics, Outcomes and Safety Registry (PKUDOS; NCT00778206) suggested that pegvaliase was more effective at lowering mean blood Phe levels than sapropterin + MNT or MNT alone at 1 and 2 years of treatment. The current work augments and complements the previous analysis by including additional follow-up from the completed studies, robust methods reflecting careful consideration of issues with the distribution of Phe, and alternative methods for adjustment that are important for control of potential confounding in comparative effectiveness. Median blood Phe levels were lower, and median intact protein intakes were higher, in the pegvaliase group (n = 183) than in the sapropterin + MNT (n = 82) and MNT (n = 67) groups at Years 1, 2, and 3. In the pegvaliase group, median blood Phe levels decreased from baseline (1244 µmol/L) to Year 1 (535 µmol/L), Year 2 (142 µmol/L), and Year 3 (167 µmol/L). In the sapropterin + MNT group, median blood Phe levels decreased from baseline (900 µmol/L) to Year 1 (588 µmol/L) and Year 2 (592 µmol/L), and increased at Year 3 (660 µmol/L). In the MNT group, median blood Phe levels decreased slightly from baseline (984 µmol/L) to Year 1 (939 µmol/L) and Year 2 (941 µmol/L), and exceeded baseline levels at Year 3 (1157 µmol/L). The model-estimated proportions of participants achieving blood Phe ≤600 µmol/L were 41%, 100%, and 100% in the pegvaliase group at Years 1, 2, and 3, respectively, compared with 55%, 58%, and 38% in the sapropterin + MNT group and 5%, 16%, and 0% in the MNT group. The estimated proportions of participants achieving more stringent blood Phe targets of ≤360 µmol/L and ≤120 µmol/L were also higher in the pegvaliase group than in the other groups at Years 2 and 3. Overall, our results indicate that, compared with standard therapy, pegvaliase induces a substantial, progressive, and sustained decrease in blood Phe levels - to a much greater extent than sapropterin + MNT or MNT alone - which is expected to improve long-term outcomes in patients with phenylketonuria.

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.

Towards precision medicine for phenylketonuria: The effect of restoring a strict metabolic control in adult patients with early-treated phenylketonuria.

BACKGROUND AND OBJECTIVE: Neonatal screening and early treatment have changed the natural history of PKU, preventing severe neurological and intellectual disability. Nevertheless, the outcome of the disease in early-treated adult patients (ETPKU) is less than optimal, the predictive value of metabolic biomarkers is feeble, and the recommended levels of blood phenylalanine (Phe) for adulthood are controversial. A crucial question whose answer will improve our understanding and treatment of PKU is whether cognitive outcomes can be modulated by levels of Phe even in early-treated adults. To address this question, we carried out an interventional study in seven ETPKU women planning a pregnancy. METHODS: They underwent an extensive neurocognitive assessment at baseline, and 3 and 6 months after having attained the blood Phe concentration recommended to prevent PKU fetopathy, but before pregnancy. RESULTS: After 3 and 6 months with a stable blood Phe level of about 240 µmol/L, all participants experienced significant improvements in almost all neurocognitive domains and tasks. IQ also increased of 11 to 21 points from the last assessment before enrolment. This pattern remained strong and consistent after correction for multiple comparisons. CONCLUSION: Our results indicate that a) strong cognitive improvement is possible even in adulthood and may be demonstrated by lowering Phe near normal levels; b) testing cognition under different metabolic conditions may unveil an individual vulnerability to Phe. These results pave the way for personalised treatment of the disease in adults with ETPKU.

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.

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.

The management of transitional care of patients affected by phenylketonuria in Italy: Review and expert opinion.

Phenylketonuria (PKU) is a metabolic inherited disorder in which transition from infancy to adult care is particularly difficult and not sufficiently regulated. According to the scientific literature, only few medical centers offer healthcare assistance for adult patients with PKU that are therefore still treated in pediatric settings. This generates psychological, emotional, and organizational discomfort among patients, leading them to discontinue the follow-up. European guidelines and national consensus documents underline this unmet need and the lack of practical recommendations for a structured transitional pathway in PKU. The aim of this review and expert opinion is to propose good practices for managing the transition period of PKU patients, based on the literature and the experience of a panel of Italian experts in PKU. The consensus of the experts was obtained through the administration of three rounds of surveys and one structured interview. The result is the first proposal of a pathway for an efficient transition of PKU patients. Key steps of the proposed pathway are the "a priori" planning involving the pediatric and adult teams, the acceptance of the patient and his/her family to the process, the preliminary definition of appropriate spaces in the structure, the organization of meetings with the joint team, and the appointment of a transition coordinator. For the first time, the involvement of decision makers and patient associations is proposed.

Importance of the long non-coding RNA (lncRNA) transcript HULC for the regulation of phenylalanine hydroxylase and treatment of phenylketonuria.

More than 1280 variants in the phenylalanine hydroxylase (PAH) gene are responsible for a broad spectrum of phenylketonuria (PKU) phenotypes. While the genotype-phenotype correlation is reaching 88%, for some inconsistent phenotypes with the same genotype additional factors like tetrahydrobiopterin (BH4), the PAH co-chaperone DNAJC12, phosphorylation of the PAH residues or epigenetic factors may play an important role. Very recently an additional player, the long non-coding RNA (lncRNA) transcript HULC, was described to regulate PAH activity and enhance residual enzyme activity of some PAH variants (e.g., the most common p.R408W) by using HULC mimics. In this review we present an overview of the lncRNA function and in particular the interplay of the HUCL transcript with the PAH and discuss potential applications for the future treatment of some PKU patients.

The Impact of Telemedicine for Monitoring and Treatment of Phenylketonuria Patients on Metabolic Outcome During Coronavirus Disease-19 Outbreak.

Introduction: The prognosis of phenylketonuria (PKU) in terms of neurocognitive outcome is directly related to lifelong phenylalanine (Phe) levels and adherence to treatment. Monitoring and treatment of PKU patients can be complicated in challenging circumstances as pandemics. This study aims to evaluate the impact of telemedicine for monitoring and treatment of PKU patients on metabolic outcome during coronavirus disease-19 (COVID-19) outbreak. Materials and Methods: Patients who were diagnosed as PKU and treated with low Phe diet, tetrahydrobiopterin (BH4), or BH4 adjunct with low Phe diet were enrolled. Study period was divided into two periods: prepandemic period wherein patients were followed up in outpatients' clinic and during pandemic wherein telemedicine was used. Demographic findings, laboratory results, and therapy responses were reviewed retrospectively and compared between the two periods. All procedures were in accordance with the ethical standards of the local ethical committee of Cerrahpasa Medical Faculty (17/11/2020-151640) and with the Helsinki Declaration of 1975, as revised in 2013. Results: Ninety-three (n = 93) patients were enrolled to this study. The ratio of the samples with Phe levels in the recommended ranges was found to be statistically higher during the pandemic wherein an online monitoring system was used in all treatment modalities (p< 0.05). The decrease in Phe washout frequency was statistically significant during the pandemic in the low Phe diet group (p < 0.05). Considering the relationship between Phe tolerance before and during the pandemic, a significant increase in Phe tolerance was noted during the pandemic in the low Phe diet group (p< 0.05). Conclusions: Telemedicine can be an appropriate and effective monitoring option for PKU patients during the COVID-19 pandemic.

Milestones in treatments for inborn errors of metabolism: Reflections on Where chemistry and medicine meet.

From Sir Archibald Garrod's initial description of the tetrad of albinism, alkaptonuria, cystinuria, and pentosuria to today, the field of medicine dedicated to inborn errors of metabolism has evolved from disease identification and mechanistic discovery to the development of therapies designed to subvert biochemical defects. In this review, we highlight major milestones in the treatment and diagnosis of inborn errors of metabolism, starting with dietary therapy for phenylketonuria in the 1950s and 1960s, and ending with current approaches in genetic manipulation.

Use of an adeno-associated virus serotype Anc80 to provide durable cure of phenylketonuria in a mouse model.

Phenylketonuria (PKU) is the most common inborn error of metabolism of the liver, and results from mutations of both alleles of the phenylalanine hydroxylase gene (PAH). As such, it is a suitable target for gene therapy via gene delivery with a recombinant adeno-associated virus (AAV) vector. Here we use the synthetic AAV vector Anc80 via systemic administration to deliver a functional copy of a codon-optimized human PAH gene, with or without an intron spacer, to the Pahenu2 mouse model of PKU. Dose-dependent transduction of the liver and expression of PAH mRNA were present with both vectors, resulting in significant and durable reduction of circulating phenylalanine, reaching near control levels in males. Coat color of treated Pahenu2 mice reflected an increase in pigmentation from brown to the black color of control animals, further indicating functional restoration of phenylalanine metabolism and its byproduct melanin. There were no adverse effects associated with administration of AAV up to 5 × 1012 VG/kg, the highest dose tested. Only minor and/or transient variations in some liver enzymes were observed in some of the AAV-dosed animals which were not associated with pathology findings in the liver. Finally, there was no impact on cell turnover or apoptosis as evaluated by Ki-67 and TUNEL staining, further supporting the safety of this approach. This study demonstrates the therapeutic potential of AAV Anc80 to safely and durably cure PKU in a mouse model, supporting development for clinical consideration.

Predictability and inconsistencies of cognitive outcome in patients with phenylketonuria and personalised therapy: the challenge for the future guidelines.

Phenylketonuria (PKU) is a prototypical model of a neurodevelopmental metabolic disease that follows a cascade of pathological events affecting brain maturation and functioning. Neonatal screening and early treatment have eradicated the classical PKU phenotype in patients with early and continuously treated phenylketonuria (ECTPKU). However, effort is required to optimise the treatment of the disease to minimise the risk of lifelong neurological, cognitive and behavioural impairment, and to solve issues on the variability in clinical outcome that are rather not understood and has yet hampered a more personalised approach to its treatment. The aim of the present review is to focus on the inconsistencies in the clinical outcome of adult patients with ECTPKU unexplained by the biochemical markers adopted for the monitoring of the disease to date. The interindividual variability of clinical outcome in late as well as in early treated patients under similar biochemical control suggests the existence of disease-independent determinants influencing the individual vulnerability to the neurotoxic effect of phenylalanine. This is further supported by the low predictive power of blood phenylalanine on the clinical outcome from the second decade of life onwards. In conclusion, individual vulnerability to the metabolic alterations of PKU contributes to the prognosis of PKU, also in patients with ECTPKU. The biological factors constitutive of this vulnerability are unknown (but have not been the object of many studies so far) and should be the target of further research as prerequisite for a personalised treatment aimed at avoiding burden and costs of overtreatment and clinical consequences and risks of undertreatment in patients with PKU.

Guide for diagnosis and treatment of hyperphenylalaninemia.

IMPORTANCE: Sapropterin hydrochloride, a natural coenzyme (6R-tetrahydrobiopterin) of phenylalanine hydroxylase, was first approved as a treatment for tetrahydrobiopterin deficiency in 1992 in Japan, and was then approved as a treatment for a tetrahydrobiopterin-responsive hyperphenylalaninemia in 2007 and 2008, in the USA and Japan, respectively. Guidelines are required on the proper use of sapropterin hydrochloride for tetrahydrobiopterin-responsive hyperphenylalaninemia. OBSERVATIONS: It is recommended that tetrahydrobiopterin-responsive hyperphenylalaninemia should be diagnosed in all cases of hyperphenylalaninemia, including phenylketonuria, by tetrahydrobiopterin administration tests rather than by phenotype or blood phenylalanine levels. CONCLUSIONS AND RELEVANCE: If tetrahydrobiopterin-responsive hyperphenylalaninemia is diagnosed, all ages can be treated with sapropterin hydrochloride. Although there are reports that sapropterin hydrochloride is effective and safe for the prevention of maternal phenylketonuria, further investigation is required.

A novel Pah-exon1 deleted murine model of phenylalanine hydroxylase (PAH) deficiency.

Phenylalanine hydroxylase (PAH) deficiency, colloquially known as phenylketonuria (PKU), is among the most common inborn errors of metabolism and in the past decade has become a target for the development of novel therapeutics such as gene therapy. PAH deficient mouse models have been key to new treatment development, but all prior existing models natively express liver PAH polypeptide as inactive or partially active PAH monomers, which complicates the experimental assessment of protein expression following therapeutic gene, mRNA, protein, or cell transfer. The mutant PAH monomers are able to form hetero-tetramers with and inhibit the overall holoenzyme activity of wild type PAH monomers produced from a therapeutic vector. Preclinical therapeutic studies would benefit from a PKU model that completely lacks both PAH activity and protein expression in liver. In this study, we employed CRISPR/Cas9-mediated gene editing in fertilized mouse embryos to generate a novel mouse model that lacks exon 1 of the Pah gene. Mice that are homozygous for the Pah exon 1 deletion are viable, severely hyperphenylalaninemic, accurately replicate phenotypic features of untreated human classical PKU and lack any detectable liver PAH activity or protein. This model of classical PKU is ideal for further development of gene and cell biologics to treat PKU.

The impact of COVID-19 pandemic on the diagnosis and management of inborn errors of metabolism: A global perspective.

Quantitative estimates for the global impact of COVID-19 on the diagnosis and management of patients with inborn errors of metabolism (IEM) are lacking. We collected relevant data from 16 specialized medical centers treating IEM patients in Europe, Asia and Africa. The median decline of reported IEM related services in March 1st-May 31st 2020 compared to the same period in 2019 were as high as 60-80% with a profound impact on patient management and care for this vulnerable patient group. More representative data along with outcome data and guidelines for managing IEM disorders under such extraordinary circumstances are needed.

Examining the blood amino acid status in pretherapeutic patients with hyperphenylalaninemia.

BACKGROUND: Hyperphenylalaninemia is the most common genetic metabolic disease. Early treatment prevents brain injury effectively. The present study aimed to detect the exact amino acid status of patients with hyperphenylalaninemia before treatment. METHODS: Data of 116 newborn patients from our Newborn Screening Center and 161 older patients from our clinic before treatment were collected. The content of 17 amino acids in their blood was determined by tandem mass spectrometry and compared with normal controls. Relationship between phenylalanine and other amino acids in patients was analyzed using the smoothing curve fitting and threshold effect analysis. RESULTS: Most amino acids in the blood of patients were within the normal range; however, they were different significantly from those of the normal children. Newborn patients showed higher phenylalanine (346.30 vs 45.90 µmol/L), valine (121.50 vs 110.30 µmol/L), citrulline, ornithine and lower tyrosine (52.97 vs 66.12 µmol/L), threonine (68.68 vs 78.21 µmol/L), glutamine levels than observed in normal newborns. Older patients showed significantly higher phenylalanine (844.00 vs 51.82 µmol/L), valine (117.60 vs 110.90 µmol/L), histidine, serine and lower tyrosine (55.97 vs 67.31 µmol/L), threonine (35.94 vs 51.89 µmol/L), alanine, asparagine, glutamic acid, methionine, arginine, glycine, ornithine, glutamine content than found in matched normal children. Tyrosine, valine, ornithine, and threonine in newborn patients and tyrosine, glycine, glutamine, and threonine in older patients had a nonlinear correlation with phenylalanine levels with obvious threshold effect and clear inflection points. CONCLUSION: Significant difference was observed in the amino acid status between pretherapeutic hyperphenylalaninemia patients and normal children. Some amino acids showed notable threshold effect with phenylalanine level in a nonlinear pattern.

Psychosocial functioning in children with phenylketonuria: Relationships between quality of life and parenting indicators.

OBJECTIVE: This study aimed to assess the impact of phenylketonuria (PKU) and its treatment on parent and child health-related quality of life (HRQoL) and to identify the parenting-related correlates of parent and child HRQoL, as well as metabolic control. METHODS: Eighteen mothers of 2- to 12-year-old children with PKU participated and completed a series of self-report questionnaires including the PKU Impact and Treatment Quality of Life Questionnaire (PKU-QOL). RESULTS: Mothers reported that the most significant impact of PKU on HRQoL was in relation to the impact of their child's anxiety during blood tests on their own HRQoL and guilt related to poor adherence to dietary restrictions and supplementation regimens. Higher reported intensity of child emotional and behavioural difficulties and parenting stress were associated with higher scores for PKU symptoms on the PKU-QOL, higher scores for emotional, social, and overall impact of PKU, and higher scores for the impact of dietary restriction. Where mothers reported greater use of overreactivity as a parenting strategy, children tended to have better lifetime phenylalanine levels; however, the overall impact of PKU and the impact of supplement administration on mothers' HRQoL were worse for these families. CONCLUSIONS: These findings have implications for a holistic family-centred approach to the care of children with PKU and their families.

[Clinical practice guidelines for phenylketonuria].

Phenylketonuria (PKU) is an autosomal recessive hereditary disease and a common disorder of amino acid metabolism. The average incidence of PKU in China is approximately 1/11 000. It is characterized by lower incidence in the South and higher incidence in the North, particularly the Northwest. PKU is a treatable disease and has been listed in the national newborn screening program. Neonates with positive indication of screening can achieve satisfactory therapeutic effect by timely control of phenylalanine intake after the definite diagnosis. This guideline aims to summarize the knowledge of medical genetics and key points of clinical management of PKU, so as to improve the diagnostic level and standardize newborn screening and clinical treatment of patients.

Neuropsychological Profile of Children with Early and Continuously Treated Phenylketonuria: Systematic Review and Future Approaches.

OBJECTIVE: To provide a comprehensive systematic review of the literature by examining studies published on all cognitive aspects of children with early and continuously treated phenylketonuria (ECT-PKU) included in the databases Medline, PsycINFO, and PsycARTICLE. METHOD: In addition to a classical approach, we summarized methodology and results of each study in order to discuss current theoretical and methodological issues. We also examined recent advances in biochemical markers and treatments of PKU, with implications for future research on metabolic control and its role as a determinant of neuropsychological outcome. RESULTS: Consistent with previous reviews, the hypothesis of a specific and central executive impairment in children with ECT-PKU was suggested. However, findings are inconclusive regarding the nature of executive impairments as well as their specificity, impact on everyday life, persistence over time, and etiology. CONCLUSION: Given the current state of the science, we suggest future directions for research that utilizes a developmental and integrative approach to examine the effects of recent advances in biochemical markers and treatment of PKU. (JINS, 2019, 25, 624-643).