Ex vivo gene editing and cell therapy for hereditary tyrosinemia type 1.

BACKGROUND: We previously demonstrated the successful use of in vivo CRISPR gene editing to delete 4-hydroxyphenylpyruvate dioxygenase (HPD) to rescue mice deficient in fumarylacetoacetate hydrolase (FAH), a disorder known as hereditary tyrosinemia type 1 (HT1). The aim of this study was to develop an ex vivo gene-editing protocol and apply it as a cell therapy for HT1. METHODS: We isolated hepatocytes from wild-type (C57BL/6J) and Fah-/- mice and then used an optimized electroporation protocol to deliver Hpd-targeting CRISPR-Cas9 ribonucleoproteins into hepatocytes. Next, hepatocytes were transiently incubated in cytokine recovery media formulated to block apoptosis, followed by splenic injection into recipient Fah-/- mice. RESULTS: We observed robust engraftment and expansion of transplanted gene-edited hepatocytes from wild-type donors in the livers of recipient mice when transient incubation with our cytokine recovery media was used after electroporation and negligible engraftment without the media (mean: 46.8% and 0.83%, respectively; p=0.0025). Thus, the cytokine recovery medium was critical to our electroporation protocol. When hepatocytes from Fah-/- mice were used as donors for transplantation, we observed 35% and 28% engraftment for Hpd-Cas9 ribonucleoproteins and Cas9 mRNA, respectively. Tyrosine, phenylalanine, and biochemical markers of liver injury normalized in both Hpd-targeting Cas9 ribonucleoprotein and mRNA groups independent of induced inhibition of Hpd through nitisinone, indicating correction of disease indicators in Fah-/- mice. CONCLUSIONS: The successful liver cell therapy for HT1 validates our protocol and, despite the known growth advantage of HT1, showcases ex vivo gene editing using electroporation in combination with liver cell therapy to cure a disease model. These advancements underscore the potential impacts of electroporation combined with transplantation as a cell therapy.

Engineering a transposon-associated TnpB-ωRNA system for efficient gene editing and phenotypic correction of a tyrosinaemia mouse model.

Transposon-associated ribonucleoprotein TnpB is known to be the ancestry endonuclease of diverse Cas12 effector proteins from type-V CRISPR system. Given its small size (408 aa), it is of interest to examine whether engineered TnpB could be used for efficient mammalian genome editing. Here, we showed that the gene editing activity of native TnpB from Deinococcus radiodurans (ISDra2 TnpB) in mouse embryos was already higher than previously identified small-sized Cas12f1. Further stepwise engineering of noncoding RNA (ωRNA or reRNA) component of TnpB significantly elevated the nuclease activity of TnpB. Notably, an optimized TnpB-ωRNA system could be efficiently delivered in vivo with single adeno-associated virus (AAV) and corrected the disease phenotype in a tyrosinaemia mouse model. Thus, the engineered miniature TnpB system represents a new addition to the current genome editing toolbox, with the unique feature of the smallest effector size that facilitate efficient AAV delivery for editing of cells and tissues.

An engineered Escherichia coli Nissle strain prevents lethal liver injury in a mouse model of tyrosinemia type 1.

BACKGROUND & AIMS: Hereditary tyrosinemia type 1 (HT1) results from the loss of fumarylacetoacetate hydrolase (FAH) activity and can lead to lethal liver injury. Therapeutic options for HT1 remain limited. In this study, we aimed to construct an engineered bacterium capable of reprogramming host metabolism and thereby provide a potential alternative approach for the treatment of HT1. METHODS: Escherichia coli Nissle 1917 (EcN) was engineered to express genes involved in tyrosine metabolism in the anoxic conditions that are characteristic of the intestine (EcN-HT). Bodyweight, survival rate, plasma (tyrosine/liver function), H&E staining and RNA sequencing were used to assess its ability to degrade tyrosine and protect against lethal liver injury in Fah-knockout (KO) mice, a well-accepted model of HT1. RESULTS: EcN-HT consumed tyrosine and produced L-DOPA (levodopa) in an in vitro system. Importantly, in Fah-KO mice, the oral administration of EcN-HT enhanced tyrosine degradation, reduced the accumulation of toxic metabolites, and protected against lethal liver injury. RNA sequencing analysis revealed that EcN-HT rescued the global gene expression pattern in the livers of Fah-KO mice, particularly of genes involved in metabolic signaling and liver homeostasis. Moreover, EcN-HT treatment was found to be safe and well-tolerated in the mouse intestine. CONCLUSIONS: This is the first report of an engineered live bacterium that can degrade tyrosine and alleviate lethal liver injury in mice with HT1. EcN-HT represents a novel engineered probiotic with the potential to treat this condition. IMPACT AND IMPLICATIONS: Patients with hereditary tyrosinemia type 1 (HT1) are characterized by an inability to metabolize tyrosine normally and suffer from liver failure, renal dysfunction, neurological impairments, and cancer. Given the overlap and complementarity between the host and microbial metabolic pathways, the gut microbiome provides a potential chance to regulate host metabolism through degradation of tyrosine and reduction of byproducts that might be toxic. Herein, we demonstrated that an engineered live bacterium, EcN-HT, could enhance tyrosine breakdown, reduce the accumulation of toxic tyrosine byproducts, and protect against lethal liver injury in Fah-knockout mice. These findings suggested that engineered live biotherapeutics that can degrade tyrosine in the gut may represent a viable and safe strategy for the prevention of lethal liver injury in HT1 as well as the mitigation of its associated pathologies.

Novel HPD mutation p.A244V compound with p.T219M causing tyrosinemia type III in a Chinese girl and review of the genotype-phenotype spectrum.

BACKGROUND: Hereditary tyrosinemia type III (HT III) is an extremely rare form of tyrosinemia, characterized by autosomal recessive inheritance and biallelic mutations in the HPD gene. The clinical presentation of HT III is variable and poorly understood, with symptoms ranging from developmental delay and intellectual impairment to seizures and intermittent ataxia. This study aimed to provide further insights into the clinical and genetic characteristics of HT III. METHODS: A 3-year-old girl, identified through newborn screening, was diagnosed with HT III using targeted next-generation sequencing. A comprehensive literature review was conducted, and the clinical, biochemical, and genetic findings of previously reported HT III patients were summarized and analyzed. RESULTS: The genetic analysis of the proband revealed compound heterozygous mutations in the HPD gene such as c.731C>T (p.A244V) and c.656C>T (p.T219M). Notably, the HPD p.A244V mutation had not been previously documented in public databases or the scientific literature. Bioinformatics analysis classified both variants as pathogenic variants. The patient exhibited persistent tyrosinemia, elevated levels of related metabolite derivatives, confirming the diagnosis of HT III. The review of previously published cases contributed to a better understanding of the clinical and genetic characteristics associated with HT III. CONCLUSION: Early diagnosis and prompt treatment in infancy are crucial for managing HT III effectively. Dietary therapy, particularly during childhood, plays a significant role in disease management. The findings from this study enhance our understanding of the genotype-phenotype associations in HT III and emphasize the importance of early intervention for improved patient outcomes.

Hereditary Tyrosinemia Type 1 Mice under Continuous Nitisinone Treatment Display Remnants of an Uncorrected Liver Disease Phenotype.

Hereditary tyrosinemia type 1 (HT1) is a genetic disorder of the tyrosine degradation pathway (TIMD) with unmet therapeutic needs. HT1 patients are unable to fully break down the amino acid tyrosine due to a deficient fumarylacetoacetate hydrolase (FAH) enzyme and, therefore, accumulate toxic tyrosine intermediates. If left untreated, they experience hepatic failure with comorbidities involving the renal and neurological system and the development of hepatocellular carcinoma (HCC). Nitisinone (NTBC), a potent inhibitor of the 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme, rescues HT1 patients from severe illness and death. However, despite its demonstrated benefits, HT1 patients under continuous NTBC therapy are at risk to develop HCC and adverse reactions in the eye, blood and lymphatic system, the mechanism of which is poorly understood. Moreover, NTBC does not restore the enzymatic defects inflicted by the disease nor does it cure HT1. Here, the changes in molecular pathways associated to the development and progression of HT1-driven liver disease that remains uncorrected under NTBC therapy were investigated using whole transcriptome analyses on the livers of Fah- and Hgd-deficient mice under continuous NTBC therapy and after seven days of NTBC therapy discontinuation. Alkaptonuria (AKU) was used as a tyrosine-inherited metabolic disorder reference disease with non-hepatic manifestations. The differentially expressed genes were enriched in toxicological gene classes related to liver disease, liver damage, liver regeneration and liver cancer, in particular HCC. Most importantly, a set of 25 genes related to liver disease and HCC development was identified that was differentially regulated in HT1 vs. AKU mouse livers under NTBC therapy. Some of those were further modulated upon NTBC therapy discontinuation in HT1 but not in AKU livers. Altogether, our data indicate that NTBC therapy does not completely resolves HT1-driven liver disease and supports the sustained risk to develop HCC over time as different HCC markers, including Moxd1, Saa, Mt, Dbp and Cxcl1, were significantly increased under NTBC.

[Clinical features and genetic analysis of a child with acute form of Tyrosinemia type I due to a novel variant of FAH gene].

OBJECTIVE: To analyze the clinical phenotype and genetic basis for a child with acute form of tyrosinemia type I (TYRSN1). METHODS: A child with TYRSN1 who presented at the Gansu Provincial Maternal and Child Health Care Hospital in October 2020 was selected as the subject. The child was subjected to tandem mass spectrometry (MS-MS) and urine gas chromatography-mass spectrometry (GC-MS) for the detection of inherited metabolic disorders, in addition with whole exome sequencing (WES). Candidate variants were validated by Sanger sequencing. RESULTS: The child's clinical features included abdominal distension, hepatomegaly, anemia and tendency of bleeding. By mass spectrometry analysis, her serum and urine tyrosine and succinylacetone levels have both exceeded the normal ranges. WES and Sanger sequencing revealed that she has harbored c.1062+5G>A and c.943T>C (p.Cys315Arg) compound heterozygous variants of the FAH gene, which were inherited from her father and mother, respectively. Among these, the c.943T>C was unreported previously. CONCLUSION: Considering her clinical phenotype and result of genetic testing, the child was diagnosed with TYRSN1 (acute type). The compound heterozygous variants of the FAH gene probably underlay the disease in this child. Above finding has further expanded the spectrum of FAH gene variants, and provided a basis for accurate treatment, genetic counseling and prenatal diagnosis for her family.

The compound heterozygous mutations of c.607G>a and c.657delC in the FAH gene are associated with renal damage with hereditary tyrosinemia type 1 (HT1).

BACKGROUND: Hereditary tyrosinemia type 1 (HT1) is a rare inherited metabolic disease characterized by severe liver and renal dysfunction. Early identification in affected children is critical for improved treatment options and prognosis. METHODS: In this study, we identified novel compound heterozygous mutations (NM_000137: c.657delC (p.K220Rfs*12) and c.607G>A (p.A203T)) in the fumarylacetoacetate hydrolase (FAH) gene in a family. We also characterized the clinical phenotype of the proband and verified the pathogenic effects of the mutations. Furthermore, we explored the pathogenic mechanism of renal injury through renal biopsy pathology and cell-based in vitro assays. Our study aims to verify the association between novel fumarylacetoacetate hydrolase (FAH) variants and HT1, confirm the pathogenic effects of the mutations and explore the pathogenic mechanism of renal injury. RESULTS: We showed these FAH mutations were inherited in an autosomal recessive manner and resulted in abnormal FAH protein expression and dysfunction, leading to fumarylacetoacetate (FAA) accumulation. The proband also showed apparent renal injury, including glomerular filtration barrier dysfunction and abnormal tubular protein reabsorption. CONCLUSIONS: These observations may provide deeper insights on disease pathogenesis and identify potential therapeutic approaches for HT1 from a genetic perspective. Similarly, we hope to provide valuable information for genetic counseling and prenatal diagnostics.

Identification and functional characterization of a novel homozygous intronic variant in the fumarylacetoacetate hydrolase gene in a Chinese patient with tyrosinemia type 1.

BACKGROUND: Hereditary tyrosinemia type 1 (HT1; OMIM# 276700) is a genetic metabolism disorder caused by disease-causing variants in the fumarylacetoacetate hydrolase (FAH) gene encoding the last enzyme of the tyrosine catabolic pathway. Herein, we describe the clinical features and genetic characteristics of HT1 in a five years and seven months old Chinese patient. METHODS: After clinical diagnosis of the proband with HT1, genetic testing was performed by Sanger sequencing of the FAH gene in all family members. Functional analysis of the disease-causing variant was performed by cDNA sequencing to understand the effect of the variant on FAH transcript. To further predict the variant effect, we used Human Splicing Finder (HSF) and PyMol in silico analysis. RESULTS: We identified a novel previously undescribed intronic variant in the FAH gene (c.914-1G>A). It was detected in a child who was homozygous for the variant and had the clinical presentation of HT1. cDNA sequencing showed that this splice-junction variant affected the transcription of FAH by formation of two different transcripts. Our observations and laboratory experiments were in line with in silico methods. CONCLUSIONS: Our study provides new insight into the HT1 variant spectrum and a better understanding of this disease in the Chinese population. This will be useful for molecular diagnosis in our country in cases where premarital screening, prenatal diagnosis and preimplantation genetic diagnosis are planned.

The mutation spectrum and ethnic distribution of non-hepatorenal tyrosinemia (types II, III).

BACKGROUND: Different types of non-hepatorenal tyrosinemia are among the rare forms of tyrosinemia. Tyrosinemia type II and III are autosomal recessive disorders caused by pathogenic variants in the tyrosine aminotransferase (TAT), and 4-hydroxyphenyl-pyruvate dioxygenas (HPPD) genes, respectively. There are still unclarified aspects in their clinical presentations, mutational spectrum, and genotype-phenotype correlation. MAIN BODY: In this study, we evaluated the spectrum of TAT and HHPD gene mutations in patients with tyrosinemia type II and III. Moreover, biochemical and clinical findings are evaluated to establish a genotype-phenotype relationship in the above-mentioned patients. Thirty-three TAT variants have been reported in 42 families, consisting of 21 missense variants, 5 frameshift variants, 4 nonsense variants, 2 variants that primarily cause splicing site, and 1 skipping complete exon (large deletion). The most common variant is p.Arg57Ter, causing a splicing defect, and resulting in premature termination of translation, which was found in 10 patients from 3 families. In HPPD gene, eleven variants in 16 patients have been reported including 7 missense variants, 2 nonsense variants, 1 splice defect, and 1 frameshift variant so far. All variants are unique, except for p.Tyr160Cys, which is a missense variant found in two different patients. Regarding genotype-phenotype correlations, in 90% of tyrosinemia type II patients, positive clinical and biochemical correlations with a detected variant are observed. In HPPD gene, due to the small number of patients, it is not possible to make a definite conclusion. CONCLUSION: This is the first large review of variants in TAT and HPPD, highlighting the wide spectrum of disease-causing mutations. Such information is beneficial for the establishment of a privileged mutation screening process in a specific region or ethnic group.

An Infant with Bilateral Keratitis: From Infectious to Genetic Diagnosis.

BACKGROUND Tyrosinemia Type II (TYRII) is a rare autosomal recessive inborn error of metabolism caused by deficiency of tyrosine aminotransferase (TAT), leading to hypertyrosinemia. TYRII patients often present in the first year of life with ocular and cutaneous findings, including corneal ulcers, pseudodendritic keratitis, and palmoplantar hyperkeratosis. The corneal involvement is often mistaken for herpes simplex virus (HSV) keratitis, which is a much commoner condition. CASE REPORT A previously healthy 10-month-old male infant was referred to Ophthalmology for acute onset photophobia. Bilateral dendritiform corneal lesions raised the suspicion for herpetic keratitis. Additionally, a papular, crusted lesion was found on his thumb after a few days of hospitalization, also raising concerns about HSV. The patient's clinical condition seemed to improve under intravenous acyclovir and supportive treatment. A conjunctival swab and crusted lesion on the thumb were tested for HSV using a polymerase chain reaction (PCR) technique, and both were negative. Nevertheless, given the clinical presentation and the favorable course of signs and symptoms, hospital discharge was planned with oral acyclovir. It was halted by an alternative diagnosis of autosomal recessive inborn error of metabolism, tyrosinemia type II, confirmed by elevated plasma tyrosine level and later by molecular analysis requested as a confirmatory investigation by the genetics medical team. CONCLUSIONS The corneal involvement in TYRII is often mistaken for HSV keratitis, and clinical course alone should not halt further investigations to rule out TYRII. Clinicians should suspect TYRII clinically when its characteristic ocular dendritiform lesions are present, namely in infancy or early childhood, and even in the absence of its typical cutaneous palmoplantar hyperkeratosis plaques.

In vivo lentiviral vector gene therapy to cure hereditary tyrosinemia type 1 and prevent development of precancerous and cancerous lesions.

Conventional therapy for hereditary tyrosinemia type-1 (HT1) with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) delays and in some cases fails to prevent disease progression to liver fibrosis, liver failure, and activation of tumorigenic pathways. Here we demonstrate cure of HT1 by direct, in vivo administration of a therapeutic lentiviral vector targeting the expression of a human fumarylacetoacetate hydrolase (FAH) transgene in the porcine model of HT1. This therapy is well tolerated and provides stable long-term expression of FAH in pigs with HT1. Genomic integration displays a benign profile, with subsequent fibrosis and tumorigenicity gene expression patterns similar to wild-type animals as compared to NTBC-treated or diseased untreated animals. Indeed, the phenotypic and genomic data following in vivo lentiviral vector administration demonstrate comparative superiority over other therapies including ex vivo cell therapy and therefore support clinical application of this approach.

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.

Neurocognitive outcome and mental health in children with tyrosinemia type 1 and phenylketonuria: A comparison between two genetic disorders affecting the same metabolic pathway.

Tyrosinemia type 1 (TT1) and phenylketonuria (PKU) are both inborn errors of phenylalanine-tyrosine metabolism. Neurocognitive and behavioral outcomes have always featured in PKU research but received less attention in TT1 research. This study aimed to investigate and compare neurocognitive, behavioral, and social outcomes of treated TT1 and PKU patients. We included 33 TT1 patients (mean age 11.24 years; 16 male), 31 PKU patients (mean age 10.84; 14 male), and 58 age- and gender-matched healthy controls (mean age 10.82 years; 29 male). IQ (Wechsler-subtests), executive functioning (the Behavioral Rating Inventory of Executive Functioning), mental health (the Achenbach-scales), and social functioning (the Social Skills Rating System) were assessed. Results of TT1 patients, PKU patients, and healthy controls were compared using Kruskal-Wallis tests with post-hoc Mann-Whitney U tests. TT1 patients showed a lower IQ and poorer executive functioning, mental health, and social functioning compared to healthy controls and PKU patients. PKU patients did not differ from healthy controls regarding these outcome measures. Relatively poor outcomes for TT1 patients were particularly evident for verbal IQ, BRIEF dimensions "working memory", "plan and organize" and "monitor", ASEBA dimensions "social problems" and "attention problems", and for the SSRS "assertiveness" scale (all p values <0.001). To conclude, TT1 patients showed cognitive impairments on all domains studied, and appeared to be significantly more affected than PKU patients. More attention should be paid to investigating and monitoring neurocognitive outcome in TT1 and research should focus on explaining the underlying pathophysiological mechanism.

A split prime editor with untethered reverse transcriptase and circular RNA template.

Delivery and optimization of prime editors (PEs) have been hampered by their large size and complexity. Although split versions of genome-editing tools can reduce construct size, they require special engineering to tether the binding and catalytic domains. Here we report a split PE (sPE) in which the Cas9 nickase (nCas9) remains untethered from the reverse transcriptase (RT). The sPE showed similar efficiencies in installing precise edits as the parental unsplit PE3 and no increase in insertion-deletion (indel) byproducts. Delivery of sPE to the mouse liver with hydrodynamic injection to modify ß-catenin drove tumor formation with similar efficiency as PE3. Delivery with two adeno-associated virus (AAV) vectors corrected the disease-causing mutation in a mouse model of type I tyrosinemia. Similarly, prime editing guide RNAs (pegRNAs) can be split into a single guide RNA (sgRNA) and a circular RNA RT template to increase flexibility and stability. Compared to previous sPEs, ours lacks inteins, protein-protein affinity modules and nuclease-sensitive pegRNA extensions, which increase construct complexity and might reduce efficiency. Our modular system will facilitate the delivery and optimization of PEs.

Clinical and biochemical footprints of inherited metabolic diseases. VIII. Neoplasias.

Cancer, caused by multiple cumulative pathogenic variants in tumor suppressor genes and proto-oncogenes, is a leading cause of mortality worldwide. The uncontrolled and rapid cell growth of the tumors requires a reprogramming of the complex cellular metabolic network to favor anabolism. Adequate management and treatment of certain inherited metabolic diseases might prevent the development of certain neoplasias, such as hepatocellular carcinoma in tyrosinemia type 1 or hepatocellular adenomas in glycogen storage disorder type 1a. We reviewed and updated the list of known metabolic etiologies associated with various types of benign and malignant neoplasias, finding 64 relevant inborn errors of metabolism. This is the eighth article of the series attempting to create a comprehensive list of clinical and metabolic differential diagnosis by system involvement.

Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo.

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.

Neonatal hyperinsulinism in transient and classical forms of tyrosinemia.

BACKGROUND: The spectrum of disorders associated with hyperinsulinemic hypoglycemia (HHI) has vastly increased over the past 20 years with identification of molecular, metabolic and cellular pathways involved in the regulation of insulin secretion and its actions. Hereditary tyrosinemia (HT1) is a rare metabolic disorder associated with accumulation of toxic metabolites of the tyrosine pathway due to a genetically mediated enzyme defect of fumarylacetoacetate hydrolase. Transient tyrosinemia of the newborn (TTN) is a benign condition with a maturational defect of the enzymes associated with tyrosine metabolism without any genetic abnormalities. RESULTS: We describe two rare cases of HHI, one in a patient with HT1 and for the first time, in a patient with TTN. Each of our patients presented in the neonatal period with persistent hypoglycemia that on biochemical evaluation was consistent with HHI. Each patient received diazoxide therapy for 3.5 months and 17 months of life, respectively and HHI resolved thereafter. CONCLUSION: Despite the fact that HHI has been described in HT1 for several decades, no specific mechanism has been delineated. Although we considered the common embryonal origin of the liver and pancreas with the hepatotoxic effect in HT1 also impacting the latter, this was not a possible explanation for TTN. The commonality between our two patients is the accumulation of certain amino acids which are known to be insulinotropic. We therefore hypothesize that the excess of amino acids such as leucine, lysine, valine and isoleucine in our patients resulted in HHI, which was transient. Both patients responded to diazoxide. This novel presentation in TTN and the reassuring response in both HT1 and TTN to diazoxide will be useful to inform physicians about managing HHI in these patients. Further studies are required to delineate the mechanism of HHI in these infants.

Generation of an induced pluripotent stem cell line SDQLCHi026-A from a hereditary tyrosinemia type I patient carrying compound heterozygote mutations in FAH gene.

Here we describe the generation of induced pluripotent stem cells (iPSCs) from a patient diagnosed as hereditary tyrosinemia type I (HT1) caused by FAH gene mutation. Induced pluripotent stem cells (iPSCs) were developed using non-integrating episomal vectors containing OCT4, SOX2, KLF4, BCL-XL and MYC. The established iPSC line (SDQLCHi026-A) displayed pluripotent cell morphology, high expression levels of pluripotency markers, differentiation potential in vitro, normal karyotype, and remaining the original FAH gene mutation.

Therapeutic Targeting of Fumaryl Acetoacetate Hydrolase in Hereditary Tyrosinemia Type I.

Fumarylacetoacetate hydrolase (FAH) is the fifth enzyme in the tyrosine catabolism pathway. A deficiency in human FAH leads to hereditary tyrosinemia type I (HT1), an autosomal recessive disorder that results in the accumulation of toxic metabolites such as succinylacetone, maleylacetoacetate, and fumarylacetoacetate in the liver and kidney, among other tissues. The disease is severe and, when untreated, it can lead to death. A low tyrosine diet combined with the herbicidal nitisinone constitutes the only available therapy, but this treatment is not devoid of secondary effects and long-term complications. In this study, we targeted FAH for the first-time to discover new chemical modulators that act as pharmacological chaperones, directly associating with this enzyme. After screening several thousand compounds and subsequent chemical redesign, we found a set of reversible inhibitors that associate with FAH close to the active site and stabilize the (active) dimeric species, as demonstrated by NMR spectroscopy. Importantly, the inhibitors are also able to partially restore the normal phenotype in a newly developed cellular model of HT1.

Induced Liver Regeneration Enhances CRISPR/Cas9-Mediated Gene Repair in Tyrosinemia Type 1.

The efficiency of gene repair by homologous recombination in the liver is enhanced by CRISP/Cas9 incision near the mutation. In this study, we explored interventions designed to further enhance in vivo hepatocyte gene repair in a model of hereditary tyrosinemia. A two-AAV system was employed: one virus carried a Staphylococcus pyogenes Cas9 (SpCas9) expression cassette and the other harbored a U6 promoter-driven sgRNA and a fragment of fumarylacetoacetate hydrolase (Fah) genomic DNA as the homologous recombination donor. In neonatal mice, a gene correction frequency of ∼10.8% of hepatocytes was achieved. The efficiency in adult mice was significantly lower at ∼1.6%. To determine whether hepatocyte replication could enhance the targeting frequency, cell division was induced with thyroid hormone T3. This more than doubled the gene correction efficiency to 3.5% (p < 0.005). To determine whether SpCas9 delivery was rate limiting, the gene repair AAV was administered to SpCas9 transgenic mice. However, this did not significantly enhance gene repair. Finally, we tested whether the Fanconi anemia (FA) DNA repair pathway was important in hepatocyte gene repair. Gene correction frequencies were significantly lower in neonatal mice lacking the FA complementation group A (Fanca) gene. Taken together, we conclude that pharmacological induction of hepatocyte replication along with manipulation of DNA repair pathways could be a useful strategy for enhancing in vivo gene correction.