Biology subjects

Schoser, B.

Publications and source records attributed to Schoser, B..

7 recordsLinked to original sources

bioRxiv2024

Mitochondrial leakage and mtDNA damage trigger early immune response in Inclusion Body Myositis.

Polymyositis with mitochondrial pathology (PM-Mito) was first identified in 1997 as a subtype of idiopathic inflammatory myopathy. Recent findings demonstrated significant molecular similarities between PM-Mito and Inclusion Body Myositis (IBM), suggesting a trajectory from early to late IBM and prompting the inclusion of PM-Mito as an IBM precursor (early IBM) within the IBM spectrum. Both PM-Mito and IBM show mitochondrial abnormalities, suggesting mitochondrial disturbance is a critical element of IBM pathogenesis. The primary objective of this cross-sectional study was to characterize the mitochondrial phenotype in PM-Mito at histological, ultrastructural, and molecular levels and to study the interplay between mitochondrial dysfunction and inflammation. Skeletal muscle biopsies of 27 patients with PM-Mito and 27 with typical IBM were included for morphological and ultrastructural analysis. Mitochondrial DNA (mtDNA) copy number and deletions were assessed by qPCR and long-range PCR, respectively. In addition, full-length single-molecule sequencing of the mtDNA enabled precise mapping of deletions. Protein and RNA levels were studied using unbiased proteomic profiling, immunoblotting, and bulk RNA sequencing. Cell-free mtDNA (cfmtDNA) was measured in the serum of IBM patients. We found widespread mitochondrial abnormalities in both PM-Mito and IBM, illustrated by elevated numbers of COX-negative and SDH-positive fibers and prominent ultrastructural abnormalities with disorganized and concentric cristae within enlarged and dysmorphic mitochondria. MtDNA copy numbers were significantly reduced, and multiple large-scale mtDNA deletions were already evident in PM-Mito, compared to healthy age-matched controls, similar to the IBM group. The activation of the canonical cGAS/STING inflammatory pathway, possibly triggered by the intracellular leakage of mitochondrial DNA, was evident in PM-Mito and IBM. Elevated levels of circulating cfmtDNA also indicated leakage of mtDNA as a likely inflammatory trigger. In PM-Mito and IBM, these findings were accompanied by dysregulation of proteins and transcripts linked to the mitochondrial membranes. In summary, we identified that mitochondrial dysfunction with multiple mtDNA deletions and depletion, disturbed mitochondrial ultrastructure, and defects of the inner mitochondrial membrane are features of PM-Mito and IBM, underlining the concept of an IBM-spectrum disease (IBM-SD). The activation of inflammatory pathways related to mtDNA release indicates a significant role of mitochondria-associated inflammation in the pathogenesis of IBM-SD. Thus, mitochondrial abnormalities precede tissue remodeling and infiltration by specific T-cell subpopulations (e.g., KLRG1+) characteristic of late IBM. This study highlights the critical role of early mitochondrial abnormalities in the pathomechanism of IBM, which may lead to new approaches to therapy.

neuroscience↗

bioRxiv2023

Preclinical studies with ground germinated barley (GGB) for oral enzyme replacement therapy (Oral-ERT) in Pompe disease knockout mice

Genetic deficiency of lysosomal acid maltase or acid -glucosidase (GAA) results in the orphan disease known as glycogen storage disease type II or acid maltase deficiency (AMD) or Pompe disease (PD), encompassing at least four clinical subtypes of varying severity. PD results from mutations in the GAA gene and deficient GAA activity, resulting in the accumulation of glycogen in tissues (primarily muscle) and characterized by progressive skeletal muscle weakness and respiratory insufficiency. The current approved enzyme replacement therapy (ERT) for PD is via intravenous infusion of a recombinant human GAA (rhGAA) secreted by CHO cells (Myozyme, Sanofi-Genzyme) given once every 2 weeks and has shown varying efficacy in patients. Although the current ERT has proven to be very efficient in rescuing cardiac abnormalities and extending the life span of infants, the response in skeletal muscle is variable. In late-onset patients, only mild improvements in motor and respiratory functions have been achieved and the current ERT is unsatisfactory in the reversal of skeletal muscle pathology. Additional challenges for ERT include insufficient targeting/uptake of enzyme into disease-relevant tissues, poor tolerability due to severe ERT-mediated anaphylactic and immunologic reactions and the prohibitively high cost of lifelong ERT ($250-500K/year adult patient). A consensus at a Nov.-2019 US Acid Maltase Deficiency Association conference suggested that a multi-pronged approach including gene therapy, diet, exercise, etc. must be evaluated for a successful treatment. Our objective is to develop an innovative and affordable approach via barley GAA (bGAA) from ground germinated barley (GGB) or liquid GGB (L-GGB) for Oral-ERT for PD or as a daily supplement to Myozyme. To this end, we hypothesize that a bGAA produced in germinated barley can be ingested daily that allows the maintenance of a therapeutic level of enzyme. We have shown in extensive preliminary data that GGB or L-GGB was (1) enzymatically active, (2) was taken up by GAA KO mice and human WBCs to reverse the enzyme defect that was blocked by mannose-6-phosphate, (3) hydrolyzed glycogen, (4) increased significant changes in the clinical phenotype towards the WT levels in GAA KO mice dose-dependently, (5) taken up by PD myoblasts, lymphoid/fibroblasts cells to reverse the defect, (6) bGAA was [~]70kD, (7) Km, Vmax, pH optima, inhibitors and kinetics was similar to human placental GAA and an rhGAA and (8) was strain specific.

genetics↗

bioRxiv2022

Metabolic, Fibrotic, and Splicing Pathways Are All Altered in Emery-Dreifuss Muscular Dystrophy Spectrum Patients to Differing Degrees

Emery-Dreifuss muscular dystrophy (EDMD) is a genetically and clinically variable disorder. Previous attempts to use gene expression changes find its pathomechanism were unavailing, so we here engaged a functional pathway analysis. RNA-Seq was performed on cells from 10 patients diagnosed with an EDMD spectrum disease with different mutations in 7 genes. Upon comparing to controls, the pathway analysis revealed that multiple genes involved in fibrosis, metabolism, myogenic signaling, and splicing were affected in all patients. Splice variant analysis revealed alterations of muscle-specific variants for several important muscle genes. Deeper analysis of metabolic pathways revealed a reduction in glycolytic and oxidative metabolism and reduced numbers of mitochondria across a larger set of 14 EDMD patients and 7 controls. Intriguingly, the gene expression signatures segregated the patients into three subgroups whose distinctions could potentially relate to differences in clinical presentation. Finally, differential expression analysis of miRNAs changing in the patients similarly highlighted fibrosis, metabolism, and myogenic signaling pathways. This pathway approach revealed a clear EDMD signature that can both be used as the basis for establishing a biomarker panel specific to EDMD and direct further investigation into its pathomechanism. Furthermore, the segregation of specific gene changes into three distinct categories that appear to correlate with clinical presentation may be developed into prognostic biomarkers, though this will first require their testing in a wider set of patients with more clinical information.

genetics↗

bioRxiv2021

Preclinical studies for plant-based oral enzyme replacement therapy (Oral-ERT) in Pompe disease knockout mice with transgenic tobacco seeds expressing human GAA (tobrhGAA)

Genetic deficiency of acid -glucosidase (GAA) results in glycogen storage disease type II (GSDII) or Pompe disease (PD) encompassing at least four clinical subtypes of varying severity (infantile; childhood, juvenile and late onset). Our objective is to develop an innovative and affordable approach for enzyme replacement therapy (ERT) via oral administration (Oral-ERT) to maintain a sustained, therapeutic level of enzyme on a daily basis to improve efficacy of treatment and quality of life for people living with Pompe disease. A consensus at a 2019 US Acid Maltase Deficiency (AMDA) conference suggested that a multi-pronged approach including gene therapy, diet, exercise, etc. must be evaluated for a successful treatment of Pompe disease. Tobacco seeds contain the metabolic machinery that is more compatible with mammalian glycosylation-phosphorylation and processing. Previously, we have shown that a lysate from transgenic tobacco seeds expressing human GAA (tobrhGAA) was enzymatically active and can correct enzyme deficiency in cultured PD cells and in adult lymphocytes of Pompe patients and in vivo in disease-relevant tissues in GAA knockout (KO) mice when administered IP. We have extended these pre-clinical studies in PD knockout (KO) mice with ground tobrhGAA seeds that supports proof-of-concept for Oral-ERT for future clinical trials. Briefly in GAA KO mice, Oral-ERT with ground tobrhGAA seeds showed significant reversal of fore-limb and hind-limb muscle weakness, increased motor coordination/balance/strength and mobility, improved spontaneous learning, increased GAA baseline activity in tissues, reduced glycogen in tissues and negible serum titers to GAA. Pharmacokinetics showed maximum serum GAA concentration (Cs) at 8-10 hr and peak urine excretion at 10-12 hr. The tobrhGAA was taken up in PD fibroblast, lymphoid and myoblast cells. Enzyme kinetics compared favorably or superior to placental hGAA, plus alglucosidase alfa or other rhGAAs for Km, Vmax, pH optima, thermal heat stability and IC50 for inhibitors. The tobrhGAA in seeds was extremely stable stored for 15 years at room temperature. NGS-genome sequencing of the tobrhGAA and wild-type plants and RNA expression profiles was performed and will be posted on our website. Thus, Oral-ERT with ground tobrhGAA seeds is an innovative approach that overcomes some of the challenges of alglucosidase alfa-ERT and provides a more effective, safe and significantly less expensive treatment.

genetics↗

bioRxiv2021

Crotamine as a vehicle for non-viral gene delivery for Pompe disease

Genetic deficiency of lysosomal acid alpha glucosidase or acid maltase (GAA) results in Pompe disease (PD), encompassing at least five clinical subtypes of varying severity. The current approved enzyme replacement therapy (ERT) for PD is via IV infusion every 2 weeks of a recombinant human GAA (rhGAA) secreted by Chinese hamster ovary (CHO) cells (alglucosidase alfa/Myozyme, Sanofi/Genzyme). Although alglucosidase alfa has proven to be efficient in rescuing cardiac abnormalities and extending the life span of the infantile form, the response in skeletal muscle is variable. ERT usually begins when the patients are symptomatic and secondary problems are already present which are compounded by low alglucosidase alfa uptake, transient nature (every 2 weeks with a rapid return to defect levels), variable glycogen reduction, autophagic accumulation, immune response and high cost. A consensus at a recent US Acid Maltase Deficiency (AMD) conference suggested that a multi-pronged approach including gene therapy, diet, exercise, etc. must be evaluated for a successful treatment of PD. Compared to replication defective viruses, non-viral gene transfer offers fewer safety concerns and, if recent studies are validated, has a wider range of cells. In order for gene therapy (GT) to succeed, the gene of interest must be delivered into the affected cell and expressed to overcome the inherited deficiency. Cell penetrating peptides (CPPs) enter eukaryotic cells through an energy-independent mechanism and efficiently carry biologically active and therapeutic molecules into cells and localize in the cytoplasm or nucleus. CPPs are usually covalently linked to the cargo, including peptides and DNA. Crotamine (Cro) from the South American rattlesnake-Crotalus durrissus terrificus venom, can bind electrostatically to plasmid DNA to deliver into cells, including muscle. We have assembled a bacterial expression vector for Cro and purified the recombinant Cro (rCro). Transient transfection in AMD fibroblasts and ex vivo in whole blood from an adult Pompe patient with rCro complexed with the pcDNA3 x hGAA cDNA demonstrated increased GAA activity. In GAA knockout (KO) mice receiving a single injection of rCro complexed to pcDNA3 x hGAA cDNA intraperitoneally (IP), we found increased GAA activity in tissues after 48 hr. After 8 treatments-IP over 55 days, we found increased vertical hang-time activity, reduced glycogen deposition, increased GAA activity/hGAA plasmid in tissues and minimal immune-reaction to rCro. A subsequent study of 5 administrations every 2 to 3 weeks showed reverse of the clinical phenotypes by running wheel activity, Rotarod, grip-strength meter, open field mobility and T-maze. Tissue culture experiments in PD fibroblast, lymphoid and skeletal muscle cell lines showed increased GAA activity after rCro transient gene delivery.

genetics↗

bioRxiv2019

Towards development of a statistical framework to evaluate myotonic dystrophy type 1 mRNA biomarkers in the context of a clinical trial

Myotonic dystrophy type 1 (DM1) is a rare genetic disorder, characterised by muscular dystrophy, myotonia, and other symptoms. DM1 is caused by the expansion of a CTG repeat in the 3-untranslated region of DMPK. Longer CTG expansions are associated with greater symptom severity and earlier age at onset. The primary mechanism of pathogenesis is thought to be mediated by a gain of function of the CUG-containing RNA, that leads to trans-dysregulation of RNA metabolism of many other genes. Specifically, the alternative splicing (AS) and alternative polyadenylation (APA) of many genes is known to be disrupted. In the context of clinical trials of emerging DM1 treatments, it is important to be able to objectively quantify treatment efficacy at the level of molecular biomarkers. We show how previously described candidate mRNA biomarkers can be used to model an effective reduction in CTG length, using modern high-dimensional statistics (machine learning), and a blood and muscle mRNA microarray dataset. We show how this model could be used to detect treatment effects in the context of a clinical trial.

bioinformatics↗

bioRxiv2019

A Multistage Sequencing Strategy Pinpoints Many Novel and Candidate Disease Alleles for Orphan Disease Emery-Dreifuss Muscular Dystrophy and Supports Gene Misregulation as its Pathomechanism

Limitations of genome-wide approaches for genetically-heterogenous orphan diseases led us to develop a new approach to identify novel Emery-Dreifuss muscular dystrophy (EDMD) candidate genes. We generated a primer library to genes: (I) linked to EDMD, (II) mutated in related muscular dystrophies, (III) highlighted from limited exome sequencing, (IV) encoding muscle-specific nuclear membrane proteins. Sequencing 56 unlinked EDMD patients yielded confirmed or strong candidate alleles from all categories, accounting for most remaining unlinked patients. Known functions of newly-linked genes argue the EDMD pathomechanism is from altered gene regulation and mechanotransduction through connectivity of candidates from the nuclear envelope to the plasma membrane.

genetics↗