Partial suppression of BCAA catabolism as a potential therapy for BCKDK deficiency.

Branched chain ketoacid dehydrogenase kinase (BCKDK) deficiency is a recently described inherited neurometabolic disorder of branched chain amino acid (BCAA) metabolism implying increased BCAA catabolism. It has been hypothesized that a severe reduction in systemic BCAA levels underlies the disease pathophysiology, and that BCAA supplementation may ameliorate disease phenotypes. To test this hypothesis, we characterized a recent mouse model of BCKDK deficiency and evaluated the efficacy of enteral BCAA supplementation in this model. Surprisingly, BCAA supplementation exacerbated neurodevelopmental deficits and did not correct biochemical abnormalities despite increasing systemic BCAA levels. These data suggest that aberrant flux through the BCAA catabolic pathway, not just BCAA insufficiency, may contribute to disease pathology. In support of this conclusion, genetic re-regulation of BCAA catabolism, through Dbt haploinsufficiency, partially rescued biochemical and behavioral phenotypes in BCKDK deficient mice. Collectively, these data raise into question assumptions widely made about the pathophysiology of BCKDK insufficiency and suggest a novel approach to develop potential therapies for this disease.

Progesterone for Neurodevelopment in Fetuses With Congenital Heart Defects: A Randomized Clinical Trial.

Importance: Neurodevelopmental outcomes for children with congenital heart defects (CHD) have improved minimally over the past 20 years. Objectives: To assess the feasibility and tolerability of maternal progesterone therapy as well as the magnitude of the effect on neurodevelopment for fetuses with CHD. Design, Setting, and Participants: This double-blinded individually randomized parallel-group clinical trial of vaginal natural progesterone therapy vs placebo in participants carrying fetuses with CHD was conducted between July 2014 and November 2021 at a quaternary care children's hospital. Participants included maternal-fetal dyads where the fetus had CHD identified before 28 weeks' gestational age and was likely to need surgery with cardiopulmonary bypass in the neonatal period. Exclusion criteria included a major genetic or extracardiac anomaly other than 22q11 deletion syndrome and known contraindication to progesterone. Statistical analysis was performed June 2022 to April 2024. Intervention: Participants were 1:1 block-randomized to vaginal progesterone or placebo by diagnosis: hypoplastic left heart syndrome (HLHS), transposition of the great arteries (TGA), and other CHD diagnoses. Treatment was administered twice daily between 28 and up to 39 weeks' gestational age. Main Outcomes and Measures: The primary outcome was the motor score of the Bayley Scales of Infant and Toddler Development-III; secondary outcomes included language and cognitive scales. Exploratory prespecified subgroups included cardiac diagnosis, fetal sex, genetic profile, and maternal fetal environment. Results: The 102 enrolled fetuses primarily had HLHS (n = 52 [50.9%]) and TGA (n = 38 [37.3%]), were more frequently male (n = 67 [65.7%]), and without genetic anomalies (n = 61 [59.8%]). The mean motor score differed by 2.5 units (90% CI, -1.9 to 6.9 units; P = .34) for progesterone compared with placebo, a value not statistically different from 0. Exploratory subgroup analyses suggested treatment heterogeneity for the motor score for cardiac diagnosis (P for interaction = .03) and fetal sex (P for interaction = .04), but not genetic profile (P for interaction = .16) or maternal-fetal environment (P for interaction = .70). Conclusions and Relevance: In this randomized clinical trial of maternal progesterone therapy, the overall effect was not statistically different from 0. Subgroup analyses suggest heterogeneity of the response to progesterone among CHD diagnosis and fetal sex. Trial Registration: ClinicalTrials.gov Identifier: NCT02133573.

Improved specificity and efficacy of base-editing therapies with hybrid guide RNAs.

Phenylketonuria (PKU), hereditary tyrosinemia type 1 (HT1), and mucopolysaccharidosis type 1 (MPSI) are autosomal recessive disorders linked to the phenylalanine hydroxylase (PAH) gene, fumarylacetoacetate hydrolase (FAH) gene, and alpha-L-iduronidase (IDUA) gene, respectively. Potential therapeutic strategies to ameliorate disease include corrective editing of pathogenic variants in the PAH and IDUA genes and, as a variant-agnostic approach, inactivation of the 4-hydroxyphenylpyruvate dioxygenase (HPD) gene, a modifier of HT1, via adenine base editing. Here we evaluated the off-target editing profiles of therapeutic lead guide RNAs (gRNAs) that, when combined with adenine base editors correct the recurrent PAH P281L variant, PAH R408W variant, or IDUA W402X variant or disrupt the HPD gene in human hepatocytes. To mitigate off-target mutagenesis, we systematically screened hybrid gRNAs with DNA nucleotide substitutions. Comprehensive and variant-aware specificity profiling of these hybrid gRNAs reveal dramatically reduced off-target editing and reduced bystander editing. Lastly, in a humanized PAH P281L mouse model, we showed that when formulated in lipid nanoparticles (LNPs) with adenine base editor mRNA, selected hybrid gRNAs revert the PKU phenotype, substantially enhance on-target editing, and reduce bystander editing in vivo. These studies highlight the utility of hybrid gRNAs to improve the safety and efficacy of base-editing therapies.

Consensus guidelines for the monitoring and management of metachromatic leukodystrophy in the United States.

Metachromatic leukodystrophy (MLD) is a fatal, progressive neurodegenerative disorder caused by biallelic pathogenic mutations in the ARSA (Arylsulfatase A) gene. With the advent of presymptomatic diagnosis and the availability of therapies with a narrow window for intervention, it is critical to define a standardized approach to diagnosis, presymptomatic monitoring, and clinical care. To meet the needs of the MLD community, a panel of MLD experts was established to develop disease-specific guidelines based on healthcare resources in the United States. This group developed a consensus opinion for best-practice recommendations, as follows: (i) Diagnosis should include both genetic and biochemical testing; (ii) Early diagnosis and treatment for MLD is associated with improved clinical outcomes; (iii) The panel supported the development of newborn screening to accelerate the time to diagnosis and treatment; (iv) Clinical management of MLD should include specialists familiar with the disease who are able to follow patients longitudinally; (v) In early onset MLD, including late infantile and early juvenile subtypes, ex vivo gene therapy should be considered for presymptomatic patients where available; (vi) In late-onset MLD, including late juvenile and adult subtypes, hematopoietic cell transplant (HCT) should be considered for patients with no or minimal disease involvement. This document summarizes current guidance on the presymptomatic monitoring of children affected by MLD as well as the clinical management of symptomatic patients. Future data-driven evidence and evolution of these recommendations will be important to stratify clinical treatment options and improve clinical care.

Effective Gene Therapy for Metachromatic Leukodystrophy Achieved with Minimal Lentiviral Genomic Integrations.

Metachromatic leukodystrophy (MLD) is a fatal lysosomal storage disease (LSD) characterized by the deficient enzymatic activity of arylsulfatase A (ARSA). Combined autologous hematopoietic stem cell transplant (HSCT) with lentiviral (LV) based gene therapy has great potential to treat MLD. However, if enzyme production is inadequate, this could result in continued loss of motor function, implying a high vector copy number (VCN) requirement for optimal enzymatic output. This may place children at increased risk for genomic toxicity due to higher VCN. We increased the expression of ARSA cDNA at single integration by generating novel LVs, optimizing ARSA expression, and enhancing safety. In addition, our vectors achieved optimal transduction in mouse and human HSC with minimal multiplicity of infection (MOI). Our top-performing vector (EA1) showed at least 4X more ARSA activity than the currently EU-approved vector and a superior ability to secrete vesicle-associated ARSA, a critical modality to transfer functional enzymes from microglia to oligodendrocytes. Three-month-old Arsa -KO MLD mice transplanted with Arsa -KO BM cells transduced with 0.6 VCN of EA1 demonstrated behavior and CNS histology matching WT mice. Our novel vector boosts efficacy while improving safety as a robust approach for treating early symptomatic MLD patients.

Lmod2 is necessary for effective skeletal muscle contraction.

Muscle contraction is a regulated process driven by the sliding of actin-thin filaments over myosin-thick filaments. Lmod2 is an actin filament length regulator and essential for life since human mutations and complete loss of Lmod2 in mice lead to dilated cardiomyopathy and death. To study the little-known role of Lmod2 in skeletal muscle, we created a mouse model with Lmod2 expressed exclusively in the heart but absent in skeletal muscle. Loss of Lmod2 in skeletal muscle results in decreased force production in fast- and slow-twitch muscles. Soleus muscle from rescued Lmod2 knockout mice have shorter thin filaments, increased Lmod3 levels, and present with a myosin fiber type switch from fast myosin heavy chain (MHC) IIA to the slower MHC I isoform. Since Lmod2 regulates thin-filament length in slow-twitch but not fast-twitch skeletal muscle and force deficits were observed in both muscle types, this work demonstrates that Lmod2 regulates skeletal muscle contraction, independent of its role in thin-filament length regulation.

NeuroTri2-VISDOT: An open-access tool to harness the power of second trimester human single cell data to inform models of Mendelian neurodevelopmental disorders.

Whole exome and genome sequencing, coupled with refined bioinformatic pipelines, have enabled improved diagnostic yields for individuals with Mendelian conditions and have led to the rapid identification of novel syndromes. For many Mendelian neurodevelopmental disorders (NDDs), there is a lack of pre-existing model systems for mechanistic work. Thus, it is critical for translational researchers to have an accessible phenotype- and genotype-informed approach for model system selection. Single-cell RNA sequencing data can be informative in such an approach, as it can indicate which cell types express a gene of interest at the highest levels across time. For Mendelian NDDs, such data for the developing human brain is especially useful. A valuable single-cell RNA sequencing dataset of the second trimester developing human brain was produced by Bhaduri et al in 2021, but access to these data can be limited by computing power and the learning curve of single-cell data analysis. To reduce these barriers for translational research on Mendelian NDDs, we have built the web-based tool, Neurodevelopment in Trimester 2 - VIsualization of Single cell Data Online Tool (NeuroTri2-VISDOT), for exploring this single-cell dataset, and we have employed it in several different settings to demonstrate its utility for the translational research community.

A base editing strategy using mRNA-LNPs for in vivo correction of the most frequent phenylketonuria variant.

The c.1222C>T (p.Arg408Trp) phenylalanine hydroxylase (PAH) variant is the most frequent cause of phenylketonuria (PKU), an autosomal recessive disorder characterized by accumulation of blood phenylalanine (Phe) to neurotoxic levels. Here we devised a therapeutic base editing strategy to correct the variant, using prime-edited hepatocyte cell lines engineered with the c.1222C>T variant to screen a variety of adenine base editors and guide RNAs in vitro, followed by assessment in c.1222C>T humanized mice in vivo. We found that upon delivery of a selected adenine base editor mRNA/guide RNA combination into mice via lipid nanoparticles (LNPs), there was sufficient PAH editing in the liver to fully normalize blood Phe levels within 48 h. This work establishes the viability of a base editing strategy to correct the most common pathogenic variant found in individuals with the most common inborn error of metabolism, albeit with potential limitations compared with other genome editing approaches.

Biochemical signatures of disease severity in multiple sulfatase deficiency.

Sulfatases catalyze essential cellular reactions, including degradation of glycosaminoglycans (GAGs). All sulfatases are post-translationally activated by the formylglycine generating enzyme (FGE) which is deficient in multiple sulfatase deficiency (MSD), a neurodegenerative lysosomal storage disease. Historically, patients were presumed to be deficient of all sulfatase activities; however, a more nuanced relationship is emerging. Each sulfatase may differ in their degree of post-translational modification by FGE, which may influence the phenotypic spectrum of MSD. Here, we evaluate if residual sulfatase activity and accumulating GAG patterns distinguish cases from controls and stratify clinical severity groups in MSD. We quantify sulfatase activities and GAG accumulation using three complementary methods in MSD participants. Sulfatases differed greatly in their tolerance of reduction in FGE-mediated activation. Enzymes that degrade heparan sulfate (HS) demonstrated lower residual activities than those that act on other GAGs. Similarly, HS-derived urinary GAG subspecies preferentially accumulated, distinguished cases from controls, and correlated with disease severity. Accumulation patterns of specific sulfatase substrates in MSD provide fundamental insights into sulfatase regulation and will serve as much-needed biomakers for upcoming clinical trials. This work highlights that biomarker investigation of an ultra-rare disease can simultaneously inform our understanding of fundamental biology and advance clinical trial readiness efforts.

Diagnostic Yield of Exome Sequencing in Pediatric Cardiomyopathy.

OBJECTIVE: To assess the diagnostic yield of exome sequencing (ES) in pediatric cardiomyopathy. STUDY DESIGN: A single-institution, retrospective chart review of 91 patients with pediatric cardiomyopathy was performed. While pediatric cardiomyopathy is often genetic in nature, no genetic test is recommended as standard of care. All our patients were diagnosed with cardiomyopathy and evaluated by a medical geneticist between January 2010 through September 2022. Demographic information and clinical data were abstracted. RESULTS: Of 91 patients with pediatric cardiomyopathy, 36 (39.6%) received a diagnosis by ES. Twenty-two (61.1%) of these diagnoses would have been missed on cardiac multigene panel testing. The diagnostic yield for cardiomyopathy presenting under 1 year of age was 38.3%, while the yield for patients over 1 year of age was 41.9%. CONCLUSIONS: ES has a high diagnostic yield in pediatric cardiomyopathy compared with a gene panel. Over 60% of patients with diagnosis by ES would not have received their molecular genetic diagnosis if only multigene panel testing was sent. Diagnostic yield did not vary significantly between the subtypes of cardiomyopathy and patient age groups, highlighting the likely clinical utility of ES for all pediatric cardiomyopathy patients.

A novel iPSC model reveals selective vulnerability of neurons in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is an ultra-rare, inherited lysosomal storage disease caused by mutations in the gene sulfatase modifying factor 1 (SUMF1). MSD is characterized by the functional deficiency of all sulfatase enzymes, leading to the storage of sulfated substrates including glycosaminoglycans (GAGs), sulfolipids, and steroid sulfates. Patients with MSD experience severe neurological impairment, hearing loss, organomegaly, corneal clouding, cardiac valve disease, dysostosis multiplex, contractures, and ichthyosis. Here, we generated a novel human model of MSD by reprogramming patient peripheral blood mononuclear cells to establish an MSD induced pluripotent stem cell (iPSC) line (SUMF1 p.A279V). We also generated an isogenic control iPSC line by correcting the pathogenic variant with CRISPR/Cas9 gene editing. We successfully differentiated these iPSC lines into neural progenitor cells (NPCs) and NGN2-induced neurons (NGN2-iN) to model the neuropathology of MSD. Mature neuronal cells exhibited decreased SUMF1 gene expression, increased lysosomal stress, impaired neurite outgrowth and maturation, reduced sulfatase activities, and GAG accumulation. Interestingly, MSD iPSCs and NPCs did not exhibit as severe of phenotypes, suggesting that as neurons differentiate and mature, they become more vulnerable to loss of SUMF1. In summary, we demonstrate that this human iPSC-derived neuronal model recapitulates the cellular and biochemical features of MSD. These cell models can be used as tools to further elucidate the mechanisms of MSD pathology and for the development of therapeutics.

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

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

Partial suppression of BCAA catabolism as a potential therapy for BCKDK deficiency.

Branched chain ketoacid dehydrogenase kinase (BCKDK) deficiency is a recently described inherited neurometabolic disorder of branched chain amino acid (BCAA) metabolism implying increased BCAA catabolism. It has been hypothesized that a severe reduction in systemic BCAA levels underlies the disease pathophysiology, and that BCAA supplementation may ameliorate disease phenotypes. To test this hypothesis, we characterized a recent mouse model of BCKDK deficiency and evaluated the efficacy of enteral BCAA supplementation in this model. Surprisingly, BCAA supplementation exacerbated neurodevelopmental deficits and did not correct biochemical abnormalities despite increasing systemic BCAA levels. These data suggest that aberrant flux through the BCAA catabolic pathway, not just BCAA insufficiency, may contribute to disease pathology. In support of this conclusion, genetic re-regulation of BCAA catabolism, through Dbt haploinsufficiency, partially rescued biochemical and behavioral phenotypes in BCKDK deficient mice. Collectively, these data raise into question assumptions widely made about the pathophysiology of BCKDK insufficiency and suggest a novel approach to develop potential therapies for this disease.

Electrocardiographic Findings in Genotype-Positive and Non-sarcomeric Children with Definite Hypertrophic Cardiomyopathy and Subclinical Variant Carriers.

In children with hypertrophic cardiomyopathy (HCM), the genotype-phenotype association of abnormal electrocardiographic (ECG) features in the backdrop of gene positivity has not been well described. This study aimed to describe the abnormal ECG findings in children with HCM harboring who have genetic variants and determine the association with major adverse cardiac events (MACE). We retrospectively analyzed 81 variants-positive, phenotype-positive (V+P+), 66 variant-positive, phenotype-negative (V+P-), and 85 non-sarcomeric subjects. We analyzed ECG findings and clinical outcomes in the three groups of subjects. Repolarization abnormalities (ST and T wave changes) and pathologic Q waves were the most common abnormalities in variant and non-sarcomeric subjects. The V+P+ group showed higher occurrence of ST segment changes and T wave abnormalities compared to V+P- group. Independent predictors of MACE included ST segment changes (OR 3.54, CI 1.20-10.47, p = 0.022). T wave changes alone did not predict outcome (OR 2.13, CI 0.75-6.07, p = 0.157), but combined repolarization abnormalities (ST+T changes) were strong predictors of MACE (OR 5.84, CI 1.43-23.7, p = 0.014) than ST segment changes alone. Maximal wall z score by echocardiography was a predictor of MACE (OR 1.21, CI 1.07-1.37, p = 0.002). Despite the presence of significant myocardial hypertrophy (z score > 4.7), voltage criteria for LVH were much less predictive. In the non-sarcomeric group, RVH was significantly associated with MACE (OR 3.85, CI 1.08-13.73, p = 0.038). These abnormal ECG findings described on the platform of known genetic status and known myocardial hypertrophy may add incremental value to the diagnosis and surveillance of disease progression in children with HCM. Select ECG findings, particularly repolarization abnormalities, may serve as predictors of MACE in children.

High density SNP array and reanalysis of genome sequencing uncovers CNVs associated with neurodevelopmental disorders in KOLF2.1J iPSCs.

The KOLF2.1J iPSC line was recently proposed as a reference iPSC to promote the standardization of research studies in the stem cell field. Due to overall good performance differentiating to neural cell lineages, high gene editing efficiency, and absence of genetic variants associated to neurological disorders KOLF2.1J iPSC line was particularly recommended for neurodegenerative disease modeling. However, our work uncovers that KOLF2.1J hPSCs carry heterozygous small copy number variants (CNVs) that cause DTNBP1, JARID2 and ASTN2 haploinsufficiencies, all of which are associated with neurological disorders. We further determine that these CNVs arose in vitro over the course of KOLF2.1J iPSC generation from a healthy donor-derived KOLF2 iPSC line and affect the expression of DNTBP1, JARID2 and ASTN2 proteins in KOLF2.1J iPSCs and neural progenitors. Therefore, our study suggests that KOLF2.1J iPSCs carry genetic variants that may be deleterious for neural cell lineages. This data is essential for a careful interpretation of neural cell studies derived from KOLF2.1J iPSCs and highlights the need for a catalogue of iPSC lines that includes a comprehensive genome characterization analysis.

Rapid and definitive treatment of phenylketonuria in variant-humanized mice with corrective editing.

Phenylketonuria (PKU), an autosomal recessive disorder caused by pathogenic variants in the phenylalanine hydroxylase (PAH) gene, results in the accumulation of blood phenylalanine (Phe) to neurotoxic levels. Current dietary and medical treatments are chronic and reduce, rather than normalize, blood Phe levels. Among the most frequently occurring PAH variants in PKU patients is the P281L (c.842C>T) variant. Using a CRISPR prime-edited hepatocyte cell line and a humanized PKU mouse model, we demonstrate efficient in vitro and in vivo correction of the P281L variant with adenine base editing. With the delivery of ABE8.8 mRNA and either of two guide RNAs in vivo using lipid nanoparticles (LNPs) in humanized PKU mice, we observe complete and durable normalization of blood Phe levels within 48 h of treatment, resulting from corrective PAH editing in the liver. These studies nominate a drug candidate for further development as a definitive treatment for a subset of PKU patients.

Characterizing asparagine synthetase deficiency variants in lymphoblastoid cell lines.

Asparagine synthetase (ASNS) catalyzes the synthesis of asparagine (Asn) from aspartate and glutamine. Biallelic mutations in the ASNS gene result in ASNS Deficiency (ASNSD). Children with ASNSD exhibit congenital microcephaly, epileptic-like seizures, and continued brain atrophy, often leading to premature mortality. This report describes a 4-year-old male with global developmental delay and seizures with two novel mutations in the ASNS gene, c.614A > C (maternal) and c.1192dupT (paternal) encoding p.H205P and p.Y398Lfs*4 variants, respectively. We employed the novel use of immortalized lymphoblastoid cell lines (LCL) to show that the proliferation of the heterozygotic parental LCL was not severely affected by culture in Asn-free medium, but growth of the child's cells was suppressed by about 50%. Asn production by the LCL from both the father and the child was significantly decreased relative to the mother's cells. mRNA and protein analysis of the paternal LCL cells for the Y398Lfs*4 variant revealed reductions in both. Attempts to ectopically express the truncated Y398Lfs*4 variant in either HEK293T or ASNS-null cells resulted in little or no detectable protein. Expression and purification of the H205P variant from HEK293T cells revealed enzymatic activity similar to wild-type ASNS. Stable expression of WT ASNS rescued the growth of ASNS-null JRS cells in Asn-free medium and the H205P variant was only slightly less effective. However, the Y398Lfs*4 variant appeared to be unstable in JRS cells. These results indicate that co-expression of the H205P and Y398Lfs*4 variants leads to a significant reduction in Asn synthesis and cellular growth.