Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic.

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.

SnapShot: Lysosomal Storage Diseases.

Lysosomal storage diseases (LSDs) represent a group of monogenic inherited metabolic disorders characterized by the progressive accumulation of undegraded substrates inside lysosomes, resulting in aberrant lysosomal activity and homeostasis. This SnapShot summarizes the intracellular localization and function of proteins implicated in LSDs. Common aspects of LSD pathogenesis and the major current therapeutic approaches are noted. To view this SnapShot, open or download the PDF.

From serendipity to therapy.

My postdoctoral training in the biosynthesis of plant polysaccharides at the University of California, Berkeley, led me, rather improbably, to study mucopolysaccharide storage disorders in the intramural program of the National Institutes of Health (NIH). I have traced the path from studies of mucopolysaccharide turnover in cultured cells to the development of therapy for patients. The key experiment started as an accident, i.e., the mixing of cells of different genotypes, resulting in correction of their biochemical defect. This serendipitous experiment led to identification of the enzyme deficiencies in the Hurler and Hunter syndromes, to an understanding of the biochemistry of lysosomal enzymes in general, and to the cell biology of receptor-mediated endocytosis and targeting to lysosomes. It paved the way for the development of enzyme replacement therapy with recombinant enzymes. I have also included studies performed after I moved to the University of California, Los Angeles (UCLA), including a recent unexpected finding in a neurodegenerative mucopolysaccharide storage disease, the Sanfilippo syndrome, with implications for therapy.

Targeting the central nervous system in lysosomal storage diseases: Strategies to deliver therapeutics across the blood-brain barrier.

Lysosomal storage diseases (LSDs) are multisystem inherited metabolic disorders caused by dysfunctional lysosomal activity, resulting in the accumulation of undegraded macromolecules in a variety of organs/tissues, including the central nervous system (CNS). Treatments include enzyme replacement therapy, stem/progenitor cell transplantation, and in vivo gene therapy. However, these treatments are not fully effective in treating the CNS as neither enzymes, stem cells, nor viral vectors efficiently cross the blood-brain barrier. Here, we review the latest advancements in improving delivery of different therapeutic agents to the CNS and comment upon outstanding questions in the field of neurological LSDs.

Preclinical studies in Krabbe disease: A model for the investigation of novel combination therapies for lysosomal storage diseases.

Krabbe disease (KD) is a lysosomal storage disease (LSD) caused by mutations in the galc gene. There are over 50 monogenetic LSDs, which largely impede the normal development of children and often lead to premature death. At present, there are no cures for LSDs and the available treatments are generally insufficient, short acting, and not without co-morbidities or long-term side effects. The last 30 years have seen significant advances in our understanding of LSD pathology as well as treatment options. Two gene therapy-based clinical trials, NCT04693598 and NCT04771416, for KD were recently started based on those advances. This review will discuss how our knowledge of KD got to where it is today, focusing on preclinical investigations, and how what was discovered may prove beneficial for the treatment of other LSDs.

Engineered arylsulfatase A with increased activity, stability and brain delivery for therapy of metachromatic leukodystrophy.

A deficiency of human arylsulfatase A (hASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disease characterized by sulfatide accumulation and central nervous system (CNS) demyelination. Efficacy of enzyme replacement therapy (ERT) is increased by genetic engineering of hASA to elevate its activity and transfer across the blood-brain barrier (BBB), respectively. To further improve the enzyme's bioavailability in the CNS, we mutated a cathepsin cleavage hot spot and obtained hASAs with substantially increased half-lives. We then combined the superstabilizing exchange E424A with the activity-promoting triple substitution M202V/T286L/R291N and the ApoEII-tag for BBB transfer in a trimodal modified neoenzyme called SuPerTurbo-ASA. Compared with wild-type hASA, half-life, activity, and M6P-independent uptake were increased more than 7-fold, about 3-fold, and more than 100-fold, respectively. ERT of an MLD-mouse model with immune tolerance to wild-type hASA did not induce antibody formation, indicating absence of novel epitopes. Compared with wild-type hASA, SuPerTurbo-ASA was 8- and 12-fold more efficient in diminishing sulfatide storage of brain and spinal cord. In both tissues, storage was reduced by ∼60%, roughly doubling clearance achieved with a 65-fold higher cumulative dose of wild-type hASA previously. Due to its enhanced therapeutic potential, SuPerTurbo-ASA might be a decisive advancement for ERT and gene therapy of MLD.

Impact of COVID-19 pandemic on healthcare delivery for lysosomal storage disorders at a tertiary care public hospital in Mumbai.

Introduction: Management of lysosomal storage disorders (LSDs) requires periodic visits for medical surveillance and hospitalizations. Management of LSDs may have been adversely impacted during the COVID-19 pandemic. Objective: To identify the factors impacting health care for patients with LSDs during the COVID-19 pandemic. Methods: An observational study was conducted in Mumbai comparing infusion practices and reasons for missed infusions for 15 months before March 2020 versus two phases during the pandemic (April 2020-March 2021 and April 2021-March 2022) in patients receiving intravenous enzyme replacement therapy (ERT) and on oral substrate reduction therapy (SRT). Results: Fifteen patients with LSDs were enrolled. Before the pandemic, 6/13 (46%) were receiving ERT at the study site, 4/13 (31%) at a local hospital, and 3/13 (23%) at home; two were on SRT. The median distance traveled for receiving ERT was 37 km, and 4.4 infusions/patient were missed. From April 2020 to March 2021, two more patients opted for home ERT infusions. The median distance traveled for receiving ERT was 37 km, and 11.6 infusions/patient were missed. From April 2021 to March 2022, one more patient opted for home ERT infusions. The median distance traveled for receiving ERT was 7 km, and 5.6 infusions/patient were missed. The pandemic also affected SRT compliance adversely. For all patients, the cause of disrupted treatment was travel curbs (69%) and fear of getting COVID-19 infection (38%). Conclusions: Treatment of LSDs was disrupted during the pandemic, with an increase in missed ERT infusions and SRT doses.

Modifying enzyme replacement therapy - A perspective.

Several diseases are caused by the lack of functional proteins, including lysosomal storage diseases or haemophilia A and B. Patients suffering from one of these diseases are treated via enzyme replacement therapies to restore the missing protein. Although this treatment strategy prevents some disease symptoms, enzyme replacement therapies are very expensive and require very frequent infusions, which can cause infusion adverse reactions and massively impair the quality of life of the patients. This review proposes a technology to sustainably produce proteins within the patient to potentially make frequent protein-infusions redundant. This technology is based on blood circulating immune cells as producers of the needed therapeutic protein. To ensure a stable protein concentration over time the cells are equipped with a system, which induces cell proliferation when low therapeutic protein levels are detected and a system inhibiting cell proliferation when high therapeutic protein levels are detected.

New tools can propel research in lysosomal storage diseases.

Historically, the clinical manifestations of lysosomal storage diseases offered an early glimpse into the essential digestive functions of the lysosome. However, it was only recently that the more subtle role of this organelle in the dynamic regulation of multiple cellular processes was appreciated. With the need for precise interrogation of lysosomal interplay in health and disease comes the demand for more sophisticated functional tools. This demand has recently been met with 1) induced pluripotent stem cell-derived models that recapitulate the disease phenotype in vitro, 2) methods for lysosome affinity purification coupled with downstream omics analysis that provide a high-resolution snapshot of lysosomal alterations, and 3) gene editing and CRISPR/Cas9-based functional genomic strategies that enable screening for genetic modifiers of the disease phenotype. These emerging methods have garnered much interest in the field of neurodegeneration, and their use in the field of metabolic disorders is now also steadily gaining momentum. Looking forward, these robust tools should accelerate basic science efforts to understand lysosomal dysfunction distal to substrate accumulation and provide translational opportunities to identify disease-modifying therapies.

Molecular Trojan Horses for treating lysosomal storage diseases.

Lysosomal storage diseases (LSDs) are caused by monogenic mutations in genes encoding for proteins related to the lysosomal function. Lysosome plays critical roles in molecule degradation and cell signaling through interplay with many other cell organelles, such as mitochondria, endoplasmic reticulum, and peroxisomes. Even though several strategies (i.e., protein replacement and gene therapy) have been attempted for LSDs with promising results, there are still some challenges when hard-to-treat tissues such as bone (i.e., cartilages, ligaments, meniscus, etc.), the central nervous system (mostly neurons), and the eye (i.e., cornea, retina) are affected. Consistently, searching for novel strategies to reach those tissues remains a priority. Molecular Trojan Horses have been well-recognized as a potential alternative in several pathological scenarios for drug delivery, including LSDs. Even though molecular Trojan Horses refer to genetically engineered proteins to overcome the blood-brain barrier, such strategy can be extended to strategies able to transport and deliver drugs to specific tissues or cells using cell-penetrating peptides, monoclonal antibodies, vesicles, extracellular vesicles, and patient-derived cells. Only some of those platforms have been attempted in LSDs. In this paper, we review the most recent efforts to develop molecular Trojan Horses and discuss how this strategy could be implemented to enhance the current efficacy of strategies such as protein replacement and gene therapy in the context of LSDs.

Hypersensitivity reaction during enzyme replacement therapy in lysosomal storage disorders. A systematic review of desensitization strategies.

Lysosomal storage diseases (LSDs) are rare genetic metabolic disorders that cause the accumulation of glycosaminoglycans in lysosomes due to enzyme deficiency or reduced function. Enzyme replacement therapy (ERT) represents the gold standard treatment, but hypersensitivity reaction can occur resulting in treatment discontinuation. Thus, desensitization procedures for different culprit recombinant enzymes can be performed to restore ERT. We searched desensitization procedures performed in LSDs and focused on skin test results, protocols and premedication performed, and breakthrough reactions occurred during infusions. Fifty-two patients have been subjected to desensitization procedures successfully. Skin tests, with the culprit recombinant enzyme, deemed positive in 29 cases, doubtful in two cases, and not performed in four patients. Moreover, 29 of the 52 desensitization protocols used at the first infusion were breakthrough reaction free. Different desensitization strategies have proved safe and effective in restoring ERT in patients with previous hypersensitivity reactions. Most of these events seem to be Type I hypersensitivity reactions (IgE-mediated). Standardized in vivo and in vitro testing is necessary to better estimate the risk of the procedure and find the safest individualized desensitization protocol.

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.

Neuronal genetic rescue normalizes brain network dynamics in a lysosomal storage disorder despite persistent storage accumulation.

Although neurologic symptoms occur in two-thirds of lysosomal storage disorders (LSDs), for most we do not understand the mechanisms underlying brain dysfunction. A major unanswered question is if the pathogenic hallmark of LSDs, storage accumulation, induces functional defects directly or is a disease bystander. Also, for most LSDs we do not know the impact of loss of function in individual cell types. Understanding these critical questions are essential to therapy development. Here, we determine the impact of genetic rescue in distinct cell types on neural circuit dysfunction in CLN3 disease, the most common pediatric dementia and a paradigmatic neurodegenerative LSD. We restored Cln3 expression via AAV-mediated gene delivery and conditional genetic rescue in a CLN3 disease mouse model. Surprisingly, we found that low-level rescue of Cln3 expression in neurons alone normalized clinically relevant electrophysiologic markers of network dysfunction, despite the presence of substantial residual histopathology, in contrast to restoring expression in astrocytes. Thus, loss of CLN3 function in neurons, not storage accumulation, underlies neurologic dysfunction in CLN3 disease. This impliesies that storage clearance may be an inappropriate target for therapy development and an ineffectual biomarker.

Intrauterine enzyme replacement therapies for lysosomal storage disorders: Current developments and promising future prospects.

Lysosomal storage disorders (LSDs) are a group of monogenic condition, with many characterized by an enzyme deficiency leading to the accumulation of an undegraded substrate within the lysosomes. For those LSDs, postnatal enzyme replacement therapy (ERT) represents the standard of care, but this treatment has limitations when administered only postnatally because, at that point, prenatal disease sequelae may be irreversible. Furthermore, most forms of ERT, specifically those administered systemically, are currently unable to access certain tissues, such as the central nervous system (CNS), and furthermore, may initiate an immune response. In utero enzyme replacement therapy (IUERT) is a novel approach to address these challenges evaluated in a first-in-human clinical trial for IUERT in LSDs (NCT04532047). IUERT has numerous advantages: in-utero intervention may prevent early pathology; the CNS can be accessed before the blood-brain barrier forms; and the unique fetal immune system enables exposure to new proteins with the potential to prevent an immune response and may induce sustained tolerance. However, there are challenges and limitations for any fetal procedure that involves two patients. This article reviews the current state of IUERT for LSDs, including its advantages, limitations, and potential future directions for definitive therapies.

Genome Editing Tools for Lysosomal Storage Disorders.

Genome editing has multiple applications in the biomedical field. They can be used to modify genomes at specific locations, being able to either delete, reduce, or even enhance gene transcription and protein expression. Here, we summarize applications of genome editing used in the field of lysosomal disorders. We focus on the development of cell lines for study of disease pathogenesis, drug discovery, and pathogenicity of specific variants. Furthermore, we highlight the main studies that use gene editing as a gene therapy platform for these disorders, both in preclinical and clinical studies. We conclude that gene editing has been able to change quickly the scenario of these disorders, allowing the development of new therapies and improving the knowledge on disease pathogenesis. Should they confirm their hype, the first gene editing-based products for lysosomal disorders could be available in the next years.

Mesenchymal stem cells for lysosomal storage and polyglutamine disorders: Possible shared mechanisms.

BACKGROUND: Mesenchymal stem cells' (MSC) therapeutic potential has been investigated for the treatment of several neurodegenerative diseases. The fact these cells can mediate a beneficial effect in different neurodegenerative contexts strengthens their competence to target diverse mechanisms. On the other hand, distinct disorders may share similar mechanisms despite having singular neuropathological characteristics. METHODS: We have previously shown that MSC can be beneficial for two disorders, one belonging to the groups of Lysosomal Storage Disorders (LSDs) - the Krabbe Disease or Globoid Cell Leukodystrophy, and the other to the family of Polyglutamine diseases (PolyQs) - the Machado-Joseph Disease or Spinocerebellar ataxia type 3. We gave also input into disease characterization since neuropathology and MSC's effects are intrinsically associated. This review aims at describing MSC's multimode of action in these disorders while emphasizing to possible mechanistic alterations they must share due to the accumulation of cellular toxic products. RESULTS: Lysosomal storage disorders and PolyQs have different aetiology and associated symptoms, but both result from the accumulation of undegradable products inside neuronal cells due to inefficient clearance by the endosomal/lysosomal pathway. Moreover, numerous cellular mechanisms that become compromised latter are also shared by these two disease groups. CONCLUSIONS: Here, we emphasize MSC's effect in improving proteostasis and autophagy cycling turnover, neuronal survival, synaptic activity and axonal transport. LSDs and PolyQs, though rare in their predominance, collectively affect many people and require our utmost dedication and efforts to get successful therapies due to their tremendous impact on patient s' lives and society.

Evolving therapies in neuronopathic LSDs: opportunities and challenges.

Lysosomal storage disorders (LSD) are multisystemic progressive disorders caused by genetic mutations involving lysosomal function. While LSDs are individually considered rare diseases, the overall true prevalence of these disorders is likely higher than our current estimates. More than two third of the LSDs have associated neurodegeneration and the neurological phenotype often defines the course of the disease and treatment outcomes. Addressing the neurological involvement in LSDs has posed a significant challenge in the rapidly evolving field of therapies for these diseases. In this review, we summarize current approaches and clinical trials available for patients with neuronopathic lysosomal storage disorders, exploring the opportunities and challenges that have emerged with each of these.

Mammalian Sulfatases: Biochemistry, Disease Manifestation, and Therapy.

Sulfatases are enzymes that catalyze the removal of sulfate from biological substances, an essential process for the homeostasis of the body. They are commonly activated by the unusual amino acid formylglycine, which is formed from cysteine at the catalytic center, mediated by a formylglycine-generating enzyme as a post-translational modification. Sulfatases are expressed in various cellular compartments such as the lysosome, the endoplasmic reticulum, and the Golgi apparatus. The substrates of mammalian sulfatases are sulfolipids, glycosaminoglycans, and steroid hormones. These enzymes maintain neuronal function in both the central and the peripheral nervous system, chondrogenesis and cartilage in the connective tissue, detoxification from xenobiotics and pharmacological compounds in the liver, steroid hormone inactivation in the placenta, and the proper regulation of skin humidification. Human sulfatases comprise 17 genes, 10 of which are involved in congenital disorders, including lysosomal storage disorders, while the function of the remaining seven is still unclear. As for the genes responsible for pathogenesis, therapeutic strategies have been developed. Enzyme replacement therapy with recombinant enzyme agents and gene therapy with therapeutic transgenes delivered by viral vectors are administered to patients. In this review, the biochemical substrates, disease manifestation, and therapy for sulfatases are summarized.

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Lysosomal storage disorders (LSDs) are a group of single-gene inherited metabolic diseases caused by defects in lysosomal enzymes or function-related proteins. Enzyme replacement therapy is the main treatment method in clinical practice, but it has a poor effect in patients with neurological symptoms. With the rapid development of multi-omics, sequencing technology, and bioengineering, gene therapy has been applied in patients with LSDs. As one of the vectors of gene therapy, adeno-associated virus (AAV) has good prospects in the treatment of genetic and metabolic diseases. More and more studies have shown that AAV-mediated gene therapy is effective in LSDs. This article reviews the application of AAV-mediated gene therapy in LSDs.

Evolutionary redesign of the lysosomal enzyme arylsulfatase A increases efficacy of enzyme replacement therapy for metachromatic leukodystrophy.

Protein engineering is a means to optimize protein therapeutics developed for the treatment of so far incurable diseases including cancers and genetic disorders. Here we report on an engineering approach in which we successfully increased the catalytic rate constant of an enzyme that is presently evaluated in enzyme replacement therapies (ERT) of a lysosomal storage disease (LSD). Although ERT is a treatment option for many LSDs, outcomes are lagging far behind expectations for most of them. This has been ascribed to insufficient enzyme activities accumulating in tissues difficult to target such as brain and peripheral nerves. We show for human arylsulfatase A (hARSA) that the activity of a therapeutic enzyme can be substantially increased by reversing activity-diminishing and by inserting activity-promoting amino acid substitutions that had occurred in the evolution of hominids and non-human mammals, respectively. The potential of this approach, here designated as evolutionary redesign, was highlighted by the observation that murinization of only 1 or 3 amino acid positions increased the hARSA activity 3- and 5-fold, with little impact on stability, respectively. The two kinetically optimized hARSA variants showed no immunogenic potential in ERT of a humanized ARSA knockout mouse model of metachromatic leukodystrophy (MLD) and reduced lysosomal storage of kidney, peripheral and central nervous system up to 3-fold more efficiently than wild-type hARSA. Due to their safety profile and higher therapeutic potential the engineered hARSA variants might represent major advances for future enzyme-based therapies of MLD and stimulate analogous approaches for other enzyme therapeutics.