RARE DISEASE
Maternal phenylketonuria
Maternal phenylketonuria
Maternal phenylketonuria
Synonyms: Hyperphenylalaninemic embryopathy, Maternal PKU, Maternal hyperphenylalaninemia, Phenylketonuric embryopathy
Synonyms: Hyperphenylalaninemic embryopathy, Maternal PKU, Maternal hyperphenylalaninemia, Phenylketonuric embryopathy
Synonyms: Hyperphenylalaninemic embryopathy, Maternal PKU, Maternal hyperphenylalaninemia, Phenylketonuric embryopathy
Drug discovery
0
drugs
With orphan designations
Overview
Maternal phenylketonuria (PKU) is an inherited metabolic disorder caused by phenylalanine hydroxylase deficiency, leading to elevated maternal blood phenylalanine (Phe) levels. Untreated, this causes maternal PKU syndrome in offspring, characterized by microcephaly, congenital heart defects, intellectual disability, and growth retardation [1][4][6]. Strict preconception and gestational Phe control (<360 μmol/L) via dietary restriction and supplementation is critical to prevent teratogenic effects [1][5][14].
Categories: rare developmental anomalies during embryogenesis, rare genetic diseases, rare inborn errors of metabolism, rare neurological diseases, rare otorhinolaryngological diseases, rare surgical maxillo-facial diseases, rare teratologic disorders
Research Papers
370 drug discovery papers about Maternal phenylketonuria, with 2 first-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
370 drug discovery papers about Maternal phenylketonuria, with 2 first-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
categories:
Small molecules
small molecules
2026-03-11 | [Linking tetrahydrobiopterin depletion to ferroptosis: A novel mechanism of neurological injury in Hyperphenylalaninemia].
Hyperphenylalaninemia (HPA) is an inherited metabolic disorder caused by deficiency of phenylalanine hydroxylase, characterized by significantly elevated phenylalanine levels. Conventional mechanisms, such as neurotransmitter deficiency and dysmyelination, fail to fully explain the progressive neurological damages associated with HPA. Meanwhile, ferroptosis, an emerging form of iron-dependent regulated cell death, has proven to play an important role in neurodegenerative diseases. We hereby propose a hypothesis that tetrahydrobiopterin (BH4) depletion in HPA may lead to the collapse of intracellular antioxidant defenses. This process could induce ferroptosis, thereby serving as a pivotal mechanism underlying HPA-related neurological injury. This review has systematically summarized the pathological mechanisms of HPA, the biological features of ferroptosis, and the bridging role of BH4 between them, thereby establishing a novel "HPA-BH4-ferroptosis" theoretical framework and providing a rationale for developing new therapeutic strategies targeting ferroptosis.
2026-01-28 | The First 1000 Days of PKU: A Narrative Review of Maternal PKU and Early Life Management After Positive Newborn Screening.
Background/Objectives: The first 1000 days of life represent a critical window for growth and neurodevelopment, during which nutrition strongly influences brain development and metabolic programming. In phenylketonuria (PKU), dietary management is essential to prevent neurological impairment and later-life risk of non-communicable diseases (NCDs). This review examines current evidence on PKU from pregnancy through complementary feeding, highlighting the impact of nutritional strategies on neurodevelopmental and metabolic outcomes. Methods: This narrative review, following PRISMA guidelines, used a systematic search of PubMed and Scopus with defined PICO questions. Original research, reviews, and guidelines on PKU nutrition during the first 1000 days were included, emphasizing neurological and metabolic outcomes. Results: Articles addressed prenatal and postnatal factors in PKU. Optimised metabolic control in women with PKU is critical to prevent maternal PKU syndrome, reducing risks of miscarriage, congenital heart defects, microcephaly, and neurocognitive impairment. Pre-conception dietary management, frequent blood Phe monitoring, supplementation with Phe-free protein substitutes (PSs), micronutrients, and emerging pharmacological therapies support maternal and foetal health. Following newborn screening, early dietary treatment in infants with PKU maintains plasma Phe within safe ranges, promoting growth and neurodevelopment. Breastfeeding, combined with Phe-free infant PSs, is feasible, and complementary feeding should be introduced carefully. Frequent monitoring and tailored dietary adjustments, including second-stage PSs, support metabolic control, while data on gut microbiota remain limited. Conclusions: Early multidisciplinary interventions are crucial to optimise metabolic and neurodevelopmental outcomes during this window of opportunity. Further research is needed to address remaining gaps and optimise PKU management across the first 1000 days.
2025-11-07 | Relationship Between Gut Microbiota and Phenylalanine Levels: A Mendelian Randomization Study.
The specific members of the gut microbiota linked to phenylketonuria remain to be identified. This study aimed to assess the association between gut microbiota on phenylalanine (Phe) levels using a two-sample Mendelian randomization (MR) approach. Summary statistics from genome-wide association studies (GWAS) related to individual gut microbiota were obtained from the MiBioGen Global Consortium database. The data set of Phe levels was derived from GWAS summary datasets. Inverse variance weighting (IVW) served as the primary method to infer the causal relationship between gut microbiota and Phe levels. Additional pleiotropy and heterogeneity tests were conducted to evaluate the reliability of the findings. The Family XIII AD3011 group had a protective effect on Phe levels (OR = 0.962, 95% CI: 0.942-0.982, p < 0.001), and these associations remained significant after FDR correction (adjusted p-value = 0.027). There was no evidence of notable heterogeneity and horizontal pleiotropy among the instrumental variables. Our data indicate that Family XIII AD3011 group is associated with reduced Phe levels, highlighting a potential link between gut microbiota and Phe levels. Although MR analysis supports a causal relationship, it may not precisely estimate the effect size, necessitating further studies to validate these findings and quantify the association.
2025-01-31 | Transitioning of protein substitutes in patients with phenylketonuria: a pilot study
Introduction In phenylketonuria (PKU), there is limited information about transitioning between protein substitutes and the influencing factors, particularly in young children. This pilot study assessed the stepwise transition from second to third-stage protein substitutes in children with PKU, aged 3–5 years. Methods Demographics, child behavior, maternal anxiety, and food neophobia scores were collected at baseline, mid-transition, and final assessment. Blood phenylalanine (Phe) was collected from 6 months pre-baseline to post-final assessment. Results Twelve children ( n = 4 males, 33%, median age 3.2 years) participated. Sixty-seven percent ( n = 8) transitioned to liquid amino acid-based protein substitute and 33% ( n = 4) to glycomacropeptide (cGMP) powder. Forty-two percent ( n = 5/12) had a smooth transition (Group 1, median 3.5 months), while the remaining faced difficulty ( n = 3, 25%, Group 2), or failed full transition ( n = 4, 33%, Group 3). In Groups 2 and 3, caregivers failed to follow instructions, demonstrating inconsistencies and child resistance. Group 2 children had significantly higher blood Phe levels (above 360 μmol/L), that was significantly higher than Groups 1 and 3 ( p < 0.01), with Groups 1 and 3 maintaining blood Phe within target ( p < 0.01). Higher maternal education and nursery/school attendance significantly influenced transition success ( p < 0.05). No significant differences were found in child neophobia, maternal anxiety, or child behavior ( p > 0.05). Mothers generally reported satisfaction with the stepwise transition process. Conclusion A stepwise transition to third-stage protein substitutes in PKU is effective, but is dependent on child metabolic control, parental education, and nursery/school support.
2024-09-06 | Long-term safety of sapropterin in paediatric and adult individuals with phenylalanine hydroxylase deficiency: Final results of the Kuvan® Adult Maternal Paediatric European Registry multinational observational study.
Phenylketonuria is a rare inherited disorder that disrupts the metabolism of phenylalanine (Phe) to tyrosine by phenylalanine hydroxylase (PAH). Sapropterin dihydrochloride (Kuvan®) is approved for use in Europe to reduce blood Phe levels and improve Phe tolerance in sapropterin-responsive individuals. KAMPER (NCT01016392) is an observational, multinational registry assessing long-term safety and efficacy of sapropterin. Five hundred and seventy-six participants with PAH deficiency were enrolled from nine European countries (69 sites; December 2009-May 2016). Participants were aged <4 years (n = 11), 4 to <12 years (n = 329), 12 to <18 years (n = 141), and ≥18 years (n = 95) at enrolment. Overall, 401 (69.6%) participants experienced a total of 1960 adverse events; 61 events in 42 participants were serious, and two were considered sapropterin-related by the investigator. Mean (standard deviation) actual dietary Phe intake increased from baseline across all age groups: 957 (799) mg/day to a maximum of 1959 (1121) mg/day over a total study period of 11 years. Most participants exhibited an increase in Phe tolerance while blood Phe levels remained in the target range for their age (120-360 μmol/L for <12 years; 120-600 μmol/L for ≥12 years). Most participants exhibited normal growth for height, weight, and body mass index. No additional safety concerns were identified. As an observational study, limitations include variability in routine care practices and inconsistent availability of data. Long-term sapropterin use demonstrates a favourable safety profile in real-world settings and increases Phe tolerance in participants with PAH deficiency while maintaining blood Phe levels in the target ranges.
proteins
2026-06-15 | Increased Brown Adipose Tissue Thermogenesis in Phenylketonuria.
Phenylketonuria (PKU), the most common autosomal‑recessive disorder of amino acid metabolism, is characterized by neurological impairment and systemic metabolic alterations caused by chronically elevated phenylalanine (Phe) levels. PKU patients have long been reported to display reduced metabolic rate and impaired thermoregulation, yet the role of brown adipose tissue (BAT) in this condition remains unknown. Here, noninvasive infrared thermography was used to assess BAT activity in a cohort primarily comprising children and adolescents including controls, mild hyperphenylalaninemia (MHPA), and PKU patients, while circulating metabolic and hormonal parameters were analyzed for associations with BAT temperature. Despite overall normothermia, individuals with PKU exhibited higher BAT temperature than both control and MHPA patients, which correlated with circulating fibroblast growth factor 21 (FGF21) and thyroid hormones. To gain mechanistic insight, rats and mice were centrally treated with FGF21, reproducing the BAT thermogenic phenotype along with decreased hypothalamic AMP‑activated protein kinase (AMPK) activity and increased sympathetic drive to BAT. Consistently, analysis of public single‑cell RNA‑sequencing data revealed convergent expression of AMPK, thyroid hormone receptor, and FGF21 receptor signaling in specific hypothalamic neuronal populations. These findings reveal enhanced BAT thermogenesis in PKU and demonstrate that Phe-induced FGF21 disrupts energy homeostasis via hypothalamic AMPK inhibition.
2025-04-29 | Evaluating adverse events of pegvaliase-pqpz in phenylketonuria treatment: A comprehensive safety assessment.
In 2018, the Food and Drug Administration approved pegvaliase-pqpz as an enzyme replacement therapy to lower blood phenylalanine levels in adults with phenylketonuria. However, its potential side effects have not been fully explored in a real-world setting. This study aimed to examine pegvaliase-pqpz-related adverse events and investigate their associated risk factors to offer important insights into clinical drug applications. We extracted reports on adverse events from Food and Drug Administration Adverse Event Reporting System database from 2018Q2 to 2024Q3. The analyses included demographic data, time-to-onset, sex, age, dosage, reporting personnel, and subgroup signals. The Reporting Odds Ratio, Proportional Reporting Ratio, Bayesian Confidence Propagation Neural Network, and Multi-item Gamma Poisson Shrinker were used for disproportionality analysis. A screening procedure was implemented for designated medical events, focusing on medication-related adverse events. A total of 6264 reports were identified. Arthralgia was the most frequently reported adverse events. Lymphadenopathy, serum sickness, and tunnel vision were identified as unexpected signals. Weibull distribution analysis revealed that the incidence of adverse events decreased over time. Our study provides a deeper insight into the safety profile of pegvaliase-pqpz, thereby aiding healthcare professionals in clinical surveillance and risk identification.
2024-05-06 | Pegvaliase therapy for phenylketonuria: Real-world case series and clinical insights.
The aim of this study is to present a series of case studies on the real-life use of pegvaliase in Italy in managing patients affected by phenylketonuria (PKU) and provide practical insight and support to healthcare professionals currently approaching and facing this novel enzyme substitution therapy. A panel of 11 PKU experts from seven leading Italian treatment centers attended online virtual meetings with the aim of reviewing their clinical and practical experiences with pegvaliase based on occurred cases. In selecting the cases, specific consideration was given to the nationwide representation of the centers involved and to the number of patients with PKU managed. Cases were thoroughly reviewed, with comprehensive discussions enabling the identification of key take-home messages regarding pegvaliase therapy. The panel discussed 18 cases, 11 males and 7 females (age range 17-43 years). At the last follow-up (up to 111 weeks after pegvaliase initiation), 11 out of 18 patients (61%) reached Phe levels below 600 μmol/l. Outcomes varied significantly across cases. All cases underscore the potential of pegvaliase in reducing Phe levels, enhancing the quality of life, and promoting social skills and independence. Additionally, the cases highlight the challenges associated with pegvaliase therapy, including managing adverse events and ensuring patient motivation and adherence. This is the first report about the Italian experience of managing patients affected by PKU with pegvaliase. Given the limited real-world data on the use of pegvaliase in PKU management, this case series offers valuable insights into the practical implementation and management of pegvaliase therapy in this Country. Continued research and data collection will be crucial to confirm and progress with this treatment. Despite potential challenges, pegvaliase therapy represents a substantial promise in managing PKU in Italy. Patient education, personalized treatment approaches, and careful monitoring are important to ensure optimal patient outcomes.
2024-03-11 | Outcomes in 14 live births resulting from Pegvaliase-treated pregnancies in PKU-affected females.
Adults with PKU have difficulty maintaining plasma phenylalanine (Phe) in the range that is safe for neurologic function. Elevated plasma Phe is a risk factor for congenital anomalies and developmental delay in offspring resulting from pregnancies with poor Phe control in women with PKU. Enzyme supplementation with pegvaliase allows adults with PKU to eat an unrestricted diet and have plasma Phe levels in a safe range for pregnancy but pegvaliase has not been approved for use in pregnant females with PKU. We report the results of chart review of 14 living offspring of females affected with PKU who were responsive to pegvaliase and chose to remain on pegvaliase throughout their pregnancy. Fourteen pregnancies (one triplet pregnancy) and their offspring were identified at eight PKU treatment centers and medical records from pregnancy and birth were submitted for this study. Institutional Review Board approval was obtained. Responses to a dataset were provided to a single center and analyzed. Six females and eight males were born without congenital anomalies and all offspring had normal growth parameters. While mothers had preexisting comorbidities, no additional comorbidities were reported in the offspring. Four of eleven infants (excluding triplet pregnancies) were delivered preterm (36%), a higher rate than the general population (12%). A single first trimester (eight weeks) miscarriage in a 40y was not counted in this cohort of 14 live born infants. This retrospective study suggests that pegvaliase is effective at maintaining safe maternal blood Phe levels during pregnancy without deleterious effects on mother or child. A tendency toward premature birth (4/11; 36%) is higher than expected.
2024-03-08 | P001: Outcomes In 14 live births resulting from pegvaliase-treated pregnancies in females with PKU*
Adults with PKU have difficulty maintaining plasma phenylalanine (Phe) in the range which is safe for neurologic function. Elevated plasma Phe is a risk factor for congenital anomalies and developmental delay in offspring collectively known as Maternal PKU Syndrome (MPKU). Features of MPKU include intrauterine growth restriction (IUGR), increased frequency of spontaneous abortion, congenital heart disease, microcephaly, and developmental delays/intellectual disability. When efficacious, enzyme supplementation with pegvaliase allows adults with PKU to eat an unrestricted diet while maintaining plasma Phe levels in a safe range for pregnancy, but pegvaliase has not been approved for use in pregnant women with PKU.
cell therapies
2025-08-13 | Engineered extracellular vesicles in female reproductive and pregnancy-related disorders.
Human reproduction involves a series of highly regulated biological processes, including oogenesis, fertilization, embryo implantation, and maternal-fetal interactions. Disruptions in these processes often result in reproductive disorders, including infertility, recurrent pregnancy loss, preeclampsia, and preterm birth. Currently available therapeutic strategies are often limited by potential harm to gametes, embryos, or fetuses and off-target effects. Engineered extracellular vesicles (EVs) have emerged as a promising approach due to their excellent biocompatibility, stability in circulation, prolonged circulation time, efficient cargo delivery, and potential for cell- or tissue-specific targeting. While still in the preclinical stage, engineered EVs are already reshaping basic research by enabling cell-specific modulation of reproductive and immune processes. By exploring engineering techniques, novel biomarkers, and new targeting approaches in animal models and clinical trials, we provide new insights into how engineered EVs could transform the treatment of female reproductive disorders. We also discuss ongoing challenges, including issues of manufacturing scalability, regulatory approval, and translating preclinical efficacy into clinical settings. With further collaboration between researchers, clinicians, and biotech innovators, engineered EVs could one day become a powerful tool to support women's reproductive health.
2014-01-20 | Adult human liver mesenchymal progenitor cells express phenylalanine hydroxylase
Phenylketonuria (PKU) is one of the most prevalent inherited metabolic diseases and is accountable for a severe encephalopathy by progressive intoxication of the brain by phenylalanine. This results from an ineffective L-phenylalanine hydroxylase enzyme (PAH) due to a mutated phenylalanine hydroxylase (PAH) gene. Neonatal screening programs allow an early dietetic treatment with restrictive phenylalanine intake. This diet prevents most of the neuropsychological disabilities but remains challenging for lifelong compliance. Adult-derived human liver progenitor cells (ADHLPC) are a pool of precursors that can differentiate into hepatocytes. We aim to study PAH expression and PAH activity in a differenciated ADHLPC. ADHLPC were isolated from human hepatocyte primary culture of two different donors and differenciated under specific culture conditions. We demonstrated the high expression of PAH and a large increase of PAH activity in differenciated LPC. The age of the donor, the cellular viability after liver digestion and cryopreservation affects PAH activity. ADHLPC might therefore be considered as a suitable source for cell therapy in PKU.
2012-12-27 | 664: Development of an artificial organ system to prevent maternal PKU syndrome
It is well known that excess Phenylalanine (Phe) is teratogenic; however, the majority of women with Phenylketonuria (PKU) are not in compliance before becoming pregnant resulting in maternal PKU syndrome (mPKU). Our objective was to develop an artificial organ system to process the excess Phe and prevent the occurrence of mPKU. Building on our In vitro studies, we encapsulated 3 different cell lines (293T, HepG2, and WRL68) within polymeric alginate microspheres for studies in PKU model mice. These microspheres prevent the cells from interaction with the host immune system and allow the cells to act as an artificial liver. Microspheres were injected intraperitioneally into C57 PAHenu2 mice. Blood Phe was measured by tandem mass spectrometry prior to injection and at several time points post-treatment. Mice were analyzed for changes in phenotype after injection including restoration of black coat color and improved fertility. Differences in Phe between types of cell lines encapsulated, number of cells injected, and mouse gender were analyzed. We observed up to a 40% reduction in Phe. Differences in treatment results by gender were also observed. Phe was found to be 40% lower in treated male mice compared to treated female mice. At 28 days post-encapsulation, the percent of cell viability was 82.79 ± 1.49. Phenotypic change was observed with 4 × 10^6 cells HepG2 cells injected. These changes in phenotype included a change from grey to black fur color and the birth of a litter after treatment. Our in vivo results demonstrate that our artificial organ system is feasible. Because we observed differences between males and females, future studies will exclusively use female PKU model mice and test the ability of microspheres to prevent mPKU. Our proof of principal in vivo assays demonstrates that an artificial organ system can reduce Phe and result in phenotypic changes in mice.
2011-01-05 | 828: Treatment of maternal PKU: safety and efficacy of a novel engineered metabolic system
Elevated phenylalanine (Phe) levels in women with phenylketonuria (PKU) during pregnancy can cause teratogenic effects including mental retardation, cardiac defects, and facial dysmorphism. Studies have shown that these outcomes can be significantly improved if maternal Phe levels are within normal limits prior to pregnancy or early in gestation. However, achieving low levels of Phe during pregnancy can be difficult. We have developed an engineered metabolic pathway to process excess Phe. Our primary objective was to determine the safety of our approach in a mouse model of PKU. Our second objective was to obtain baseline efficacy data for future optimization. A stable cell line overexpressing phenylalanine hydroxylase was encapsulated within semi-permeable alginate microspheres. These encapsulated cells were injected intraperitoneally into PAHenu2 mice (n=5). Mice were observed for signs of illness and were bled at days 0, 1, and 28. Blood was assayed in duplicate by tandem mass spectroscopy to determine the Phe concentration. To control for the effects of the alginate microspheres, background strain, heterozygous, and homozygous mice were also injected with empty microspheres. All of the mice survived the encapsulated cell injection and remained alive at 28 days post-injection. Mice treated with empty microspheres did not exhibit any significant changes in Phe. In PKU model mice, Phe was significantly reduced by 15% on Day 1 and remained at this level on Day 28. Our approach was well-tolerated and safe in an animal model. While further optimization of our system is necessary to reduce Phe to normal levels, we were encouraged by the ability of the encapsulated cells to significantly reduce by Phe in vivo and for this reduction to be maintained for 28 days. After further optimization, we will insert encapsulated cells or empty spheres into female PKU model mice before breeding, and at 7, and 14 days post conception and determine whether there are cognitive and physical differences between pups born to treated and untreated dams.
2009-07-25 | Cell encapsulation as a potential nondietary therapy for maternal phenylketonuria
Objective The objective of this work was to determine whether cells overexpressing phenylalanine (Phe) hydroxylase (PAH) can significantly reduce Phe in vitro for potential use as a therapy for preventing maternal phenylketonuria. Study Design Human 293T and WRL68 cell lines were transiently and stably transfected to overexpress PAH. Cells were encapsulated within microspheres of sodium alginate. Timed measurements of Phe in media were performed using tandem mass spectrometry. Results Both nonencapsulated and encapsulated transiently transfected cells overexpressing PAH significantly reduced the Phe concentration in media by approximately 50% in comparison to mock-transfected cells. Cell line clones stably expressing PAH significantly decreased the Phe concentration in the media by up to 85% compared with media alone. Conclusion Both unencapsulated and encapsulated cells overexpressing PAH significantly reduce Phe in vitro. Studies using phenylketonuria model mice will be important in determining the ability of our therapy to prevent the teratogenic effects of elevated maternal Phe in maternal phenylketonuria. The objective of this work was to determine whether cells overexpressing phenylalanine (Phe) hydroxylase (PAH) can significantly reduce Phe in vitro for potential use as a therapy for preventing maternal phenylketonuria. Human 293T and WRL68 cell lines were transiently and stably transfected to overexpress PAH. Cells were encapsulated within microspheres of sodium alginate. Timed measurements of Phe in media were performed using tandem mass spectrometry. Both nonencapsulated and encapsulated transiently transfected cells overexpressing PAH significantly reduced the Phe concentration in media by approximately 50% in comparison to mock-transfected cells. Cell line clones stably expressing PAH significantly decreased the Phe concentration in the media by up to 85% compared with media alone. Both unencapsulated and encapsulated cells overexpressing PAH significantly reduce Phe in vitro. Studies using phenylketonuria model mice will be important in determining the ability of our therapy to prevent the teratogenic effects of elevated maternal Phe in maternal phenylketonuria.
gene therapies
2023-10-11 | Preimplantation Genetic Testing (PGT) for Non-Lethal Correctable Conditions Through Population Carrier Screening
An increasing number of patients without family history of genetic disease are referred for preimplantation genetic testing of monogenic disorders (PGT-M) after ascertainment through population (expanded) carrier screening (ECS), increasing the proportion of non-lethal correctable genetic conditions in PGT-M indication profile. The aim of this paper is to analyze the current dynamics of these changes in our PGT-M series, which is the world’s largest PGT-M experience. This was analyzed using our PGT-M for two prevalent examples of non-lethal correctable genetic conditions, phenylketonuria (PKU) and hereditary hearing loss (HHL) during the period of 2005-2022. During this period, 91 PGT-M cycles were performed for PKU, involving testing for 108 mutant alleles in PAH gene, and 262 PGT-M cycles for HHL, involving testing for up to 300 mutations in 13 different genes causing hearing loss. PGT-M for these non-lethal correctable conditions resulted in the birth of 54 babies free of PKU and 134 children free of HHL. Dynamics of PGT-M referrals during this period of 17 years demonstrated the shift from retrospective to prospective application of PGT-M for both conditions, with 87% of prospective PGT-M performed for PKU, and 82% for HHL, performed for couples who had no affected relatives with PKU or HHL. These at-risk couples were ascertained through ECS in the last decade, making it possible to provide the option of prospective PGT-M for these non-lethal correctable conditions.
2023-02-27 | Applying the CRISPR/Cas9 for Treating Human and Animal Diseases – Comprehensive Review
Abstract Recently, genome editing tools have been extensively used in many biomedical sciences. The gene editing system is applied to modify the dnA sequences in the cellular system to comprehend their physiological response. A developing genome editing technology like clustered regularly short palindromic repeats (CRISPR) is widely used in medical sciences. CRISPR and CRISPR-associated protein 9 (CRISPR/Cas9) system is being exploited to edit any DNA mutations related to inherited ailments to investigate in animals ( in vivo ) and cell lines ( in vitro ). Remarkably, CRISPR/Cas9 could be employed to examine treatments of many human genetic diseases such as cystic fibrosis, tyrosinemia, phenylketonuria, muscular dystrophy, Parkinson’s disease, retinoschisis, hemophilia, β-thalassemia and atherosclerosis. Moreover, CRISPR/Cas9 was used for disease resistance such as tuberculosis, Johne’s diseases, chronic enteritis, and brucellosis in animals. Finally, this review discusses existing progress in treating hereditary diseases using CRISPR/Cas9 technology and the high points accompanying obstacles.
2017-10-19 | Blood phenylalanine reduction corrects CNS dopamine and serotonin deficiencies and partially improves behavioral performance in adult phenylketonuric mice
Central nervous system (CNS) deficiencies of the monoamine neurotransmitters dopamine and serotonin have been implicated in the pathophysiology of neuropsychiatric dysfunction in human phenylketonuria (PKU). In this study, we confirmed the occurrence of brain dopamine and serotonin deficiencies in association with severe behavioral alterations and cognitive impairments in hyperphenylalaninemic C57BL/6-Pahenu2/enu2 mice, a model of human PKU. Phenylalanine-reducing treatments, including either dietary phenylalanine restriction or liver-directed gene therapy, initiated during adulthood were associated with increased brain monoamine content along with improvements in nesting behavior but without a change in the severe cognitive deficits exhibited by these mice. At euthanasia, there was in Pahenu2/enu2 brain a significant reduction in the protein abundance and maximally stimulated activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase 2 (TPH2), the rate limiting enzymes catalyzing neuronal dopamine and serotonin synthesis respectively, in comparison to levels seen in wild type brain. Phenylalanine-reducing treatments initiated during adulthood did not affect brain TH or TPH2 content or maximal activity. Despite this apparent fixed deficit in striatal TH and TPH2 activities, initiation of phenylalanine-reducing treatments yielded substantial correction of brain monoamine neurotransmitter content, suggesting that phenylalanine-mediated competitive inhibition of already constitutively reduced TH and TPH2 activities is the primary cause of brain monoamine deficiency in Pahenu2 mouse brain. We propose that CNS monoamine deficiency may be the cause of the partially reversible adverse behavioral effects associated with chronic HPA in Pahenu2 mice, but that phenylalanine-reducing treatments initiated during adulthood are unable to correct the neuropathology and attendant cognitive deficits that develop during juvenile life in late-treated Pahenu2/enu2 mice.
2017-04-20 | Low-Dose Gene Therapy for Murine PKU Using Episomal Naked DNA Vectors Expressing PAH from Its Endogenous Liver Promoter
Limited duration of transgene expression, insertional mutagenesis, and size limitations for transgene cassettes pose challenges and risk factors for many gene therapy vectors. Here, we report on physiological expression of liver phenylalanine hydroxylase (PAH) by delivery of naked DNA/minicircle (MC)-based vectors for correction of homozygous enu2 mice, a model of human phenylketonuria (PKU). Because MC vectors lack a defined size limit, we constructed a MC vector expressing a codon-optimized murine Pah cDNA that includes a truncated intron and is under the transcriptional control of a 3.6-kb native Pah promoter/enhancer sequence. This vector, delivered via hydrodynamic injection, yielded therapeutic liver PAH activity and sustained correction of blood phenylalanine comparable to viral or synthetic liver promoters. Therapeutic efficacy was seen with vector copy numbers of <1 vector genome per diploid hepatocyte genome and was achieved at a vector dose that was significantly lowered. Partial hepatectomy and subsequent liver regeneration was associated with >95% loss of vector genomes and PAH activity in liver, demonstrating that MC vectors had not integrated into the liver genome. In conclusion, MC vectors, which do not have a defined size-limitation, offer a favorable safety profile for hepatic gene therapy due to their non-integration in combination with native promoters. Limited duration of transgene expression, insertional mutagenesis, and size limitations for transgene cassettes pose challenges and risk factors for many gene therapy vectors. Here, we report on physiological expression of liver phenylalanine hydroxylase (PAH) by delivery of naked DNA/minicircle (MC)-based vectors for correction of homozygous enu2 mice, a model of human phenylketonuria (PKU). Because MC vectors lack a defined size limit, we constructed a MC vector expressing a codon-optimized murine Pah cDNA that includes a truncated intron and is under the transcriptional control of a 3.6-kb native Pah promoter/enhancer sequence. This vector, delivered via hydrodynamic injection, yielded therapeutic liver PAH activity and sustained correction of blood phenylalanine comparable to viral or synthetic liver promoters. Therapeutic efficacy was seen with vector copy numbers of <1 vector genome per diploid hepatocyte genome and was achieved at a vector dose that was significantly lowered. Partial hepatectomy and subsequent liver regeneration was associated with >95% loss of vector genomes and PAH activity in liver, demonstrating that MC vectors had not integrated into the liver genome. In conclusion, MC vectors, which do not have a defined size-limitation, offer a favorable safety profile for hepatic gene therapy due to their non-integration in combination with native promoters.
2016-10-27 | CRISPR RNA-guided FokI nucleases repair a PAH variant in a phenylketonuria model
The CRISPR/Cas9 system is a recently developed genome editing technique. In this study, we used a modified CRISPR system, which employs the fusion of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9) to correct the most common variant (allele frequency 21.4%) in the phenylalanine hydroxylase (PAH) gene - c.1222C>T (p.Arg408Trp) - as an approach toward curing phenylketonuria (PKU). PKU is the most common inherited diseases in amino acid metabolism. It leads to severe neurological and neuropsychological symptoms if untreated or late diagnosed. Correction of the disease-causing variants could rescue residual PAH activity and restore normal function. Co-expression of a single guide RNA plasmid, a FokI-dCas9-zsGreen1 plasmid, and the presence of a single-stranded oligodeoxynucleotide in PAH_c.1222C>T COS-7 cells - an in vitro model for PKU - corrected the PAH variant and restored PAH activity. Also in this system, the HDR enhancer RS-1 improved correction efficiency. This proof-of-concept indicates the potential of the FokI-dCas9 system for precision medicine, in particular for targeting PKU and other monogenic metabolic diseases.
small molecules
2026-03-11 | [Linking tetrahydrobiopterin depletion to ferroptosis: A novel mechanism of neurological injury in Hyperphenylalaninemia].
Hyperphenylalaninemia (HPA) is an inherited metabolic disorder caused by deficiency of phenylalanine hydroxylase, characterized by significantly elevated phenylalanine levels. Conventional mechanisms, such as neurotransmitter deficiency and dysmyelination, fail to fully explain the progressive neurological damages associated with HPA. Meanwhile, ferroptosis, an emerging form of iron-dependent regulated cell death, has proven to play an important role in neurodegenerative diseases. We hereby propose a hypothesis that tetrahydrobiopterin (BH4) depletion in HPA may lead to the collapse of intracellular antioxidant defenses. This process could induce ferroptosis, thereby serving as a pivotal mechanism underlying HPA-related neurological injury. This review has systematically summarized the pathological mechanisms of HPA, the biological features of ferroptosis, and the bridging role of BH4 between them, thereby establishing a novel "HPA-BH4-ferroptosis" theoretical framework and providing a rationale for developing new therapeutic strategies targeting ferroptosis.
2026-01-28 | The First 1000 Days of PKU: A Narrative Review of Maternal PKU and Early Life Management After Positive Newborn Screening.
Background/Objectives: The first 1000 days of life represent a critical window for growth and neurodevelopment, during which nutrition strongly influences brain development and metabolic programming. In phenylketonuria (PKU), dietary management is essential to prevent neurological impairment and later-life risk of non-communicable diseases (NCDs). This review examines current evidence on PKU from pregnancy through complementary feeding, highlighting the impact of nutritional strategies on neurodevelopmental and metabolic outcomes. Methods: This narrative review, following PRISMA guidelines, used a systematic search of PubMed and Scopus with defined PICO questions. Original research, reviews, and guidelines on PKU nutrition during the first 1000 days were included, emphasizing neurological and metabolic outcomes. Results: Articles addressed prenatal and postnatal factors in PKU. Optimised metabolic control in women with PKU is critical to prevent maternal PKU syndrome, reducing risks of miscarriage, congenital heart defects, microcephaly, and neurocognitive impairment. Pre-conception dietary management, frequent blood Phe monitoring, supplementation with Phe-free protein substitutes (PSs), micronutrients, and emerging pharmacological therapies support maternal and foetal health. Following newborn screening, early dietary treatment in infants with PKU maintains plasma Phe within safe ranges, promoting growth and neurodevelopment. Breastfeeding, combined with Phe-free infant PSs, is feasible, and complementary feeding should be introduced carefully. Frequent monitoring and tailored dietary adjustments, including second-stage PSs, support metabolic control, while data on gut microbiota remain limited. Conclusions: Early multidisciplinary interventions are crucial to optimise metabolic and neurodevelopmental outcomes during this window of opportunity. Further research is needed to address remaining gaps and optimise PKU management across the first 1000 days.
2025-11-07 | Relationship Between Gut Microbiota and Phenylalanine Levels: A Mendelian Randomization Study.
The specific members of the gut microbiota linked to phenylketonuria remain to be identified. This study aimed to assess the association between gut microbiota on phenylalanine (Phe) levels using a two-sample Mendelian randomization (MR) approach. Summary statistics from genome-wide association studies (GWAS) related to individual gut microbiota were obtained from the MiBioGen Global Consortium database. The data set of Phe levels was derived from GWAS summary datasets. Inverse variance weighting (IVW) served as the primary method to infer the causal relationship between gut microbiota and Phe levels. Additional pleiotropy and heterogeneity tests were conducted to evaluate the reliability of the findings. The Family XIII AD3011 group had a protective effect on Phe levels (OR = 0.962, 95% CI: 0.942-0.982, p < 0.001), and these associations remained significant after FDR correction (adjusted p-value = 0.027). There was no evidence of notable heterogeneity and horizontal pleiotropy among the instrumental variables. Our data indicate that Family XIII AD3011 group is associated with reduced Phe levels, highlighting a potential link between gut microbiota and Phe levels. Although MR analysis supports a causal relationship, it may not precisely estimate the effect size, necessitating further studies to validate these findings and quantify the association.
2025-01-31 | Transitioning of protein substitutes in patients with phenylketonuria: a pilot study
Introduction In phenylketonuria (PKU), there is limited information about transitioning between protein substitutes and the influencing factors, particularly in young children. This pilot study assessed the stepwise transition from second to third-stage protein substitutes in children with PKU, aged 3–5 years. Methods Demographics, child behavior, maternal anxiety, and food neophobia scores were collected at baseline, mid-transition, and final assessment. Blood phenylalanine (Phe) was collected from 6 months pre-baseline to post-final assessment. Results Twelve children ( n = 4 males, 33%, median age 3.2 years) participated. Sixty-seven percent ( n = 8) transitioned to liquid amino acid-based protein substitute and 33% ( n = 4) to glycomacropeptide (cGMP) powder. Forty-two percent ( n = 5/12) had a smooth transition (Group 1, median 3.5 months), while the remaining faced difficulty ( n = 3, 25%, Group 2), or failed full transition ( n = 4, 33%, Group 3). In Groups 2 and 3, caregivers failed to follow instructions, demonstrating inconsistencies and child resistance. Group 2 children had significantly higher blood Phe levels (above 360 μmol/L), that was significantly higher than Groups 1 and 3 ( p < 0.01), with Groups 1 and 3 maintaining blood Phe within target ( p < 0.01). Higher maternal education and nursery/school attendance significantly influenced transition success ( p < 0.05). No significant differences were found in child neophobia, maternal anxiety, or child behavior ( p > 0.05). Mothers generally reported satisfaction with the stepwise transition process. Conclusion A stepwise transition to third-stage protein substitutes in PKU is effective, but is dependent on child metabolic control, parental education, and nursery/school support.
2024-09-06 | Long-term safety of sapropterin in paediatric and adult individuals with phenylalanine hydroxylase deficiency: Final results of the Kuvan® Adult Maternal Paediatric European Registry multinational observational study.
Phenylketonuria is a rare inherited disorder that disrupts the metabolism of phenylalanine (Phe) to tyrosine by phenylalanine hydroxylase (PAH). Sapropterin dihydrochloride (Kuvan®) is approved for use in Europe to reduce blood Phe levels and improve Phe tolerance in sapropterin-responsive individuals. KAMPER (NCT01016392) is an observational, multinational registry assessing long-term safety and efficacy of sapropterin. Five hundred and seventy-six participants with PAH deficiency were enrolled from nine European countries (69 sites; December 2009-May 2016). Participants were aged <4 years (n = 11), 4 to <12 years (n = 329), 12 to <18 years (n = 141), and ≥18 years (n = 95) at enrolment. Overall, 401 (69.6%) participants experienced a total of 1960 adverse events; 61 events in 42 participants were serious, and two were considered sapropterin-related by the investigator. Mean (standard deviation) actual dietary Phe intake increased from baseline across all age groups: 957 (799) mg/day to a maximum of 1959 (1121) mg/day over a total study period of 11 years. Most participants exhibited an increase in Phe tolerance while blood Phe levels remained in the target range for their age (120-360 μmol/L for <12 years; 120-600 μmol/L for ≥12 years). Most participants exhibited normal growth for height, weight, and body mass index. No additional safety concerns were identified. As an observational study, limitations include variability in routine care practices and inconsistent availability of data. Long-term sapropterin use demonstrates a favourable safety profile in real-world settings and increases Phe tolerance in participants with PAH deficiency while maintaining blood Phe levels in the target ranges.
proteins
2026-06-15 | Increased Brown Adipose Tissue Thermogenesis in Phenylketonuria.
Phenylketonuria (PKU), the most common autosomal‑recessive disorder of amino acid metabolism, is characterized by neurological impairment and systemic metabolic alterations caused by chronically elevated phenylalanine (Phe) levels. PKU patients have long been reported to display reduced metabolic rate and impaired thermoregulation, yet the role of brown adipose tissue (BAT) in this condition remains unknown. Here, noninvasive infrared thermography was used to assess BAT activity in a cohort primarily comprising children and adolescents including controls, mild hyperphenylalaninemia (MHPA), and PKU patients, while circulating metabolic and hormonal parameters were analyzed for associations with BAT temperature. Despite overall normothermia, individuals with PKU exhibited higher BAT temperature than both control and MHPA patients, which correlated with circulating fibroblast growth factor 21 (FGF21) and thyroid hormones. To gain mechanistic insight, rats and mice were centrally treated with FGF21, reproducing the BAT thermogenic phenotype along with decreased hypothalamic AMP‑activated protein kinase (AMPK) activity and increased sympathetic drive to BAT. Consistently, analysis of public single‑cell RNA‑sequencing data revealed convergent expression of AMPK, thyroid hormone receptor, and FGF21 receptor signaling in specific hypothalamic neuronal populations. These findings reveal enhanced BAT thermogenesis in PKU and demonstrate that Phe-induced FGF21 disrupts energy homeostasis via hypothalamic AMPK inhibition.
2025-04-29 | Evaluating adverse events of pegvaliase-pqpz in phenylketonuria treatment: A comprehensive safety assessment.
In 2018, the Food and Drug Administration approved pegvaliase-pqpz as an enzyme replacement therapy to lower blood phenylalanine levels in adults with phenylketonuria. However, its potential side effects have not been fully explored in a real-world setting. This study aimed to examine pegvaliase-pqpz-related adverse events and investigate their associated risk factors to offer important insights into clinical drug applications. We extracted reports on adverse events from Food and Drug Administration Adverse Event Reporting System database from 2018Q2 to 2024Q3. The analyses included demographic data, time-to-onset, sex, age, dosage, reporting personnel, and subgroup signals. The Reporting Odds Ratio, Proportional Reporting Ratio, Bayesian Confidence Propagation Neural Network, and Multi-item Gamma Poisson Shrinker were used for disproportionality analysis. A screening procedure was implemented for designated medical events, focusing on medication-related adverse events. A total of 6264 reports were identified. Arthralgia was the most frequently reported adverse events. Lymphadenopathy, serum sickness, and tunnel vision were identified as unexpected signals. Weibull distribution analysis revealed that the incidence of adverse events decreased over time. Our study provides a deeper insight into the safety profile of pegvaliase-pqpz, thereby aiding healthcare professionals in clinical surveillance and risk identification.
2024-05-06 | Pegvaliase therapy for phenylketonuria: Real-world case series and clinical insights.
The aim of this study is to present a series of case studies on the real-life use of pegvaliase in Italy in managing patients affected by phenylketonuria (PKU) and provide practical insight and support to healthcare professionals currently approaching and facing this novel enzyme substitution therapy. A panel of 11 PKU experts from seven leading Italian treatment centers attended online virtual meetings with the aim of reviewing their clinical and practical experiences with pegvaliase based on occurred cases. In selecting the cases, specific consideration was given to the nationwide representation of the centers involved and to the number of patients with PKU managed. Cases were thoroughly reviewed, with comprehensive discussions enabling the identification of key take-home messages regarding pegvaliase therapy. The panel discussed 18 cases, 11 males and 7 females (age range 17-43 years). At the last follow-up (up to 111 weeks after pegvaliase initiation), 11 out of 18 patients (61%) reached Phe levels below 600 μmol/l. Outcomes varied significantly across cases. All cases underscore the potential of pegvaliase in reducing Phe levels, enhancing the quality of life, and promoting social skills and independence. Additionally, the cases highlight the challenges associated with pegvaliase therapy, including managing adverse events and ensuring patient motivation and adherence. This is the first report about the Italian experience of managing patients affected by PKU with pegvaliase. Given the limited real-world data on the use of pegvaliase in PKU management, this case series offers valuable insights into the practical implementation and management of pegvaliase therapy in this Country. Continued research and data collection will be crucial to confirm and progress with this treatment. Despite potential challenges, pegvaliase therapy represents a substantial promise in managing PKU in Italy. Patient education, personalized treatment approaches, and careful monitoring are important to ensure optimal patient outcomes.
2024-03-11 | Outcomes in 14 live births resulting from Pegvaliase-treated pregnancies in PKU-affected females.
Adults with PKU have difficulty maintaining plasma phenylalanine (Phe) in the range that is safe for neurologic function. Elevated plasma Phe is a risk factor for congenital anomalies and developmental delay in offspring resulting from pregnancies with poor Phe control in women with PKU. Enzyme supplementation with pegvaliase allows adults with PKU to eat an unrestricted diet and have plasma Phe levels in a safe range for pregnancy but pegvaliase has not been approved for use in pregnant females with PKU. We report the results of chart review of 14 living offspring of females affected with PKU who were responsive to pegvaliase and chose to remain on pegvaliase throughout their pregnancy. Fourteen pregnancies (one triplet pregnancy) and their offspring were identified at eight PKU treatment centers and medical records from pregnancy and birth were submitted for this study. Institutional Review Board approval was obtained. Responses to a dataset were provided to a single center and analyzed. Six females and eight males were born without congenital anomalies and all offspring had normal growth parameters. While mothers had preexisting comorbidities, no additional comorbidities were reported in the offspring. Four of eleven infants (excluding triplet pregnancies) were delivered preterm (36%), a higher rate than the general population (12%). A single first trimester (eight weeks) miscarriage in a 40y was not counted in this cohort of 14 live born infants. This retrospective study suggests that pegvaliase is effective at maintaining safe maternal blood Phe levels during pregnancy without deleterious effects on mother or child. A tendency toward premature birth (4/11; 36%) is higher than expected.
2024-03-08 | P001: Outcomes In 14 live births resulting from pegvaliase-treated pregnancies in females with PKU*
Adults with PKU have difficulty maintaining plasma phenylalanine (Phe) in the range which is safe for neurologic function. Elevated plasma Phe is a risk factor for congenital anomalies and developmental delay in offspring collectively known as Maternal PKU Syndrome (MPKU). Features of MPKU include intrauterine growth restriction (IUGR), increased frequency of spontaneous abortion, congenital heart disease, microcephaly, and developmental delays/intellectual disability. When efficacious, enzyme supplementation with pegvaliase allows adults with PKU to eat an unrestricted diet while maintaining plasma Phe levels in a safe range for pregnancy, but pegvaliase has not been approved for use in pregnant women with PKU.
cell therapies
2025-08-13 | Engineered extracellular vesicles in female reproductive and pregnancy-related disorders.
Human reproduction involves a series of highly regulated biological processes, including oogenesis, fertilization, embryo implantation, and maternal-fetal interactions. Disruptions in these processes often result in reproductive disorders, including infertility, recurrent pregnancy loss, preeclampsia, and preterm birth. Currently available therapeutic strategies are often limited by potential harm to gametes, embryos, or fetuses and off-target effects. Engineered extracellular vesicles (EVs) have emerged as a promising approach due to their excellent biocompatibility, stability in circulation, prolonged circulation time, efficient cargo delivery, and potential for cell- or tissue-specific targeting. While still in the preclinical stage, engineered EVs are already reshaping basic research by enabling cell-specific modulation of reproductive and immune processes. By exploring engineering techniques, novel biomarkers, and new targeting approaches in animal models and clinical trials, we provide new insights into how engineered EVs could transform the treatment of female reproductive disorders. We also discuss ongoing challenges, including issues of manufacturing scalability, regulatory approval, and translating preclinical efficacy into clinical settings. With further collaboration between researchers, clinicians, and biotech innovators, engineered EVs could one day become a powerful tool to support women's reproductive health.
2014-01-20 | Adult human liver mesenchymal progenitor cells express phenylalanine hydroxylase
Phenylketonuria (PKU) is one of the most prevalent inherited metabolic diseases and is accountable for a severe encephalopathy by progressive intoxication of the brain by phenylalanine. This results from an ineffective L-phenylalanine hydroxylase enzyme (PAH) due to a mutated phenylalanine hydroxylase (PAH) gene. Neonatal screening programs allow an early dietetic treatment with restrictive phenylalanine intake. This diet prevents most of the neuropsychological disabilities but remains challenging for lifelong compliance. Adult-derived human liver progenitor cells (ADHLPC) are a pool of precursors that can differentiate into hepatocytes. We aim to study PAH expression and PAH activity in a differenciated ADHLPC. ADHLPC were isolated from human hepatocyte primary culture of two different donors and differenciated under specific culture conditions. We demonstrated the high expression of PAH and a large increase of PAH activity in differenciated LPC. The age of the donor, the cellular viability after liver digestion and cryopreservation affects PAH activity. ADHLPC might therefore be considered as a suitable source for cell therapy in PKU.
2012-12-27 | 664: Development of an artificial organ system to prevent maternal PKU syndrome
It is well known that excess Phenylalanine (Phe) is teratogenic; however, the majority of women with Phenylketonuria (PKU) are not in compliance before becoming pregnant resulting in maternal PKU syndrome (mPKU). Our objective was to develop an artificial organ system to process the excess Phe and prevent the occurrence of mPKU. Building on our In vitro studies, we encapsulated 3 different cell lines (293T, HepG2, and WRL68) within polymeric alginate microspheres for studies in PKU model mice. These microspheres prevent the cells from interaction with the host immune system and allow the cells to act as an artificial liver. Microspheres were injected intraperitioneally into C57 PAHenu2 mice. Blood Phe was measured by tandem mass spectrometry prior to injection and at several time points post-treatment. Mice were analyzed for changes in phenotype after injection including restoration of black coat color and improved fertility. Differences in Phe between types of cell lines encapsulated, number of cells injected, and mouse gender were analyzed. We observed up to a 40% reduction in Phe. Differences in treatment results by gender were also observed. Phe was found to be 40% lower in treated male mice compared to treated female mice. At 28 days post-encapsulation, the percent of cell viability was 82.79 ± 1.49. Phenotypic change was observed with 4 × 10^6 cells HepG2 cells injected. These changes in phenotype included a change from grey to black fur color and the birth of a litter after treatment. Our in vivo results demonstrate that our artificial organ system is feasible. Because we observed differences between males and females, future studies will exclusively use female PKU model mice and test the ability of microspheres to prevent mPKU. Our proof of principal in vivo assays demonstrates that an artificial organ system can reduce Phe and result in phenotypic changes in mice.
2011-01-05 | 828: Treatment of maternal PKU: safety and efficacy of a novel engineered metabolic system
Elevated phenylalanine (Phe) levels in women with phenylketonuria (PKU) during pregnancy can cause teratogenic effects including mental retardation, cardiac defects, and facial dysmorphism. Studies have shown that these outcomes can be significantly improved if maternal Phe levels are within normal limits prior to pregnancy or early in gestation. However, achieving low levels of Phe during pregnancy can be difficult. We have developed an engineered metabolic pathway to process excess Phe. Our primary objective was to determine the safety of our approach in a mouse model of PKU. Our second objective was to obtain baseline efficacy data for future optimization. A stable cell line overexpressing phenylalanine hydroxylase was encapsulated within semi-permeable alginate microspheres. These encapsulated cells were injected intraperitoneally into PAHenu2 mice (n=5). Mice were observed for signs of illness and were bled at days 0, 1, and 28. Blood was assayed in duplicate by tandem mass spectroscopy to determine the Phe concentration. To control for the effects of the alginate microspheres, background strain, heterozygous, and homozygous mice were also injected with empty microspheres. All of the mice survived the encapsulated cell injection and remained alive at 28 days post-injection. Mice treated with empty microspheres did not exhibit any significant changes in Phe. In PKU model mice, Phe was significantly reduced by 15% on Day 1 and remained at this level on Day 28. Our approach was well-tolerated and safe in an animal model. While further optimization of our system is necessary to reduce Phe to normal levels, we were encouraged by the ability of the encapsulated cells to significantly reduce by Phe in vivo and for this reduction to be maintained for 28 days. After further optimization, we will insert encapsulated cells or empty spheres into female PKU model mice before breeding, and at 7, and 14 days post conception and determine whether there are cognitive and physical differences between pups born to treated and untreated dams.
2009-07-25 | Cell encapsulation as a potential nondietary therapy for maternal phenylketonuria
Objective The objective of this work was to determine whether cells overexpressing phenylalanine (Phe) hydroxylase (PAH) can significantly reduce Phe in vitro for potential use as a therapy for preventing maternal phenylketonuria. Study Design Human 293T and WRL68 cell lines were transiently and stably transfected to overexpress PAH. Cells were encapsulated within microspheres of sodium alginate. Timed measurements of Phe in media were performed using tandem mass spectrometry. Results Both nonencapsulated and encapsulated transiently transfected cells overexpressing PAH significantly reduced the Phe concentration in media by approximately 50% in comparison to mock-transfected cells. Cell line clones stably expressing PAH significantly decreased the Phe concentration in the media by up to 85% compared with media alone. Conclusion Both unencapsulated and encapsulated cells overexpressing PAH significantly reduce Phe in vitro. Studies using phenylketonuria model mice will be important in determining the ability of our therapy to prevent the teratogenic effects of elevated maternal Phe in maternal phenylketonuria. The objective of this work was to determine whether cells overexpressing phenylalanine (Phe) hydroxylase (PAH) can significantly reduce Phe in vitro for potential use as a therapy for preventing maternal phenylketonuria. Human 293T and WRL68 cell lines were transiently and stably transfected to overexpress PAH. Cells were encapsulated within microspheres of sodium alginate. Timed measurements of Phe in media were performed using tandem mass spectrometry. Both nonencapsulated and encapsulated transiently transfected cells overexpressing PAH significantly reduced the Phe concentration in media by approximately 50% in comparison to mock-transfected cells. Cell line clones stably expressing PAH significantly decreased the Phe concentration in the media by up to 85% compared with media alone. Both unencapsulated and encapsulated cells overexpressing PAH significantly reduce Phe in vitro. Studies using phenylketonuria model mice will be important in determining the ability of our therapy to prevent the teratogenic effects of elevated maternal Phe in maternal phenylketonuria.
gene therapies
2023-10-11 | Preimplantation Genetic Testing (PGT) for Non-Lethal Correctable Conditions Through Population Carrier Screening
An increasing number of patients without family history of genetic disease are referred for preimplantation genetic testing of monogenic disorders (PGT-M) after ascertainment through population (expanded) carrier screening (ECS), increasing the proportion of non-lethal correctable genetic conditions in PGT-M indication profile. The aim of this paper is to analyze the current dynamics of these changes in our PGT-M series, which is the world’s largest PGT-M experience. This was analyzed using our PGT-M for two prevalent examples of non-lethal correctable genetic conditions, phenylketonuria (PKU) and hereditary hearing loss (HHL) during the period of 2005-2022. During this period, 91 PGT-M cycles were performed for PKU, involving testing for 108 mutant alleles in PAH gene, and 262 PGT-M cycles for HHL, involving testing for up to 300 mutations in 13 different genes causing hearing loss. PGT-M for these non-lethal correctable conditions resulted in the birth of 54 babies free of PKU and 134 children free of HHL. Dynamics of PGT-M referrals during this period of 17 years demonstrated the shift from retrospective to prospective application of PGT-M for both conditions, with 87% of prospective PGT-M performed for PKU, and 82% for HHL, performed for couples who had no affected relatives with PKU or HHL. These at-risk couples were ascertained through ECS in the last decade, making it possible to provide the option of prospective PGT-M for these non-lethal correctable conditions.
2023-02-27 | Applying the CRISPR/Cas9 for Treating Human and Animal Diseases – Comprehensive Review
Abstract Recently, genome editing tools have been extensively used in many biomedical sciences. The gene editing system is applied to modify the dnA sequences in the cellular system to comprehend their physiological response. A developing genome editing technology like clustered regularly short palindromic repeats (CRISPR) is widely used in medical sciences. CRISPR and CRISPR-associated protein 9 (CRISPR/Cas9) system is being exploited to edit any DNA mutations related to inherited ailments to investigate in animals ( in vivo ) and cell lines ( in vitro ). Remarkably, CRISPR/Cas9 could be employed to examine treatments of many human genetic diseases such as cystic fibrosis, tyrosinemia, phenylketonuria, muscular dystrophy, Parkinson’s disease, retinoschisis, hemophilia, β-thalassemia and atherosclerosis. Moreover, CRISPR/Cas9 was used for disease resistance such as tuberculosis, Johne’s diseases, chronic enteritis, and brucellosis in animals. Finally, this review discusses existing progress in treating hereditary diseases using CRISPR/Cas9 technology and the high points accompanying obstacles.
2017-10-19 | Blood phenylalanine reduction corrects CNS dopamine and serotonin deficiencies and partially improves behavioral performance in adult phenylketonuric mice
Central nervous system (CNS) deficiencies of the monoamine neurotransmitters dopamine and serotonin have been implicated in the pathophysiology of neuropsychiatric dysfunction in human phenylketonuria (PKU). In this study, we confirmed the occurrence of brain dopamine and serotonin deficiencies in association with severe behavioral alterations and cognitive impairments in hyperphenylalaninemic C57BL/6-Pahenu2/enu2 mice, a model of human PKU. Phenylalanine-reducing treatments, including either dietary phenylalanine restriction or liver-directed gene therapy, initiated during adulthood were associated with increased brain monoamine content along with improvements in nesting behavior but without a change in the severe cognitive deficits exhibited by these mice. At euthanasia, there was in Pahenu2/enu2 brain a significant reduction in the protein abundance and maximally stimulated activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase 2 (TPH2), the rate limiting enzymes catalyzing neuronal dopamine and serotonin synthesis respectively, in comparison to levels seen in wild type brain. Phenylalanine-reducing treatments initiated during adulthood did not affect brain TH or TPH2 content or maximal activity. Despite this apparent fixed deficit in striatal TH and TPH2 activities, initiation of phenylalanine-reducing treatments yielded substantial correction of brain monoamine neurotransmitter content, suggesting that phenylalanine-mediated competitive inhibition of already constitutively reduced TH and TPH2 activities is the primary cause of brain monoamine deficiency in Pahenu2 mouse brain. We propose that CNS monoamine deficiency may be the cause of the partially reversible adverse behavioral effects associated with chronic HPA in Pahenu2 mice, but that phenylalanine-reducing treatments initiated during adulthood are unable to correct the neuropathology and attendant cognitive deficits that develop during juvenile life in late-treated Pahenu2/enu2 mice.
2017-04-20 | Low-Dose Gene Therapy for Murine PKU Using Episomal Naked DNA Vectors Expressing PAH from Its Endogenous Liver Promoter
Limited duration of transgene expression, insertional mutagenesis, and size limitations for transgene cassettes pose challenges and risk factors for many gene therapy vectors. Here, we report on physiological expression of liver phenylalanine hydroxylase (PAH) by delivery of naked DNA/minicircle (MC)-based vectors for correction of homozygous enu2 mice, a model of human phenylketonuria (PKU). Because MC vectors lack a defined size limit, we constructed a MC vector expressing a codon-optimized murine Pah cDNA that includes a truncated intron and is under the transcriptional control of a 3.6-kb native Pah promoter/enhancer sequence. This vector, delivered via hydrodynamic injection, yielded therapeutic liver PAH activity and sustained correction of blood phenylalanine comparable to viral or synthetic liver promoters. Therapeutic efficacy was seen with vector copy numbers of <1 vector genome per diploid hepatocyte genome and was achieved at a vector dose that was significantly lowered. Partial hepatectomy and subsequent liver regeneration was associated with >95% loss of vector genomes and PAH activity in liver, demonstrating that MC vectors had not integrated into the liver genome. In conclusion, MC vectors, which do not have a defined size-limitation, offer a favorable safety profile for hepatic gene therapy due to their non-integration in combination with native promoters. Limited duration of transgene expression, insertional mutagenesis, and size limitations for transgene cassettes pose challenges and risk factors for many gene therapy vectors. Here, we report on physiological expression of liver phenylalanine hydroxylase (PAH) by delivery of naked DNA/minicircle (MC)-based vectors for correction of homozygous enu2 mice, a model of human phenylketonuria (PKU). Because MC vectors lack a defined size limit, we constructed a MC vector expressing a codon-optimized murine Pah cDNA that includes a truncated intron and is under the transcriptional control of a 3.6-kb native Pah promoter/enhancer sequence. This vector, delivered via hydrodynamic injection, yielded therapeutic liver PAH activity and sustained correction of blood phenylalanine comparable to viral or synthetic liver promoters. Therapeutic efficacy was seen with vector copy numbers of <1 vector genome per diploid hepatocyte genome and was achieved at a vector dose that was significantly lowered. Partial hepatectomy and subsequent liver regeneration was associated with >95% loss of vector genomes and PAH activity in liver, demonstrating that MC vectors had not integrated into the liver genome. In conclusion, MC vectors, which do not have a defined size-limitation, offer a favorable safety profile for hepatic gene therapy due to their non-integration in combination with native promoters.
2016-10-27 | CRISPR RNA-guided FokI nucleases repair a PAH variant in a phenylketonuria model
The CRISPR/Cas9 system is a recently developed genome editing technique. In this study, we used a modified CRISPR system, which employs the fusion of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9) to correct the most common variant (allele frequency 21.4%) in the phenylalanine hydroxylase (PAH) gene - c.1222C>T (p.Arg408Trp) - as an approach toward curing phenylketonuria (PKU). PKU is the most common inherited diseases in amino acid metabolism. It leads to severe neurological and neuropsychological symptoms if untreated or late diagnosed. Correction of the disease-causing variants could rescue residual PAH activity and restore normal function. Co-expression of a single guide RNA plasmid, a FokI-dCas9-zsGreen1 plasmid, and the presence of a single-stranded oligodeoxynucleotide in PAH_c.1222C>T COS-7 cells - an in vitro model for PKU - corrected the PAH variant and restored PAH activity. Also in this system, the HDR enhancer RS-1 improved correction efficiency. This proof-of-concept indicates the potential of the FokI-dCas9 system for precision medicine, in particular for targeting PKU and other monogenic metabolic diseases.
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