Comparison of different promoters to improve AAV vector-mediated gene therapy for neuronopathic Gaucher disease.
Ontology highlight
ABSTRACT:
SUBMITTER: Massaro G
PROVIDER: S-EPMC11336133 | biostudies-literature | 2024 Aug
REPOSITORIES: biostudies-literature
Publications
Comparison of different promoters to improve AAV vector-mediated gene therapy for neuronopathic Gaucher disease.
Massaro Giulia G Geard Amy F AF Nelvagal Hemanth R HR Gore Katrina K Clemo Nadine K NK Waddington Simon N SN Rahim Ahad A AA
Human molecular genetics 20240801 17
Gaucher Disease (GD) is an inherited metabolic disorder caused by mutations in the GBA1 gene. It can manifest with severe neurodegeneration and visceral pathology. The most acute neuronopathic form (nGD), for which there are no curative therapeutic options, is characterised by devastating neuropathology and death during infancy. In this study, we investigated the therapeutic benefit of systemically delivered AAV9 vectors expressing the human GBA1 gene at two different doses comparing a neuronal- ...[more]
Similar Datasets
Project description:Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.
| S-EPMC10284706 | biostudies-literature
Project description:ObjectiveGaucher disease (GD) is a lysosomal storage disease characterized by a deficiency of glucocerebrosidase. Although enzyme-replacement and substrate-reduction therapies are available, their efficacies in treating the neurological manifestations of GD are negligible. Pharmacological chaperone therapy is hypothesized to offer a new strategy for treating the neurological manifestations of this disease. Specifically, ambroxol, a commonly used expectorant, has been proposed as a candidate pharmacological chaperone. The purpose of this study was to evaluate the safety, tolerability, and neurological efficacy of ambroxol in patients with neuronopathic GD.MethodsThis open-label pilot study included five patients who received high-dose oral ambroxol in combination with enzyme replacement therapy. Safety was assessed by adverse event query, physical examination, electrocardiography, laboratory studies, and drug concentration. Biochemical efficacy was assessed through evidence of glucocerebrosidase activity in the lymphocytes and glucosylsphingosine levels in the cerebrospinal fluid. Neurological efficacy was evaluated using the Unified Myoclonus Rating Scale, Gross Motor Function Measure, Functional Independence Measure, seizure frequency, pupillary light reflex, horizontal saccadic latency, and electrophysiologic studies.ResultsHigh-dose oral ambroxol had good safety and tolerability, significantly increased lymphocyte glucocerebrosidase activity, permeated the blood-brain barrier, and decreased glucosylsphingosine levels in the cerebrospinal fluid. Myoclonus, seizures, and pupillary light reflex dysfunction markedly improved in all patients. Relief from myoclonus led to impressive recovery of gross motor function in two patients, allowing them to walk again.InterpretationPharmacological chaperone therapy with high-dose oral ambroxol shows promise in treating neuronopathic GD, necessitating further clinical trials.
| S-EPMC4774255 | biostudies-literature
Project description:Neuronopathic Gaucher disease (nGD) has a very wide clinical and genotypic spectrum. However, there is no consensus definition of nGD, including no description of how best to diagnostically separate the acute form-Gaucher type 2-from the subacute or chronic form-Gaucher type 3. In this article, we define the various forms of Gaucher disease with particular emphasis on the presence of gaze palsy in all patients with nGD. This consensus definition will help in both clinical diagnosis and appropriate patient recruitment to upcoming clinical trials.
| S-EPMC7540563 | biostudies-literature
Project description:Gaucher disease (GD) is an inherited disorder caused by recessive mutations in the GBA1 gene that encodes the lysosomal enzyme β-glucocerebrosidase (β-GC). β-GC hydrolyzes glucosylceramide (GluCer) into glucose and ceramide in the lysosome, and the loss of its activity leads to GluCer accumulation in different tissues. In severe cases, enzymatic deficiency triggers inflammation, organomegaly, bone disease, and neurodegeneration. Neuronopathic Gaucher disease (nGD) encompasses two different forms of the disease, characterized by chronic or acute damage to the central nervous system (CNS). The cellular and molecular studies that uncover the pathological mechanisms of nGD mainly focus on lysosomal dysfunction since the lysosome is the key organelle affected in GD. However, new studies show alterations in other organelles that contribute to nGD pathology. For instance, abnormal accumulation of GluCer in lysosomes due to the loss of β-GC activity leads to excessive calcium release from the endoplasmic reticulum (ER), activating the ER-associated degradation pathway and the unfolded protein response. Recent evidence indicates mitophagy is altered in nGD, resulting in the accumulation of dysfunctional mitochondria, a critical factor in disease progression. Additionally, nGD patients present alterations in mitochondrial morphology, membrane potential, ATP production, and increased reactive oxygen species (ROS) levels. Little is known about potential dysfunction in other organelles of the secretory pathway, such as the Golgi apparatus and exosomes. This review focuses on collecting evidence regarding organelle dysfunction beyond lysosomes in nGD. We briefly describe cellular and animal models and signaling pathways relevant to uncovering the pathological mechanisms and new therapeutic targets in GD.
| S-EPMC9381931 | biostudies-literature
Project description:Gaucher disease (OMIM #230800) is caused by β-glucosidase deficiency and primarily involves the mononuclear phagocyte system (also called Reticuloendothelial System or Macrophage System). The disease is classified into three main phenotypes based on the presence or absence of neurological manifestations: non-neuronopathic (type 1), acute neuronopathic (type 2) and chronic neuronopathic (type 3). Typical manifestations include hepatosplenomegaly, skeletal deformities, hematological abnormalities, interstitial lung fibrosis and neurodegeneration in neuronopathic cases. Mesenteric lymphadenopathy with resultant protein losing enteropathy (PLE) has only been rarely described. Mesenteric lymphadenopathy may lead to intestinal lymphatic obstruction and secondary lymphangiectasia resulting in chronic diarrhea, abdominal pain and weight loss. Fecal protein loss with secondary hypoalbuminemia can be significant. We report a male with Chronic Neuronopathic Gaucher disease (GD) (homozygous for c.1448T > C (NM_000157.3) GBA mutation) who at 16 years of age developed intractable abdominal pain, diarrhea and weight loss. This was caused by PLE secondary to intestinal lymphangiectasia caused by calcified mesenteric lymphadenopathy despite prior long term enzyme replacement therapy (ERT) and/or substrate reduction therapy (SRT). His older similarly affected sister who had been receiving treatment with ERT and/or SRT remains stable on these treatments with no evidence of mesenteric lymphadenopathy. Medical management with total parenteral nutrition, daily medium chain triglyceride-oil (MCT) supplementation, low dose oral budesonide, continued oral SRT and an increased dose of parenteral ERT has stabilized his condition with resolution of the gastrointestinal symptoms and appropriate weight gain.
| S-EPMC4789350 | biostudies-literature
Project description:Aldehyde dehydrogenase family 1, member L1 (ALDH1L1) is a recently characterized pan-astrocytic marker that is more homogenously expressed throughout the brain than the classic astrocytic marker, glial fibrillary acidic protein. We generated putative promoter sequence variants of the rat ALDH1L1 gene for use in adeno-associated viral vector-mediated gene transfer, with an aim to achieve selective regulation of transgene expression in astrocytes in the rat brain. Unexpectedly, ALDH1L1 promoter variants mediated transcriptional activity exclusively in neurons in the substantia nigra pars compacta as assessed by luciferase reporter expression at 3 weeks postvector infusion. This selectivity for neurons in the substantia nigra pars compacta also persisted in the context of adeno-associated viral serotype 5, 8 or 9 vector-mediated gene delivery. An in vivo promoter comparison showed the highest performing ALDH1L1 promoter variant mediated higher transgene expression than the neuronal-specific synapsin 1 and tyrosine hydroxylase promoters. The ALDH1L1 promoter was also transcriptionally active in dentate granule neurons following intrahippocampal adeno-associated viral vector infusion, whereas transgene expression was detected in both striatal neurons and astrocytes following vector infusion into the striatum. Our results demonstrate the potential suitability of the ALDH1L1 promoter as a new tool in the development of gene therapy and disease modelling applications.
| S-EPMC5130080 | biostudies-literature
Project description:Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the glucosidase, beta, acid (GBA) gene that encodes the lysosomal enzyme glucosylceramidase (GCase). GCase deficiency leads to characteristic visceral pathology and, in some patients, lethal neurological manifestations. Here, we report the generation of mouse models with the severe neuronopathic form of GD. To circumvent the lethal skin phenotype observed in several of the previous GCase-deficient animals, we genetically engineered a mouse model with strong reduction in GCase activity in all tissues except the skin. These mice exhibit rapid motor dysfunction associated with severe neurodegeneration and apoptotic cell death within the brain, reminiscent of neuronopathic GD. In addition, we have created a second mouse model, in which GCase deficiency is restricted to neural and glial cell progenitors and progeny. These mice develop similar pathology as the first mouse model, but with a delayed onset and slower disease progression, which indicates that GCase deficiency within microglial cells that are of hematopoietic origin is not the primary determinant of the CNS pathology. These findings also demonstrate that normal microglial cells cannot rescue this neurodegenerative disease. These mouse models have significant implications for the development of therapy for patients with neuronopathic GD.
| S-EPMC2077282 | biostudies-literature
Project description:BackgroundThe variants of neuronopathic Gaucher disease may be viewed as a clinical phenotypic continuum divided into acute and chronic forms. The chronic neuronopathic form of Gaucher disease is characterized by a later onset of neurological symptoms and protracted neurological and visceral involvement. The first-choice treatment for nonneuronopathic Gaucher disease is enzyme replacement therapy with recombinant analogues of the deficient human enzyme glucocerebrosidase. Enzyme replacement therapy has been shown to improve hematological and bone manifestations associated with Gaucher disease, but, as with most proteins, recombinant enzymes cannot cross the blood-brain barrier, which prevents effects on neurological manifestations. Substrate reduction therapy with miglustat (N-butyldeoxynojirimycin) inhibits glucosylceramide synthase, which catalyzes the first step in glycosphingolipid synthesis. Because miglustat can cross the blood-brain barrier, it has been suggested that, combined with enzyme replacement therapy, it might be effective in treating neurological symptoms in patients with neuronopathic Gaucher disease.Case presentationWe report observed effects of combined enzyme replacement therapy and substrate reduction therapy in a 7-year-old Caucasian boy with neuronopathic Gaucher disease who was homozygous for L444P mutations. He had received enzyme replacement therapy from the age of 18 months, and concomitant miglustat treatment was commenced, with dosing according to body surface area uptitrated over 1 month with dietary modifications when he reached the age of 30 months. He experienced mild diarrhea after commencing miglustat therapy, which decreased in frequency/severity over time. His splenomegaly was reduced, and his hematological values and plasma angiotensin-converting enzyme activity normalized. Plasma chitotriosidase also showed substantial and sustained decreases. After 5 years of combination therapy, the patient showed no signs of neurological impairment.ConclusionsThis case supports the concept that oral miglustat in combination with intravenous enzyme replacement therapy may be beneficial in preventing neurological signs in patients with chronic neuronopathic Gaucher disease. The importance of dietary modifications has also been confirmed. Further follow-up studies are needed to better define the therapeutic effect of combined treatment in this Gaucher disease subtype.
| S-EPMC5248516 | biostudies-literature
Project description:Gaucher disease (GD), resulting from biallelic mutations in the gene GBA1, is a monogenic recessively inherited Mendelian disorder with a wide range of phenotypic presentations. The more severe forms of the disease, acute neuronopathic GD (GD2) and chronic neuronopathic GD (GD3), also have a continuum of disease severity with an overlap in manifestations and limited genotype-phenotype correlation. In very young patients, assigning a definitive diagnosis can sometimes be challenging. Several recent studies highlight specific features of neuronopathic GD that may provide diagnostic clues. Distinguishing between the different GD types has important therapeutic implications. Currently there are limited treatment options specifically for neuronopathic GD due to the difficulty in delivering therapies across the blood-brain barrier. In this work, we present both classic and newly appreciated aspects of the Gaucher phenotype that can aid in discriminating between acute and chronic neuronopathic GD, and highlight the continuing therapeutic challenges.
| S-EPMC7884077 | biostudies-literature
Project description:Adeno Associated Virus (AAV)-mediated gene expression in the brain is widely applied in the preclinical setting to investigate the therapeutic potential of specific molecular targets, characterize various cellular functions, and model central nervous system (CNS) diseases. In therapeutic applications in the clinical setting, gene therapy offers several advantages over traditional pharmacological based therapies, including the ability to directly manipulate disease mechanisms, selectively target disease-afflicted regions, and achieve long-term therapeutic protein expression in the absence of repeated administration of pharmacological agents. Next to the gold-standard iodixanol-based AAV vector production, we recently published a protocol for AAV production based on chloroform-precipitation, which allows for fast in-house production of small quantities of AAV vector without the need for specialized equipment. To validate our recent protocol, we present here a direct side-by-side comparison between vectors produced with either method in a series of in vitro and in vivo assays with a focus on transgene expression, cell loss, and neuroinflammatory responses in the brain. We do not find differences in transduction efficiency nor in any other parameter in our in vivo and in vitro panel of assessment. These results suggest that our novel protocol enables most standardly equipped laboratories to produce small batches of high quality and high titer AAV vectors for their experimental needs.
| S-EPMC7726153 | biostudies-literature