The parent and family impact of CLN3 disease: an observational survey-based study.

BACKGROUND: CLN3 disease (also known as CLN3 Batten disease or Juvenile Neuronal Ceroid Lipofuscinosis) is a rare pediatric neurodegenerative disorder caused by biallelic mutations in CLN3. While extensive efforts have been undertaken to understand CLN3 disease etiology, pathology, and clinical progression, little is known about the impact of CLN3 disease on parents and caregivers. Here, we investigated CLN3 disease progression, clinical care, and family experiences using semi-structured interviews with 39 parents of individuals with CLN3 disease. Analysis included response categorization by independent observers and quantitative methods. RESULTS: Parents reported patterns of disease progression that aligned with previous reports. Insomnia and thought- and mood-related concerns were reported frequently. "Decline in visual acuity" was the first sign/symptom noticed by n = 28 parents (70%). A minority of parents reported "behavioral issues" (n = 5, 12.5%), "communication issues" (n = 3, 7.5%), "cognitive decline" (n = 1, 2.5%), or "seizures" (n = 1, 2.5%) as the first sign/symptom. The mean time from the first signs or symptoms to a diagnosis of CLN3 disease was 2.8 years (SD = 4.1). Misdiagnosis was common, being reported by n = 24 participants (55.8%). Diagnostic tests and treatments were closely aligned with observed symptoms. Desires for improved or stabilized vision (top therapeutic treatment concern for n = 14, 32.6%), cognition (n = 8, 18.6%), and mobility (n = 3, 7%) dominated parental concerns and wishes for therapeutic correction. Family impacts were common, with n = 34 (81%) of respondents reporting a financial impact on the family and n = 20 (46.5%) reporting marital strain related to the disease. CONCLUSIONS: Collectively, responses demonstrated clear patterns of disease progression, a strong desire for therapies to treat symptoms related to vision and cognition, and a powerful family impact driven by the unrelenting nature of disease progression.

Sortilin inhibition treats multiple neurodegenerative lysosomal storage disorders.

Lysosomal storage disorders (LSDs) are a genetically and clinically diverse group of diseases characterized by lysosomal dysfunction. Batten disease is a family of severe LSDs primarily impacting the central nervous system. Here we show that AF38469, a small molecule inhibitor of sortilin, improves lysosomal and glial pathology across multiple LSD models. Live-cell imaging and comparative transcriptomics demonstrates that the transcription factor EB (TFEB), an upstream regulator of lysosomal biogenesis, is activated upon treatment with AF38469. Utilizing CLN2 and CLN3 Batten disease mouse models, we performed a short-term efficacy study and show that treatment with AF38469 prevents the accumulation of lysosomal storage material and the development of neuroinflammation, key disease associated pathologies. Tremor phenotypes, an early behavioral phenotype in the CLN2 disease model, were also completely rescued. These findings reveal sortilin inhibition as a novel and highly efficacious therapeutic modality for the treatment of multiple forms of Batten disease.

Therapeutic Role of Pharmacological Chaperones in Lysosomal Storage Disorders: A Review of the Evidence and Informed Approach to Reclassification.

The treatment landscape for lysosomal storage disorders (LSDs) is rapidly evolving. An increase in the number of preclinical and clinical studies in the last decade has demonstrated that pharmacological chaperones are a feasible alternative to enzyme replacement therapy (ERT) for individuals with LSDs. A systematic search was performed to retrieve and critically assess the evidence from preclinical and clinical applications of pharmacological chaperones in the treatment of LSDs and to elucidate the mechanisms by which they could be effective in clinical practice. Publications were screened according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) reporting guidelines. Fifty-two articles evaluating 12 small molecules for the treatment of seven LSDs are included in this review. Overall, a substantial amount of preclinical and clinical data support the potential of pharmacological chaperones as treatments for Fabry disease, Gaucher disease, and Pompe disease. Most of the available clinical evidence evaluated migalastat for the treatment of Fabry disease. There was a lack of consistency in the terminology used to describe pharmacological chaperones in the literature. Therefore, the new small molecule chaperone (SMC) classification system is proposed to inform a standardized approach for new, emerging small molecule therapies in LSDs.

A novel porcine model of CLN3 Batten disease recapitulates clinical phenotypes.

Mouse models of CLN3 Batten disease, a rare lysosomal storage disorder with no cure, have improved our understanding of CLN3 biology and therapeutics through their ease of use and a consistent display of cellular pathology. However, the translatability of murine models is limited by disparities in anatomy, body size, life span and inconsistent subtle behavior deficits that can be difficult to detect in CLN3 mutant mouse models, thereby limiting their use in preclinical studies. Here, we present a longitudinal characterization of a novel miniswine model of CLN3 disease that recapitulates the most common human pathogenic variant, an exon 7-8 deletion (CLN3Δex7/8). Progressive pathology and neuron loss is observed in various regions of the CLN3Δex7/8 miniswine brain and retina. Additionally, mutant miniswine present with retinal degeneration and motor abnormalities, similar to deficits seen in humans diagnosed with the disease. Taken together, the CLN3Δex7/8 miniswine model shows consistent and progressive Batten disease pathology, and behavioral impairment mirroring clinical presentation, demonstrating its value in studying the role of CLN3 and safety/efficacy of novel disease-modifying therapeutics.

Protracted CLN3 Batten disease in mice that genetically model an exon-skipping therapeutic approach.

Genetic mutations that disrupt open reading frames and cause translation termination are frequent causes of human disease and are difficult to treat due to protein truncation and mRNA degradation by nonsense-mediated decay, leaving few options for traditional drug targeting. Splice-switching antisense oligonucleotides offer a potential therapeutic solution for diseases caused by disrupted open reading frames by inducing exon skipping to correct the open reading frame. We have recently reported on an exon-skipping antisense oligonucleotide that has a therapeutic effect in a mouse model of CLN3 Batten disease, a fatal pediatric lysosomal storage disease. To validate this therapeutic approach, we generated a mouse model that constitutively expresses the Cln3 spliced isoform induced by the antisense molecule. Behavioral and pathological analyses of these mice demonstrate a less severe phenotype compared with the CLN3 disease mouse model, providing evidence that antisense oligonucleotide-induced exon skipping can have therapeutic efficacy in treating CLN3 Batten disease. This model highlights how protein engineering through RNA splicing modulation can be an effective therapeutic approach.

Immune transgene-dependent myocarditis in macaques after systemic administration of adeno-associated virus expressing human acid alpha-glucosidase.

Immune responses to human non-self transgenes can present challenges in preclinical studies of adeno-associated virus (AAV) gene therapy candidates in nonhuman primates. Although anti-transgene immune responses are usually mild and non-adverse, they can confound pharmacological readouts and complicate translation of results between species. We developed a gene therapy candidate for Pompe disease consisting of AAVhu68, a clade F AAV closely related to AAV9, that expresses an engineered human acid-alpha glucosidase (hGAA) tagged with an insulin-like growth factor 2 variant (vIGF2) peptide for enhanced cell uptake. Rhesus macaques were administered an intravenous dose of 1x1013 genome copies (GC)/kg, 5x1013 GC/kg, or 1 x 1014 GC/kg of AAVhu68.vIGF2.hGAA. Some unusually severe adaptive immune responses to hGAA presented, albeit with a high degree of variability between animals. Anti-hGAA responses ranged from absent to severe cytotoxic T-cell-mediated myocarditis with elevated troponin I levels. Cardiac toxicity was not dose dependent and affected five out of eleven animals. Upon further investigation, we identified an association between toxicity and a major histocompatibility complex class I haplotype (Mamu-A002.01) in three of these animals. An immunodominant peptide located in the C-terminal region of hGAA was subsequently identified via enzyme-linked immunospot epitope mapping. Another notable observation in this preclinical safety study cohort pertained to the achievement of robust and safe gene transfer upon intravenous administration of 5x1013 GC/kg in one animal with a low pre-existing neutralizing anti-capsid antibodies titer (1:20). Collectively, these findings may have significant implications for gene therapy inclusion criteria.

Early postnatal administration of an AAV9 gene therapy is safe and efficacious in CLN3 disease.

CLN3 disease, caused by biallelic mutations in the CLN3 gene, is a rare pediatric neurodegenerative disease that has no cure or disease modifying treatment. The development of effective treatments has been hindered by a lack of etiological knowledge, but gene replacement has emerged as a promising therapeutic platform for such disorders. Here, we utilize a mouse model of CLN3 disease to test the safety and efficacy of a cerebrospinal fluid-delivered AAV9 gene therapy with a study design optimized for translatability. In this model, postnatal day one administration of the gene therapy virus resulted in robust expression of human CLN3 throughout the CNS over the 24-month duration of the study. A range of histopathological and behavioral parameters were assayed, with the therapy consistently and persistently rescuing a number of hallmarks of disease while being safe and well-tolerated. Together, the results show great promise for translation of the therapy into the clinic, prompting the launch of a first-in-human clinical trial (NCT03770572).

Magnetic resonance brain volumetry biomarkers of CLN2 Batten disease identified with miniswine model.

Late-infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease (Batten disease) is a rare pediatric disease, with symptom development leading to clinical diagnosis. Early diagnosis and effective tracking of disease progression are required for treatment. We hypothesize that brain volumetry is valuable in identifying CLN2 disease at an early stage and tracking disease progression in a genetically modified miniswine model. CLN2R208X/R208X miniswine and wild type controls were evaluated at 12- and 17-months of age, correlating to early and late stages of disease progression. Magnetic resonance imaging (MRI) T1- and T2-weighted data were acquired. Total intercranial, gray matter, cerebrospinal fluid, white matter, caudate, putamen, and ventricle volumes were calculated and expressed as proportions of the intracranial volume. The brain regions were compared between timepoints and cohorts using Gardner-Altman plots, mean differences, and confidence intervals. At an early stage of disease, the total intracranial volume (- 9.06 cm3), gray matter (- 4.37% 95 CI - 7.41; - 1.83), caudate (- 0.16%, 95 CI - 0.24; - 0.08) and putamen (- 0.11% 95 CI - 0.23; - 0.02) were all notably smaller in CLN2R208X/R208X miniswines versus WT, while cerebrospinal fluid was larger (+ 3.42%, 95 CI 2.54; 6.18). As the disease progressed to a later stage, the difference between the gray matter (- 8.27%, 95 CI - 10.1; - 5.56) and cerebrospinal fluid (+ 6.88%, 95 CI 4.31; 8.51) continued to become more pronounced, while others remained stable. MRI brain volumetry in this miniswine model of CLN2 disease is sensitive to early disease detection and longitudinal change monitoring, providing a valuable tool for pre-clinical treatment development and evaluation.

Sex-split analysis of pathology and motor-behavioral outcomes in a mouse model of CLN8-Batten disease reveals an increased disease burden and trajectory in female Cln8mnd mice.

BACKGROUND: CLN8-Batten disease (CLN8 disease) is a rare neurodegenerative disorder characterized phenotypically by progressive deterioration of motor and cognitive abilities, visual symptoms, epileptic seizures, and premature death. Mutations in CLN8 results in characteristic Batten disease symptoms and brain-wide pathology including accumulation of lysosomal storage material, gliosis, and neurodegeneration. Recent investigations of other subforms of Batten disease (CLN1, CLN3, CLN6) have emphasized the influence of biological sex on disease and treatment outcomes; however, little is known about sex differences in the CLN8 subtype. To determine the impact of sex on CLN8 disease burden and progression, we utilized a Cln8mnd mouse model to measure the impact and progression of histopathological and behavioral outcomes between sexes. RESULTS: Several notable sex differences were observed in the presentation of brain pathology, including Cln8mnd female mice consistently presenting with greater GFAP+ astrocytosis and CD68+ microgliosis in the somatosensory cortex, ventral posteromedial/ventral posterolateral nuclei of the thalamus, striatum, and hippocampus when compared to Cln8mnd male mice. Furthermore, sex differences in motor-behavioral assessments revealed Cln8mnd female mice experience poorer motor performance and earlier death than their male counterparts. Cln8mnd mice treated with an AAV9-mediated gene therapy were also examined to assess sex differences on therapeutics outcomes, which revealed no appreciable differences between the sexes when responding to the therapy. CONCLUSIONS: Taken together, our results provide further evidence of biologic sex as a modifier of Batten disease progression and outcome, thus warranting consideration when conducting investigations and monitoring therapeutic impact.

Glycerophosphoinositol is Elevated in Blood Samples From CLN3 Δex7-8 pigs, Cln3 Δex7-8 Mice, and CLN3-Affected Individuals.

Introduction: CLN3 Batten disease is a rare pediatric neurodegenerative lysosomal disorder caused by biallelic disease-associated variants in CLN3. Despite decades of intense research, specific biofluid biomarkers of disease status have not been reported, hindering clinical development of therapies. Thus, we sought to determine whether individuals with CLN3 Batten disease have elevated levels of specific metabolites in blood. Methods: We performed an exhaustive metabolomic screen using serum samples from a novel minipig model of CLN3 Batten disease and validated findings in CLN3 pig serum and CSF and Cln3 mouse serum. We further validate biomarker candidates with a retrospective analysis of plasma and CSF samples collected from participants in a natural history study. Plasma samples were evaluated from 22 phenotyped individuals with CLN3 disease, 15 heterozygous carriers, and 6 non-affected non-carriers (NANC). Results: CLN3 pig serum samples from 4 ages exhibited large elevations in 4 glycerophosphodiester species: glycerophosphoinositol (GPI), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), and glycerophosphoserine (GPS). GPI and GPE exhibited the largest elevations, with similar elevations found in CLN3 pig CSF and Cln3 mouse serum. In plasma samples from individuals with CLN3 disease, glycerophosphoethanolamine and glycerophosphoinositol were significantly elevated with glycerophosphoinositol exhibiting the clearest separation (mean 0.1338 vs 0.04401 nmol/mL for non-affected non-carriers). Glycerophosphoinositol demonstrated excellent sensitivity and specificity as a biomarker, with a receiver operating characteristic area under the curve of 0.9848 (P = .0003). Conclusions: GPE and GPI could have utility as biomarkers of CLN3 disease status. GPI, in particular, shows consistent elevations across a diverse cohort of individuals with CLN3. This raises the potential to use these biomarkers as a blood-based diagnostic test or as an efficacy measure for disease-modifying therapies.

A Novel Porcine Model of CLN2 Batten Disease that Recapitulates Patient Phenotypes.

CLN2 Batten disease is a lysosomal disorder in which pathogenic variants in CLN2 lead to reduced activity in the enzyme tripeptidyl peptidase 1. The disease typically manifests around 2 to 4 years of age with developmental delay, ataxia, seizures, inability to speak and walk, and fatality between 6 and 12 years of age. Multiple Cln2 mouse models exist to better understand the etiology of the disease; however, these models are unable to adequately recapitulate the disease due to differences in anatomy and physiology, limiting their utility for therapeutic testing. Here, we describe a new CLN2R208X/R208X porcine model of CLN2 disease. We present comprehensive characterization showing behavioral, pathological, and visual phenotypes that recapitulate those seen in CLN2 patients. CLN2R208X/R208X miniswine present with gait abnormalities at 6 months of age, ERG waveform declines at 6-9 months, vision loss at 11 months, cognitive declines at 12 months, seizures by 15 months, and early death at 18 months due to failure to thrive. CLN2R208X/R208X miniswine also showed classic storage material accumulation and glial activation in the brain at 6 months, and cortical atrophy at 12 months. Thus, the CLN2R208X/R208X miniswine model is a valuable resource for biomarker discovery and therapeutic development in CLN2 disease.

Promoting validation and cross-phylogenetic integration in model organism research.

Model organism (MO) research provides a basic understanding of biology and disease due to the evolutionary conservation of the molecular and cellular language of life. MOs have been used to identify and understand the function of orthologous genes, proteins, cells and tissues involved in biological processes, to develop and evaluate techniques and methods, and to perform whole-organism-based chemical screens to test drug efficacy and toxicity. However, a growing richness of datasets and the rising power of computation raise an important question: How do we maximize the value of MOs? In-depth discussions in over 50 virtual presentations organized by the National Institutes of Health across more than 10 weeks yielded important suggestions for improving the rigor, validation, reproducibility and translatability of MO research. The effort clarified challenges and opportunities for developing and integrating tools and resources. Maintenance of critical existing infrastructure and the implementation of suggested improvements will play important roles in maintaining productivity and facilitating the validation of animal models of human biology and disease.

Transmembrane Batten Disease Proteins Interact With a Shared Network of Vesicle Sorting Proteins, Impacting Their Synaptic Enrichment.

Batten disease is unique among lysosomal storage disorders for the early and profound manifestation in the central nervous system, but little is known regarding potential neuron-specific roles for the disease-associated proteins. We demonstrate substantial overlap in the protein interactomes of three transmembrane Batten proteins (CLN3, CLN6, and CLN8), and that their absence leads to synaptic depletion of key partners (i.e., SNAREs and tethers) and altered synaptic SNARE complexing in vivo, demonstrating a novel shared etiology.

CLN7 gene therapy: hope for an ultra-rare condition.

CLN7 Batten disease, also known as variant late infantile neuronal ceroid lipofuscinosis type 7 (vLINCL7), is an ultra-rare form of Batten disease that presents early in life with severe neurological symptoms, including visual deficits, motor problems, and frequent seizures. There is high unmet need for disease-modifying therapies, as no existing treatment can halt progression or prevent premature death. In this issue of the JCI, Chen et al. present an AAV gene therapy for CLN7 that shows marked benefit in a mouse model of CLN7 Batten disease, paving the way for a phase I trial. The candidate gene therapy shows benefit for histopathology, behavioral abnormalities, and survival in mice and offers an acceptable safety profile in both mice and rats. Questions remain regarding dose, scaling, and timing of administration for patients, but this work is a substantial step forward for a very challenging disease.

Phosphorylation-dependent proteome of Marcks in ependyma during aging and behavioral homeostasis in the mouse forebrain.

Ependymal cells (ECs) line the ventricular surfaces of the mammalian central nervous system (CNS) and their development is indispensable to structural integrity and functions of the CNS. We previously reported that EC-specific genetic deletion of the myristoylated alanine-rich protein kinase C substrate (Marcks) disrupts barrier functions and elevates oxidative stress and lipid droplet accumulation in ECs causing precocious cellular aging. However, little is known regarding the mechanisms that mediate these changes in ECs. To gain insight into Marcks-mediated mechanisms, we performed mass spectrometric analyses on Marcks-associated proteins in young and aged ECs in the mouse forebrain using an integrated approach. Network analysis on annotated proteins revealed that the identified Marcks-associated complexes are in part involved in protein transport mechanisms in young ECs. In fact, we found perturbed intracellular vesicular trafficking in cultured ECs with selective deletion of Marcks (Marcks-cKO mice), or upon pharmacological alteration to phosphorylation status of Marcks. In comparison, Marcks-associated protein complexes in aged ECs appear to be involved in regulation of lipid metabolism and responses to oxidative stress. Confirming this, we found elevated signatures of inflammation in the cerebral cortices and the hippocampi of young Marcks-cKO mice. Interestingly, behavioral testing using a water maze task indicated that spatial learning and memory is diminished in young Marcks-cKO mice similar to aged wildtype mice. Taken together, our study provides first line of evidence for potential mechanisms that may mediate differential Marcks functions in young and old ECs, and their effect on forebrain homeostasis during aging.

On the cusp of cures: Breakthroughs in Batten disease research.

Batten disease is a family of rare, lysosomal disorders caused by mutations in one of at least 13 genes, which encode a diverse set of lysosomal and extralysosomal proteins. Despite decades of research, the development of effective therapies has remained intractable. But now, the field is experiencing rapid, unprecedented progress on multiple fronts. New tools are providing insights into previously unsolvable problems, with molecular functions now known for nine Batten disease proteins. Protein interactome data are uncovering potential functional overlap between several Batten disease proteins, providing long-sought links between seemingly disparate proteins. Understanding of cellular etiology is elucidating contributions from and interactions between various CNS cell types. Collectively, this explosion in insight is hastening an unparalleled period of therapeutic breakthroughs, with multiple therapies showing great promise in preclinical and clinical studies. The coming years will provide a continuation of this rapid progress, with the promise of effective treatments giving patients hope.

Collagen has a unique SEC24 preference for efficient export from the endoplasmic reticulum.

SEC24 is mainly involved in cargo sorting during COPII vesicle assembly. There are four SEC24 paralogs (A-D) in vertebrates, which are classified into two subgroups (SEC24A/B and SEC24C/D). Pathological mutations in SEC24D cause osteogenesis imperfecta with craniofacial dysplasia in humans. sec24d mutant fish also recapitulate the phenotypes. Consistent with the skeletal phenotypes, the secretion of collagen was severely defective in mutant fish, emphasizing the importance of SEC24D in collagen secretion. However, SEC24D patient-derived fibroblasts show only a mild secretion phenotype, suggesting tissue-specificity in the secretion process. Using Sec24d KO mice and cultured cells, we show that SEC24A and SEC24B also contribute to endoplasmic reticulum (ER) export of procollagen. In contrast, fibronectin 1 requires either SEC24C or SEC24D for ER export. On the basis of our results, we propose that procollagen interacts with multiple SEC24 paralogs for efficient export from the ER, and that this is the basis for tissue-specific phenotypes resulting from SEC24 paralog deficiency.

Endolysosomal N-glycan processing is critical to attain the most active form of the enzyme acid alpha-glucosidase.

Acid alpha-glucosidase (GAA) is a lysosomal glycogen-catabolizing enzyme, the deficiency of which leads to Pompe disease. Pompe disease can be treated with systemic recombinant human GAA (rhGAA) enzyme replacement therapy (ERT), but the current standard of care exhibits poor uptake in skeletal muscles, limiting its clinical efficacy. Furthermore, it is unclear how the specific cellular processing steps of GAA after delivery to lysosomes impact its efficacy. GAA undergoes both proteolytic cleavage and glycan trimming within the endolysosomal pathway, yielding an enzyme that is more efficient in hydrolyzing its natural substrate, glycogen. Here, we developed a tool kit of modified rhGAAs that allowed us to dissect the individual contributions of glycan trimming and proteolysis on maturation-associated increases in glycogen hydrolysis using in vitro and in cellulo enzyme processing, glycopeptide analysis by MS, and high-pH anion-exchange chromatography with pulsed amperometric detection for enzyme kinetics. Chemical modifications of terminal sialic acids on N-glycans blocked sialidase activity in vitro and in cellulo, thereby preventing downstream glycan trimming without affecting proteolysis. This sialidase-resistant rhGAA displayed only partial activation after endolysosomal processing, as evidenced by reduced catalytic efficiency. We also generated enzymatically deglycosylated rhGAA that was shown to be partially activated despite not undergoing proteolytic processing. Taken together, these data suggest that an optimal rhGAA ERT would require both N-glycan and proteolytic processing to attain the most efficient enzyme for glycogen hydrolysis and treatment of Pompe disease. Future studies should examine the amenability of next-generation ERTs to both types of cellular processing.

Intracranial delivery of AAV9 gene therapy partially prevents retinal degeneration and visual deficits in CLN6-Batten disease mice.

Batten disease is a family of rare, fatal, neuropediatric diseases presenting with memory/learning decline, blindness, and loss of motor function. Recently, we reported the use of an AAV9-mediated gene therapy that prevents disease progression in a mouse model of CLN6-Batten disease (Cln6 nclf ), restoring lifespans in treated animals. Despite the success of our viral-mediated gene therapy, the dosing strategy was optimized for delivery to the brain parenchyma and may limit the therapeutic potential to other disease-relevant tissues, such as the eye. Here, we examine whether cerebrospinal fluid (CSF) delivery of scAAV9.CB.CLN6 is sufficient to ameliorate visual deficits in Cln6 nclf mice. We show that intracerebroventricular (i.c.v.) delivery of scAAV9.CB.CLN6 completely prevents hallmark Batten disease pathology in the visual processing centers of the brain, preserving neurons of the superior colliculus, thalamus, and cerebral cortex. Importantly, i.c.v.-delivered scAAV9.CB.CLN6 also expresses in many cells throughout the central retina, preserving many photoreceptors typically lost in Cln6 nclf mice. Lastly, scAAV9.CB.CLN6 treatment partially preserved visual acuity in Cln6 nclf mice as measured by optokinetic response. Taken together, we report the first instance of CSF-delivered viral gene reaching and rescuing pathology in both the brain parenchyma and retinal neurons, thereby partially slowing visual deterioration.