Development, validation and application of single molecule molecular inversion probe based novel integrated genetic screening method for 29 common lysosomal storage disorders in India.

BACKGROUND: Current clinical diagnosis pathway for lysosomal storage disorders (LSDs) involves sequential biochemical enzymatic tests followed by DNA sequencing, which is iterative, has low diagnostic yield and is costly due to overlapping clinical presentations. Here, we describe a novel low-cost and high-throughput sequencing assay using single-molecule molecular inversion probes (smMIPs) to screen for causative single nucleotide variants (SNVs) and copy number variants (CNVs) in genes associated with 29 common LSDs in India. RESULTS: 903 smMIPs were designed to target exon and exon-intron boundaries of targeted genes (n = 23; 53.7 kb of the human genome) and were equimolarly pooled to create a sequencing library. After extensive validation in a cohort of 50 patients, we screened 300 patients with either biochemical diagnosis (n = 187) or clinical suspicion (n = 113) of LSDs. A diagnostic yield of 83.4% was observed in patients with prior biochemical diagnosis of LSD. Furthermore, diagnostic yield of 73.9% (n = 54/73) was observed in patients with high clinical suspicion of LSD in contrast with 2.4% (n = 1/40) in patients with low clinical suspicion of LSD. In addition to detecting SNVs, the assay could detect single and multi-exon copy number variants with high confidence. Critically, Niemann-Pick disease type C and neuronal ceroid lipofuscinosis-6 diseases for which biochemical testing is unavailable, could be diagnosed using our assay. Lastly, we observed a non-inferior performance of the assay in DNA extracted from dried blood spots in comparison with whole blood. CONCLUSION: We developed a flexible and scalable assay to reliably detect genetic causes of 29 common LSDs in India. The assay consolidates the detection of multiple variant types in multiple sample types while having improved diagnostic yield at same or lower cost compared to current clinical paradigm.

Elevated cholesterol in ATAD3 mutants is a compensatory mechanism that leads to membrane cholesterol aggregation.

Aberrant cholesterol metabolism causes neurological disease and neurodegeneration, and mitochondria have been linked to perturbed cholesterol homeostasis via the study of pathological mutations in the ATAD3 gene cluster. However, whether the cholesterol changes were compensatory or contributory to the disorder was unclear, and the effects on cell membranes and the wider cell were also unknown. Using patient-derived cells, we show that cholesterol perturbation is a conserved feature of pathological ATAD3 variants that is accompanied by an expanded lysosome population containing membrane whorls characteristic of lysosomal storage diseases. Lysosomes are also more numerous in Drosophila neural progenitor cells expressing mutant Atad3, which exhibit abundant membrane-bound cholesterol aggregates, many of which co-localize with lysosomes. By subjecting the Drosophila Atad3 mutant to nutrient restriction and cholesterol supplementation, we show that the mutant displays heightened cholesterol dependence. Collectively, these findings suggest that elevated cholesterol enhances tolerance to pathological ATAD3 variants; however, this comes at the cost of inducing cholesterol aggregation in membranes, which lysosomal clearance only partly mitigates.

Biomarkers for gene therapy clinical trials of lysosomal storage disorders.

Lysosomal storage disorders (LSDs) are multisystemic progressive disorders caused by defects in proteins involved in lysosomal function. Different gene therapy strategies are under clinical investigation in several LSDs to overcome the limitations of available treatments. However, LSDs are slowly progressive diseases that require long-term studies to establish the efficacy of experimental treatments. Biomarkers can be reliable substitutes for clinical responses and improve the efficiency of clinical trials, especially when long-term disease interventions are evaluated. In this review, we summarize both available and future biomarkers for LSDs and discuss their strengths and weaknesses.

Anti-amyloid treatment is broadly effective in neuronopathic mucopolysaccharidoses and synergizes with gene therapy in MPS-IIIA.

Mucopolysaccharidoses (MPSs) are childhood diseases caused by inherited deficiencies in glycosaminoglycan degradation. Most MPSs involve neurodegeneration, which to date is untreatable. Currently, most therapeutic strategies aim at correcting the primary genetic defect. Among these strategies, gene therapy has shown great potential, although its clinical application is challenging. We have shown previously in an MPS-IIIA mouse model that the molecular tweezer (MT) CLR01, a potent, broad-spectrum anti-amyloid small molecule, inhibits secondary amyloid storage, facilitates amyloid clearance, and protects against neurodegeneration. Here, we demonstrate that combining CLR01 with adeno-associated virus (AAV)-mediated gene therapy, targeting both the primary and secondary pathologic storage in MPS-IIIA mice, results in a synergistic effect that improves multiple therapeutic outcomes compared to each monotherapy. Moreover, we demonstrate that CLR01 is effective therapeutically in mouse models of other forms of neuronopathic MPS, MPS-I, and MPS-IIIC. These strongly support developing MTs as an effective treatment option for neuronopathic MPSs, both on their own and in combination with gene therapy, to improve therapeutic efficacy and translation into clinical application.

Hematopoietic stem cell transplantation leads to biochemical and functional correction in two mouse models of acid ceramidase deficiency.

Farber disease (FD) and spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) are ultra-rare lysosomal storage disorders caused by deficient acid ceramidase (ACDase) activity. Although both conditions are caused by mutations in the ASAH1 gene, clinical presentations differ considerably. FD patients usually die in childhood, while SMA-PME patients can live until adulthood. There is no treatment for FD or SMA-PME. Hematopoietic stem cell transplantation (HSCT) and gene therapy strategies for the treatment of ACDase deficiency are being investigated. We have previously generated and characterized mouse models of both FD and SMA-PME that recapitulate the symptoms described in patients. Here, we show that HSCT improves lifespan, behavior, hematopoietic system anomalies, and plasma cytokine levels and significantly reduces histiocytic infiltration and ceramide accumulation throughout the tissues investigated, including the CNS, in both models of ACDase-deficient mice. HSCT was also successful in preventing lesion development and significant demyelination of the spinal cord seen in SMA-PME mice. Importantly, we note that only early and generally pre-symptomatic treatment was effective, and kidney impairment was not improved in either model.

An empowered, clinically viable hematopoietic stem cell gene therapy for the treatment of multisystemic mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a rare X-linked recessive lysosomal storage disorder due to a mutation in the lysosomal enzyme iduronate-2-sulfatase (IDS) gene. IDS deficiency leads to a progressive, multisystem accumulation of glycosaminoglycans (GAGs) and results in central nervous system (CNS) manifestations in the severe form. We developed up to clinical readiness a new hematopoietic stem cell (HSC) gene therapy approach for MPS II that benefits from a novel highly effective transduction protocol. We first provided proof of concept of efficacy of our approach aimed at enhanced IDS enzyme delivery to the CNS in a murine study of immediate translational value, employing a lentiviral vector (LV) encoding a codon-optimized human IDS cDNA. Then the therapeutic LV was tested for its ability to efficiently and safely transduce bona fide human HSCs in clinically relevant conditions according to a standard vs. a novel protocol that demonstrated superior ability to transduce bona fide long-term repopulating HSCs. Overall, these results provide strong proof of concept for the clinical translation of this approach for the treatment of Hunter syndrome.

Brain Disorders Due to Lysosomal Dysfunction.

Recent studies of autophagic and lysosomal pathways have significantly changed our understanding of lysosomes; once thought to be simple degradative and recycling centers, lysosomes are now known to be organelles capable of influencing signal transduction, via the mammalian target of rapamycin complex 1 (mTORC1), and regulating gene expression, via transcription factor EB (TFEB) and other transcription factors. These pathways are particularly relevant to maintaining brain homeostasis, as dysfunction of the endolysosomal and autophagic pathways has been associated with common neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's, and lysosomal storage disorders, a group of inherited disorders characterized by the intralysosomal buildup of partially degraded metabolites. This review focuses on the cellular biology of lysosomes and discusses the possible mechanisms by which disruption of their function contributes to neurodegeneration. We also review and discuss how targeting TFEB and lysosomes may offer innovative therapeutic approaches for treating a wide range of neurological conditions.

First-in-human single-dose study of nizubaglustat, a dual inhibitor of ceramide glucosyltransferase and non-lysosomal glucosylceramidase: Safety, tolerability, pharmacokinetics, and pharmacodynamics of single ascending and multiple doses in healthy adults.

Nizubaglustat is a novel, orally available, brain penetrant, potent, and selective dual inhibitor of ceramide glucosyltranferase and non-lysosomal neutral glucosylceramidase (NLGase), which is currently under development for the treatment of subjects with neurological manifestations in primary and secondary gangliosidoses. The objectives of this first-in-human study were to evaluate the safety and tolerability, pharmacokinetics, and pharmacodynamics (PD) of single oral doses of nizubaglustat after single (1, 3, and 9 mg) and multiple oral doses (9 mg once per day (QD) over 14 days) in healthy adults. Nizubaglustat was rapidly absorbed and systemic exposure was dose-proportional. Steady-state was achieved after three days of QD multiple dosing with minimal accumulation. Renal clearance accounted for around 15% of nizubaglustat elimination. Following multiple dosing, plasma concentrations of glucosylceramide (GlcCer), lactosylceramide (LacCer), and monosialodihexosylganglioside (GM3) decreased to a nadir at Day 10. PD target engagement of GCS inhibition was shown by a median decrease from baseline of plasma concentrations of GlcCer, LacCer, and GM3 ganglioside by 70%, 50%, and 48%, respectively. NLGase inhibition was also manifested by increased concentrations of GlcCer in cerebrospinal fluid from Day 1 to Day 14. Nizubaglustat was safe and well-tolerated at all doses tested. Consistent with the high selectivity, and the absence of intestinal disaccharidases inhibition, no cases of diarrhea were reported. No decreased appetite or weight loss was noted. Only treatment-emergent adverse events with preferred terms belonging to the system organ class skin and subcutaneous disorders of mild intensity were reported as drug-related in the nizubaglustat arm, in line with the pharmacological mechanism targeting glucosylceramide metabolism. Taken together, these data support QD dosing of nizubaglustat and its ongoing development in patients with primary and secondary forms of gangliosidoses.

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.

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.

Muscle-specific, liver-detargeted adeno-associated virus gene therapy rescues Pompe phenotype in adult and neonate Gaa-/- mice.

Pompe disease (PD) is a neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency. Reduced GAA activity leads to pathological glycogen accumulation in cardiac and skeletal muscles responsible for severe heart impairment, respiratory defects, and muscle weakness. Enzyme replacement therapy with recombinant human GAA (rhGAA) is the standard-of-care treatment for PD, however, its efficacy is limited due to poor uptake in muscle and the development of an immune response. Multiple clinical trials are ongoing in PD with adeno-associated virus (AAV) vectors based on liver- and muscle-targeting. Current gene therapy approaches are limited by liver proliferation, poor muscle targeting, and the potential immune response to the hGAA transgene. To generate a treatment tailored to infantile-onset PD, we took advantage of a novel AAV capsid able to increase skeletal muscle targeting compared to AAV9 while reducing liver overload. When combined with a liver-muscle tandem promoter (LiMP), and despite the extensive liver-detargeting, this vector had a limited immune response to the hGAA transgene. This combination of capsid and promoter with improved muscle expression and specificity allowed for glycogen clearance in cardiac and skeletal muscles of Gaa-/- adult mice. In neonate Gaa-/- , complete rescue of glycogen content and muscle strength was observed 6 months after AAV vector injection. Our work highlights the importance of residual liver expression to control the immune response toward a potentially immunogenic transgene expressed in muscle. In conclusion, the demonstration of the efficacy of a muscle-specific AAV capsid-promoter combination for the full rescue of PD manifestation in both neonate and adult Gaa-/- provides a potential therapeutic avenue for the infantile-onset form of this devastating disease.

A phase III, open-label clinical trial evaluating pegunigalsidase alfa administered every 4 weeks in adults with Fabry disease previously treated with other enzyme replacement therapies.

Pegunigalsidase alfa, a PEGylated α-galactosidase A enzyme replacement therapy (ERT) for Fabry disease, has a longer plasma half-life than other ERTs administered intravenously every 2 weeks (E2W). BRIGHT (NCT03180840) was a phase III, open-label study in adults with Fabry disease, previously treated with agalsidase alfa or beta E2W for ≥3 years, who switched to 2 mg/kg pegunigalsidase alfa every 4 weeks (E4W) for 52 weeks. Primary objective assessed safety, including number of treatment-emergent adverse events (TEAEs). Thirty patients were enrolled (24 males); 23 previously received agalsidase beta. Pegunigalsidase alfa plasma concentrations remained above the lower limit of quantification throughout the 4-week dosing interval. Thirty-three of 182 TEAEs (in 9 patients) were considered treatment-related; all were mild/moderate. No patients developed de novo anti-drug antibodies (ADAs). In the efficacy analysis (n = 29), median (inter-quartile range) eGFR change from baseline over 52 weeks was -1.9 (-5.9; 1.8) mL/min/1.73 m2 (n = 28; males [n = 22]: -2.4 [-5.2; 3.2]; females [n = 6]: -0.7 [-9.2; 2.0]). Overall, median eGFR slope was -1.9 (-8.3; 1.9) mL/min/1.73 m2/year (ADA-negative [n = 20]: -1.2 [-6.4; 2.6]; ADA-positive [n = 9]: -8.4 [-11.6; -1.0]). Lyso-Gb3 concentrations were low and stable in females, with a slight increase in males (9/24 ADA-positive). The BRIGHT study results suggest that 2 mg/kg pegunigalsidase alfa E4W is tolerated well in stable adult patients with Fabry disease. Due to the low number of patients in this study, more research is needed to demonstrate the effects of pegunigalsidase alfa given E4W. Further evidence, outside of this clinical trial, should be factored in for physicians to prolong the biweekly ERT intervals to E4W. TAKE-HOME MESSAGE: Treatment with 2 mg/kg pegunigalsidase alfa every 4 weeks could offer a new treatment option for patients with Fabry disease.

Discovery and characterization of novel TRPML1 agonists.

Screening a library of >100,000 compounds identified the substituted tetrazole compound 1 as a selective TRPML1 agonist. Both enantiomers of compound 1 were separated and profiled in vitro and in vivo. Their selectivity, ready availability and CNS penetration should enable them to serve as the tool compounds of choice in future TRPML1 channel activation studies. SAR studies on conformationally locked macrocyclic analogs further improved the TRPML1 agonist potency while retaining the selectivity.

Ellagic acid and its metabolites urolithins A/B ameliorate most common disease phenotypes in cellular and mouse models for lysosomal storage disorders by enhancing extracellular vesicle secretion.

Niemann Pick diseases types A (NPDA) and C (NPDC) are lysosomal storage disorders (LSDs) leading to cognitive impairment, neurodegeneration, and early death. NPDA and NPDC have different genetic origins, being caused by mutations in the acid sphingomyelinase (ASM) or the cholesterol transport protein NPC1, respectively. However, they share a common pathological hallmark in the accumulation of lipids in the endolysosomal compartment. Here, we tested the hypothesis that polyphenols reduce lipid overload in NPD cells by enhancing the secretion of extracellular vesicles (ECVs). We show that among the polyphenols tested, the ellagic acid metabolites, urolithin A and B, were the safest and most efficient in increasing ECV secretion. They reduced levels of accumulating lipids and lysosomal size and permeabilization in cultured bone marrow-derived macrophages and neurons from ASMko and NPC1 mutant mice, which mimic NPDA and NPDC, respectively. Moreover, oral treatment with ellagic acid reduced lipid levels, ameliorated lysosomal alterations, and diminished microglia activation in the brain of NPD mice. These results support the therapeutic value of ECV secretion and polyphenols for NPDs, which may also help treat other LSDs characterized by intracellular lipid overload.

Influence of initial clinical suspicion on the diagnostic yield of laboratory enzymatic testing in lysosomal storage disorders. Experience from a multispecialty hospital.

Lysosomal storage disorders (LSD) are a group of inherited metabolic diseases mainly caused by a deficiency of lysosomal hydrolases, resulting in a gradual accumulation of non-degraded substrates in different tissues causing the characteristic clinical manifestations of such disorders. Confirmatory tests of suspected LSD individuals include enzymatic and genetic testing. A well-oriented clinical suspicion can improve the cost-effectiveness of confirmatory tests and reduce the time expended to achieve the diagnosis. Thus, this work aims to retrospectively study the influence of clinical orientation on the diagnostic yield of enzymatic tests in LSD by retrieving clinical, biochemical, and genetic data obtained from subjects with suspicion of LSD. Our results suggest that the clinical manifestations at the time of diagnosis and the initial clinical suspicion can have a great impact on the diagnostic yield of enzymatic tests, and that clinical orientation performed in specialized clinical departments can contribute to improve it. In addition, the analysis of enzymatic tests as the first step in the diagnostic algorithm can correctly guide subsequent confirmatory genetic tests, in turn increasing their diagnostic yield. In summary, our results suggest that initial clinical suspicion plays a crucial role on the diagnostic yield of confirmatory enzymatic tests in LSD.

Potent and Selective Cell-Active Iminosugar Inhibitors of Human α-N-Acetylgalactosaminidase (α-NAGAL).

Glycoside hydrolases (GHs) are a class of enzymes with emerging roles in a range of disease. Selective GH inhibitors are sought to better understand their functions and assess the therapeutic potential of modulating their activities. Iminosugars are a promising class of GH inhibitors but typically lack the selectivity required to accurately perturb biological systems. Here, we describe a concise synthesis of iminosugar inhibitors of N-acetyl-α-galactosaminidase (α-NAGAL), the GH responsible for cleaving terminal α-N-acetylgalactosamine residues from glycoproteins and other glycoconjugates. Starting from non-carbohydrate precursors, this modular synthesis supported the identification of a potent (490 nM) and α-NAGAL selective (∼200-fold) guanidino-containing derivative DGJNGuan. To illustrate the cellular activity of this new inhibitor, we developed a quantitative fluorescence image-based method to measure levels of the Tn-antigen, a cellular glycoprotein substrate of α-NAGAL. Using this assay, we show that DGJNGuan exhibits excellent inhibition of α-NAGAL within cells using patient derived fibroblasts (EC50 =150 nM). Moreover, in vitro and in cell assays to assess levels of lysosomal ß-hexosaminidase substrate ganglioside GM2 show that DGJNGuan is selective whereas DGJNAc exhibits off-target inhibition both in vitro and within cells. DGJNGuan is a readily produced and selective tool compound that should prove useful for investigating the physiological roles of α-NAGAL.

Analysis of urinary oligosaccharide excretion patterns by UHPLC/HRAM mass spectrometry for screening of lysosomal storage disorders.

Oligosaccharidoses, sphingolipidoses and mucolipidoses are lysosomal storage disorders (LSDs) in which defective breakdown of glycan-side chains of glycosylated proteins and glycolipids leads to the accumulation of incompletely degraded oligosaccharides within lysosomes. In metabolic laboratories, these disorders are commonly diagnosed by thin-layer chromatography (TLC) but more recently also mass spectrometry-based approaches have been published. To expand the possibilities to screen for these diseases, we developed an ultra-high-performance liquid chromatography (UHPLC) with a high-resolution accurate mass (HRAM) mass spectrometry (MS) screening platform, together with an open-source iterative bioinformatics pipeline. This pipeline generates comprehensive biomarker profiles and allows for extensive quality control (QC) monitoring. Using this platform, we were able to identify α-mannosidosis, ß-mannosidosis, α-N-acetylgalactosaminidase deficiency, sialidosis, galactosialidosis, fucosidosis, aspartylglucosaminuria, GM1 gangliosidosis, GM2 gangliosidosis (M. Sandhoff) and mucolipidosis II/III in patient samples. Aberrant urinary oligosaccharide excretions were also detected for other disorders, including NGLY1 congenital disorder of deglycosylation, sialic acid storage disease, MPS type IV B and GSD II (Pompe disease). For the latter disorder, we identified heptahexose (Hex7), as a potential urinary biomarker, in addition to glucose tetrasaccharide (Glc4), for the diagnosis and monitoring of young onset cases of Pompe disease. Occasionally, so-called "neonate" biomarker profiles were observed in young patients, which were probably due to nutrition. Our UHPLC/HRAM-MS screening platform can easily be adopted in biochemical laboratories and allows for simple and robust screening and straightforward interpretation of the screening results to detect disorders in which aberrant oligosaccharides accumulate.

Advances in therapies for neurological lysosomal storage disorders.

Lysosomal Storage Disorders (LSDs) are a diverse group of inherited, monogenic diseases caused by functional defects in specific lysosomal proteins. The lysosome is a cellular organelle that plays a critical role in catabolism of waste products and recycling of macromolecules in the body. Disruption to the normal function of the lysosome can result in the toxic accumulation of storage products, often leading to irreparable cellular damage and organ dysfunction followed by premature death. The majority of LSDs have no curative treatment, with many clinical subtypes presenting in early infancy and childhood. Over two-thirds of LSDs present with progressive neurodegeneration, often in combination with other debilitating peripheral symptoms. Consequently, there is a pressing unmet clinical need to develop new therapeutic interventions to treat these conditions. The blood-brain barrier is a crucial hurdle that needs to be overcome in order to effectively treat the central nervous system (CNS), adding considerable complexity to therapeutic design and delivery. Enzyme replacement therapy (ERT) treatments aimed at either direct injection into the brain, or using blood-brain barrier constructs are discussed, alongside more conventional substrate reduction and other drug-related therapies. Other promising strategies developed in recent years, include gene therapy technologies specifically tailored for more effectively targeting treatment to the CNS. Here, we discuss the most recent advances in CNS-targeted treatments for neurological LSDs with a particular emphasis on gene therapy-based modalities, such as Adeno-Associated Virus and haematopoietic stem cell gene therapy approaches that encouragingly, at the time of writing are being evaluated in LSD clinical trials in increasing numbers. If safety, efficacy and improved quality of life can be demonstrated, these therapies have the potential to be the new standard of care treatments for LSD patients.

Early recognition of CLN3 disease facilitated by visual electrophysiology and multimodal imaging.

BACKGROUND: Neuronal ceroid lipofuscinosis is a group of neurodegenerative disorders with varying visual dysfunction. CLN3 is a subtype which commonly presents with visual decline. Visual symptomatology can be indistinct making early diagnosis difficult. This study reports ocular biomarkers of CLN3 patients to assist clinicians in early diagnosis, disease monitoring, and future therapy. METHODS: Retrospective review of 5 confirmed CLN3 patients in our eye clinic. Best corrected visual acuity (BCVA), electroretinogram (ERG), ultra-widefield (UWF) fundus photography and fundus autofluorescence (FAF), and optical coherence tomography (OCT) studies were undertaken. RESULTS: Five unrelated children, 4 females and 1 male, with median age of 6.2 years (4.6-11.7) at first assessment were investigated at the clinic from 2016 to 2021. Four homozygous and one heterozygous pathogenic CLN3 variants were found. Best corrected visual acuities (BCVAs) ranged from 0.18 to 0.88 logMAR at first presentation. Electronegative ERGs were identified in all patients. Bull's eye maculopathies found in all patients. Hyper-autofluorescence ring surrounding hypo-autofluorescence fovea on FAF was found. Foveal ellipsoid zone (EZ) disruptions were found in all patients with additional inner and outer retinal microcystic changes in one patient. Neurological problems noted included autism, anxiety, motor dyspraxia, behavioural issue, and psychomotor regression. CONCLUSIONS: CLN3 patients presented at median age 6.2 years with visual decline. Early onset maculopathy with an electronegative ERG and variable cognitive and motor decline should prompt further investigations including neuropaediatric evaluation and genetic assessment for CLN3 disease. The structural parameters such as EZ and FAF will facilitate ocular monitoring.

Fluoxetine ameliorates mucopolysaccharidosis type IIIA.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is an autosomal recessive disorder caused by mutations in SGSH involved in the degradation of heparan sulfate. MPS-IIIA presents severe neurological symptoms such as progressive developmental delay and cognitive decline, for which there is currently no treatment. Brain targeting represents the main challenge for therapeutics to treat MPS-IIIA, and the development of small-molecule-based treatments able to reach the CNS could be a relevant advance for therapy. Using cell-based high content imaging to survey clinically approved drugs in MPS-IIIA cells, we identified fluoxetine, a selective serotonin reuptake inhibitor. Fluoxetine increases lysosomal and autophagic functions via TFEB activation through a RagC-dependent mechanism. Mechanistically, fluoxetine increases lysosomal exocytosis in mouse embryonic fibroblasts from MPS-IIIA mice, suggesting that this process may be responsible for heparan sulfate clearance. In vivo, fluoxetine ameliorates somatic and brain pathology in a mouse model of MPS-IIIA by decreasing the accumulation of glycosaminoglycans and aggregated autophagic substrates, reducing inflammation, and slowing down cognitive deterioration. We repurposed fluoxetine for potential therapeutics to treat human MPS-IIIA disease.