Early diagnosis and treatment by newborn screening (NBS) or family history is associated with improved visual outcomes for long-chain 3-hydroxyacylCoA dehydrogenase deficiency (LCHADD) chorioretinopathy.

Long chain 3-hydroxyacyl-CoA dehydrogenase (LCHADD) is the only fatty acid oxidation disorder to develop a progressive chorioretinopathy resulting in vision loss; newborn screening (NBS) for this disorder began in the United States around 2004. We compared visual outcomes among 40 participants with LCHADD or trifunctional protein deficiency diagnosed symptomatically to those who were diagnosed via NBS or a family history. Participants completed ophthalmologic testing including measures of visual acuity, electroretinograms (ERG), fundal imaging, contrast sensitivity, and visual fields. Records were reviewed to document medical and treatment history. Twelve participants presented symptomatically with hypoglycemia, failure to thrive, liver dysfunction, cardiac arrest, or rhabdomyolysis. Twenty eight were diagnosed by NBS or due to a family history of LCHADD. Participants diagnosed symptomatically were older but had similar percent males and genotypes as those diagnosed by NBS. Treatment consisted of fasting avoidance, dietary long-chain fat restriction, MCT, C7, and/or carnitine supplementation. Visual acuity, rod- and cone-driven amplitudes on ERG, contrast sensitivity scores, and visual fields were all significantly worse among participants diagnosed symptomatically compared to NBS. In mixed-effects models, both age and presentation (symptomatic vs. NBS) were significant independent factors associated with visual outcomes. This suggests that visual outcomes were improved by NBS, but there was still lower visual function with advancing age in both groups. Early diagnosis and treatment by NBS is associated with improved visual outcomes and retinal function compared to participants who presented symptomatically. Despite the impact of early intervention, chorioretinopathy was greater with advancing age, highlighting the need for novel treatments.

Cardiac phenotype in adolescents and young adults with long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) deficiency.

PURPOSE: Long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency (LCHADD) is a rare fatty acid oxidation disorder characterized by recurrent episodes of metabolic decompensation and rhabdomyolysis, as well as retinopathy, peripheral neuropathy, and cardiac involvement, such as infantile dilated cardiomyopathy. Because LCHADD patients are surviving longer, we sought to characterize LCHADD-associated major cardiac involvement in adolescence and young adulthood. METHODS: A retrospective cohort of 16 adolescent and young adult participants with LCHADD was reviewed for cardiac phenotype. RESULTS: Major cardiac involvement occurred in 9 of 16 participants, including sudden death, out-of-hospital cardiac arrest, acute cardiac decompensations with heart failure and/or in-hospital cardiac arrest, end-stage dilated cardiomyopathy, and moderate restrictive cardiomyopathy. Sudden cardiac arrest was more common in males and those with a history of infant cardiomyopathy. CONCLUSION: The cardiac manifestations of LCHADD in adolescence and early adulthood are complex and distinct from the phenotype seen in infancy. Life-threatening arrhythmia occurs at substantial rates in LCHADD, often in the absence of metabolic decompensation or rhabdomyolysis. The potential risk factors identified here-male sex and history of infant cardiomyopathy-may hint at strategies for risk stratification and possibly the prevention of these events.

A proposal for an updated staging system for LCHADD retinopathy.

OBJECTIVE: To develop an updated staging system for long-chain 3-hydroxyacyl coenzyme A dehydrogenase deficiency (LCHADD) chorioretinopathy based on contemporary multimodal imaging and electrophysiology. METHODS: We evaluated forty cases of patients with genetically confirmed LCHADD or trifunctional protein deficiency (TFPD) enrolled in a prospective natural history study. Wide-field fundus photographs, fundus autofluorescence (FAF), optical coherence tomography (OCT), and full-field electroretinogram (ffERG) were reviewed and graded for severity. RESULTS: Two independent experts first graded fundus photos and electrophysiology to classify the stage of chorioretinopathy based upon an existing published system. With newer imaging modalities and improved electrophysiology, many patients did not fit cleanly into a single traditional staging group. Therefore, we developed a novel staging system that better delineated the progression of LCHADD retinopathy. We maintained the four previous delineated stages but created substages A and B in stages 2 to 3 to achieve better differentiation. DISCUSSION: Previous staging systems of LCHADD chorioretinopathy relied on only on the assessment of standard 30 to 45-degree fundus photographs, visual acuity, fluorescein angiography (FA), and ffERG. Advances in recordings of ffERG and multimodal imaging with wider fields of view, allow better assessment of retinal changes. Following these advanced assessments, seven patients did not fit neatly into the original classification system and were therefore recategorized under the new proposed system. CONCLUSION: The new proposed staging system improves the classification of LCHADD chorioretinopathy, with the potential to lead to a deeper understanding of the disease's progression and serve as a more reliable reference point for future therapeutic research.

Evaluating change in diet with pegvaliase treatment in adults with phenylketonuria: Analysis of phase 3 clinical trial data.

Phenylketonuria (PKU), a genetic disorder characterized by phenylalanine hydroxylase (PAH) deficiency and phenylalanine (Phe) accumulation, is primarily managed with a protein-restricted diet and PKU-specific medical foods. Pegvaliase is an enzyme substitution therapy approved for individuals with PKU and uncontrolled blood Phe concentrations (>600 µmol/L) despite prior management. This analysis assessed the effect of pegvaliase on dietary intake using data from the Phase 3 PRISM-1 (NCT01819727), PRISM-2 (NCT01889862), and 165-304 (NCT03694353) clinical trials. Participants (N = 250) had a baseline diet assessment, blood Phe ≥600 µmol/L, and had discontinued sapropterin; they were not required to follow a Phe-restricted diet. Outcomes were analyzed by baseline dietary group, categorized as >75%, some (>0% but ≤75%), or no protein intake from medical food. At baseline, mean age was 29.1 years, 49.2% were female, mean body mass index was 28.4 kg/m2, and mean blood Phe was 1237.0 µmol/L. Total protein intake was stable up to 48 months for all 3 baseline dietary groups. Over this time, intact protein intake increased in all groups, and medical protein intake decreased in those who consumed any medical protein at baseline. Of participants consuming some or >75% medical protein at baseline, 49.1% and 34.1% were consuming no medical protein at last assessment, respectively. Following a first hypophenylalaninemia (HypoPhe; 2 consecutive blood Phe measurements <30 µmol/L) event, consumption of medical protein decreased and consumption of intact protein increased. Substantial and sustained Phe reductions were achieved in all 3 baseline dietary groups. The probability of achieving sustained Phe response (SPR) at ≤600 µmol/L was significantly greater for participants consuming medical protein versus no medical protein in an unadjusted analysis, but no statistically significant difference between groups was observed for probability of achieving SPR ≤360 or SPR ≤120 µmol/L. Participants with alopecia (n = 49) had longer pegvaliase treatment durations, reached HypoPhe sooner, and spent longer in HypoPhe than those who did not have alopecia. Most (87.8%) had an identifiable blood Phe drop before their first alopecia episode, and 51.0% (n = 21/41) of first alopecia episodes with known duration resolved before the end of the HypoPhe episode. In conclusion, pegvaliase treatment allowed adults with PKU to lower their blood Phe, reduce their reliance on medical protein, and increase their intact and total protein intake. Results also suggest that HypoPhe does not increase the risk of protein malnutrition in adults with PKU receiving pegvaliase.

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.

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.

Complete correction of murine phenylketonuria by selection-enhanced hepatocyte transplantation.

BACKGROUND AND AIMS: Hepatocyte transplantation for genetic liver diseases has several potential advantages over gene therapy. However, the low efficiency of cell engraftment has limited its clinical implementation. This problem could be overcome by selectively expanding transplanted donor cells until they replace enough of the liver mass to achieve therapeutic benefit. We previously described a gene therapy method to selectively expand hepatocytes deficient in cytochrome p450 reductase (Cypor) using acetaminophen (APAP). Because Cypor is required for the transformation of APAP to a hepatotoxic metabolite, Cypor-deficient cells are protected from toxicity and are able to expand following APAP-induced liver injury. Here, we apply this selection system to correct a mouse model of phenylketonuria by cell transplantation. APPROACH AND RESULTS: Hepatocytes from a wild-type donor animal were edited in vitro to create Cypor deficiency and then transplanted into phenylketonuric animals. Following selection with APAP, blood phenylalanine concentrations were fully normalized and remained stable following APAP withdrawal. Cypor-deficient hepatocytes expanded from < 1% to ~14% in corrected animals, and they showed no abnormalities in blood chemistries, liver histology, or drug metabolism. CONCLUSIONS: We conclude that APAP-mediated selection of transplanted hepatocytes is a potential therapeutic for phenylketonuria with long-term efficacy and a favorable safety profile.

Complete correction of murine phenylketonuria by selection-enhanced hepatocyte transplantation.

Hepatocyte transplantation for genetic liver diseases has several potential advantages over gene therapy. However, low efficiency of cell engraftment has limited its clinical implementation. This problem could be overcome by selectively expanding transplanted donor cells until they replace enough of the liver mass to achieve therapeutic benefit. We previously described a gene therapy method to selectively expand hepatocytes deficient in cytochrome p450 reductase (Cypor) using acetaminophen (APAP). Because Cypor is required for the transformation of APAP to a hepatotoxic metabolite, Cypor deficient cells are protected from toxicity and are able to expand following APAP-induced liver injury. Here, we apply this selection system to correct a mouse model of phenylketonuria (PKU) by cell transplantation. Hepatocytes from a wildtype donor animal were edited in vitro to create Cypor deficiency and then transplanted into PKU animals. Following selection with APAP, blood phenylalanine concentrations were fully normalized and remained stable following APAP withdrawal. Cypor-deficient hepatocytes expanded from <1% to ~14% in corrected animals, and they showed no abnormalities in blood chemistries, liver histology, or drug metabolism. We conclude that APAP-mediated selection of transplanted hepatocytes is a potential therapeutic for PKU with long-term efficacy and a favorable safety profile.

Expert Consensus on the Long-Term Effectiveness of Medical Nutrition Therapy and Its Impact on the Outcomes of Adults with Phenylketonuria.

Many adults with phenylketonuria (PKU) rely on medical nutrition therapy (MNT; low phenylalanine (Phe) diet with protein substitutes/medical foods) to maintain blood Phe concentrations within recommended ranges and prevent PKU-associated comorbidities. Despite disease detection through newborn screening and introduction of MNT as early as birth, adherence to MNT often deteriorates from childhood onwards, complicating the assessment of its effectiveness in the long term. Via a modified Delphi process, consensus (≥70% agreement) was sought on 19 statements among an international, multidisciplinary 13-member expert panel. After three iterative voting rounds, the panel achieved consensus on 17 statements related to the limitations of the long-term effectiveness of MNT (7), the burden of long-term reliance on MNT (4), and its potential long-term detrimental health effects (6). According to the expert panel, the effectiveness of MNT is limited in the long term, is associated with a high treatment burden, and demonstrates that adults with PKU are often unable to achieve metabolic control through dietary management alone, creating an unmet need in the adult PKU population.

A G1528C Hadha knock-in mouse model recapitulates aspects of human clinical phenotypes for long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency.

Long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is a fatty acid oxidation disorder (FAOD) caused by a pathogenic variant, c.1528 G > C, in HADHA encoding the alpha subunit of trifunctional protein (TFPα). Individuals with LCHADD develop chorioretinopathy and peripheral neuropathy not observed in other FAODs in addition to the more ubiquitous symptoms of hypoketotic hypoglycemia, rhabdomyolysis and cardiomyopathy. We report a CRISPR/Cas9 generated knock-in murine model of G1528C in Hadha that recapitulates aspects of the human LCHADD phenotype. Homozygous pups are less numerous than expected from Mendelian probability, but survivors exhibit similar viability with wildtype (WT) littermates. Tissues of LCHADD homozygotes express TFPα protein, but LCHADD mice oxidize less fat and accumulate plasma 3-hydroxyacylcarnitines compared to WT mice. LCHADD mice exhibit lower ketones with fasting, exhaust earlier during treadmill exercise and develop a dilated cardiomyopathy compared to WT mice. In addition, LCHADD mice exhibit decreased visual performance, decreased cone function, and disruption of retinal pigment epithelium. Neurological function is affected, with impaired motor function during wire hang test and reduced open field activity. The G1528C knock-in mouse exhibits a phenotype similar to that observed in human patients; this model will be useful to explore pathophysiology and treatments for LCHADD in the future.

Resting and total energy expenditure of patients with long-chain fatty acid oxidation disorders (LC-FAODs).

The basis of medical nutrition therapy for patients with LC-FAODs is to provide adequate energy to maintain anabolism and prevent catabolism. In practice, energy needs are estimated based on formulas derived from normal populations but it is unknown if energy expenditure among patients with LC-FAODs is similar to the normal population. We measured resting energy expenditure (REE), total energy expenditure (TEE) and body composition in 31 subjects with LC-FAODs ranging in age from 7 to 64 years. Measured REE was lower than estimated REE by various prediction equations and measured TEE was lower than estimated TEE. It is possible that the lower energy expenditure based on prediction formulas from the normal population is due to differences in body composition; we compared body composition to normal data from the 2017-18 National Health and Nutrition Examination Survey (NHANES). Fat free mass and fat mass was similar between subjects with an LC-FAOD and NHANES normal data suggesting no difference in body composition. We then compared measured REE and TEE to normal published data from the Dietary Reference Intakes (DRI). Measured REE and TEE were significantly lower among subjects with LC-FAODs compared to normal published energy expenditure data. Our results suggests patients with a LC-FAOD exhibit a lower REE and therefore actually have a slightly lower TEE than estimated. Current prediction equations may overestimate energy expenditure of patients with a LC-FAOD.

Reinstitution of pegvaliase therapy during lactation.

Pegvaliase, an injectable form of phenylalanine ammonia lyase, is an enzyme substitution therapy for adults with phenylketonuria (PKU). Experience with pegvaliase during lactation is scarce. Limited evidence suggests that pegvaliase does not pass into breast milk. The case presented here describes the pregnancy and lactation experience of a woman with PKU who was treated with pegvaliase prior to pregnancy, discontinued pegvaliase and was treated with a phenylalanine-restricted diet in preparation for and during pregnancy, and then reinstituted pegvaliase two weeks after giving birth and throughout lactation. No pegvaliase activity was detected in pumped breast milk samples prior to reinstituting pegvaliase, and at doses of 80, 110 and 140 mg/week during lactation. The phenylalanine content of breast milk samples collected during pegvaliase therapy were not significantly different than controls, and the infant has grown and developed normally, indicating that pegvaliase therapy during lactation is safe.

Management of early treated adolescents and young adults with phenylketonuria: Development of international consensus recommendations using a modified Delphi approach.

BACKGROUND: Early treated patients with phenylketonuria (PKU) often become lost to follow-up from adolescence onwards due to the historical focus of PKU care on the pediatric population and lack of programs facilitating the transition to adulthood. As a result, evidence on the management of adolescents and young adults with PKU is limited. METHODS: Two meetings were held with a multidisciplinary international panel of 25 experts in PKU and comorbidities frequently experienced by patients with PKU. Based on the outcomes of the first meeting, a set of statements were developed. During the second meeting, these statements were voted on for consensus generation (≥70% agreement), using a modified Delphi approach. RESULTS: A total of 37 consensus recommendations were developed across five areas that were deemed important in the management of adolescents and young adults with PKU: (1) general physical health, (2) mental health and neurocognitive functioning, (3) blood Phe target range, (4) PKU-specific challenges, and (5) transition to adult care. The consensus recommendations reflect the personal opinions and experiences from the participating experts supported with evidence when available. Overall, clinicians managing adolescents and young adults with PKU should be aware of the wide variety of PKU-associated comorbidities, initiating screening at an early age. In addition, management of adolescents/young adults should be a joint effort between the patient, clinical center, and parents/caregivers supporting adolescents with gradually gaining independent control of their disease during the transition to adulthood. CONCLUSIONS: A multidisciplinary international group of experts used a modified Delphi approach to develop a set of consensus recommendations with the aim of providing guidance and offering tools to clinics to aid with supporting adolescents and young adults with PKU.

Biochemical and behavioural profile of NTBC treated Tyrosinemie type 1 mice.

BACKGROUND: Tyrosinemia type 1 (HT1) is a rare metabolic disorder caused by a defect in the tyrosine catabolic pathway. Since HT1 patients are treated with NTBC, outcome improved and life expectancy greatly increased. However extensive neurocognitive and behavioural problems have been described, which might be related to treatment with NTBC, the biochemical changes induced by NTBC, or metabolites accumulating due to the enzymatic defect characterizing the disease. OBJECTIVE: To study the possible pathophysiological mechanisms of brain dysfunction in HT1, we assessed blood and brain LNAA, and brain monoamine neurotransmitter metabolite levels in relation to behavioural and cognitive performance of HT1 mice. DESIGN: C57BL/6 littermates were divided in three different experimental groups: HT1, heterozygous and wild-type mice (n = 10; 5 male). All groups were treated with NTBC and underwent cognitive and behavioural testing. One week after behavioural testing, blood and brain material were collected to measure amino acid profiles and brain monoaminergic neurotransmitter levels. RESULTS: Irrespective of the genetic background, NTBC treatment resulted in a clear increase in brain tyrosine levels, whereas all other brain LNAA levels tended to be lower than their reference values. Despite these changes in blood and brain biochemistry, no significant differences in brain monoamine neurotransmitter (metabolites) were found and all mice showed normal behaviour and learning and memory. CONCLUSION: Despite the biochemical changes, NTBC and genotype of the mice were not associated with poorer behavioural and cognitive function of the mice. Further research involving dietary treatment of FAH-/- are warranted to investigate whether this reveals the cognitive impairments that have been seen in treated HT1 patients.

Modeling the cognitive effects of diet discontinuation in adults with phenylketonuria (PKU) using pegvaliase therapy in PAH-deficient mice.

Existing phenylalanine hydroxylase (PAH)-deficient mice strains are useful models of untreated or late-treated human phenylketonuria (PKU), as most contemporary therapies can only be initiated after weaning and the pups have already suffered irreversible consequences of chronic hyperphenylalaninemia (HPA) during early brain development. Therefore, we sought to evaluate whether enzyme substitution therapy with pegvaliase initiated near birth and administered repetitively to C57Bl/6-Pahenu2/enu2 mice would prevent HPA-related behavioral and cognitive deficits and form a model for early-treated PKU. The main results of three reported experiments are: 1) lifelong weekly pegvaliase treatment prevented the cognitive deficits associated with HPA in contrast to persisting deficits in mice treated with pegvaliase only as adults. 2) Cognitive deficits reappear in mice treated with weekly pegvaliase from birth but in which pegvaliase is discontinued at 3 months age. 3) Twice weekly pegvaliase injection also prevented cognitive deficits but again cognitive deficits emerged in early-treated animals following discontinuation of pegvaliase treatment during adulthood, particularly in females. In all studies, pegvaliase treatment was associated with complete correction of brain monoamine neurotransmitter content and with improved overall growth of the mice as measured by body weight. Mean total brain weight however remained low in all PAH deficient mice regardless of treatment. Application of enzyme substitution therapy with pegvaliase, initiated near birth and continued into adulthood, to PAH-deficient Pahenu2/enu2 mice models contemporary early-treated human PKU. This model will be useful for exploring the differential pathophysiologic effects of HPA at different developmental stages of the murine brain.

A non-interventional observational study to identify and validate clinical outcome assessments for adults with phenylketonuria for use in clinical trials.

INTRODUCTION: Current clinical outcome assessments (COAs) are not effectively capturing the complex array of symptoms of adults with phenylketonuria (PKU). This study aimed to identify concepts of interest relevant to adults with PKU. Based on these concepts, COAs for patient-reported outcomes (PROs), observer-reported outcomes (ObsROs), and clinician-reported outcomes (ClinROs) were selected or developed and content validity was assessed. MATERIALS AND METHODS: Concept-elicitation interviews were conducted with an international cohort of adults with PKU (n = 30), family member observers (n = 14), and clinical experts (n = 8). Observers and clinical experts were included to overcome the risk of lack of self-awareness among adults with PKU. The concepts of interests endorsed by ≥30% of patients, observers, and/or clinical experts were selected, mapped to items in existing COAs, and used to develop global impression items for patients, observers, and clinicians. Next, the content validity of the COAs and global impression items was evaluated by cognitive interviews with patients (n = 22), observers (n = 11), and clinical experts (n = 8). All patients were categorized according to blood phenylalanine (Phe) levels (i.e., <600 µmol/L, 600-1200 µmol/L, and >1200 µmol/L). RESULTS: Concepts of interests were identified across four domains: emotional, cognitive, physical, and behavioral. After mapping, eight existing COAs were selected based on the concept coverage (six PROs, one ObsRO, and one ClinRO). The six PRO measures were considered as potentially fit-for-purpose. The ObsRO measure was not deemed relevant for use in observers of adults with PKU and only a subscale of the ClinRO measure was considered valid for assessing adults with PKU by clinicians. Due to the lack of existing COAs covering all concepts of interests, global impression items for symptom severity and change in symptoms were developed, which were limited to one question covering in total 14 concepts. Upon validation, some of the patient and observer global impression items were excluded as they were subject to lack of insight or could not be reported by observers. Due to the limited interaction time between clinician and patient, use of the clinician global impression items was not supported. CONCLUSION: Existing COAs relevant to adults with PKU were selected and PKU-specific global impression items were developed by mapping the most frequently identified concepts of interests from internationally-conducted in-depth interviews. Future studies should address the appropriateness of the selected COAs and global impression items to assess if these can be used as efficacy endpoints in PKU clinical trials.

Therapeutic liver repopulation by transient acetaminophen selection of gene-modified hepatocytes.

Gene therapy by integrating vectors is promising for monogenic liver diseases, especially in children where episomal vectors remain transient. However, reaching the therapeutic threshold with genome-integrating vectors is challenging. Therefore, we developed a method to expand hepatocytes bearing therapeutic transgenes. The common fever medicine acetaminophen becomes hepatotoxic via cytochrome p450 metabolism. Lentiviral vectors with transgenes linked in cis to a Cypor shRNA were administered to neonatal mice. Hepatocytes lacking the essential cofactor of Cyp enzymes, NADPH-cytochrome p450 reductase (Cypor), were selected in vivo by acetaminophen administration, replacing up to 50% of the hepatic mass. Acetaminophen treatment of the mice resulted in over 30-fold expansion of transgene-bearing hepatocytes and achieved therapeutic thresholds in hemophilia B and phenylketonuria. We conclude that therapeutically modified hepatocytes can be selected safely and efficiently in preclinical models with a transient regimen of moderately hepatotoxic acetaminophen.

Pharmacokinetic, pharmacodynamic, and immunogenic rationale for optimal dosing of pegvaliase, a PEGylated bacterial enzyme, in adult patients with phenylketonuria.

Phenylketonuria (PKU), a deficiency in the activity of the enzyme phenylalanine hydroxylase, leads to toxic levels of phenylalanine (Phe) in the blood and brain. Pegvaliase (recombinant Anabaena variabilis phenylalanine ammonia lyase conjugated with polyethylene glycol) is approved to manage PKU in patients aged greater than or equal to 18 years in the United States and in patients aged greater than or equal to 16 years in the European Union. Pharmacokinetic, pharmacodynamic, and immunogenicity results from five open-label pegvaliase trials were assessed. Studies with induction/titration/maintenance (I/T/M) dosing regimens demonstrated pharmacokinetic stabilization and sustained efficacy associated with maintenance doses (20, 40, or 60 mg/day). Immune-mediated pegvaliase clearance was high during induction/titration phases when the early immune response was peaking. The combination of low drug dosage and high drug clearance led to low drug exposure and minimal decreases in blood Phe levels during induction/titration. Higher drug exposure and substantial reductions in blood Phe levels were observed later in treatment as drug clearance was reduced due to the maturation of the immune response, which allowed for increased dosing to target levels. The incidence of hypersensitivity reactions was temporally associated with the peaking of the early antidrug immune response and decreased with time as immune response matured after the first 6 months of treatment. These results support an I/T/M dosing regimen and suggest a strategy for administration of other nonhuman biologics to achieve efficacy and improve tolerability.

Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing.

Phenylketonuria (PKU) is a metabolic disorder whereby phenylalanine metabolism is deficient due to allelic variations in the gene for phenylalanine hydroxylase (PAH). There is no cure for PKU other than orthotopic liver transplantation, and the standard of care for patients is limited to dietary restrictions and key amino acid supplementation. Therefore, Pah was edited in pig fibroblasts for the generation of PKU clone piglets that harbor a common and severe human mutation, R408W. Additionally, the proximal region to the mutation was further humanized by introducing 5 single nucleotide polymorphisms (SNPs) to allow for development of gene editing machinery that could be translated directly from the pig model to human PKU patients that harbor at least one classic R408W allele. Resulting piglets were hypopigmented (a single Ossabaw piglet) and had low birthweight (all piglets). The piglets had similar levels of PAH expression, but no detectable enzymatic activity, consistent with the human phenotype. The piglets were fragile and required extensive neonatal care to prevent failure to thrive and early demise. Phenylalanine levels rose sharply when dietary Phe was unrestricted but could be rapidly reduced with a low Phe diet. Fibroblasts isolated from R408W piglets show susceptibility to correction using CRISPR or TALEN, with subsequent homology-directed recombination to correct Pah. This pig model of PKU provides a powerful new tool for development of all classes of therapeutic candidates to treat or cure PKU, as well as unique value for proof-of-concept studies for in vivo human gene editing platforms in the context of this humanized PKU allele.

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.