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"Richards, Amy"
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Biophysical and Structural Characterization of Polyethylenimine-Mediated siRNA Delivery in Vitro
The goals of this study were as follows: 1) to evaluate the efficacy of different polyethylenimine (PEI) structures for siRNA delivery in a model system, and 2) to determine the biophysical and structural characteristics of PEI that relate to siRNA delivery.
Biophysical characterization (effective diameter and zeta potential), cytotoxicities, relative binding affinities and in vitro transfection efficiencies were determined using nano-complexes formed from PEI's of 800, 25,000, (both branched) and 22,000 (linear) molecular weights at varying N:P ratios and siRNA concentrations. The HR5-CL11 cell line stably expressing luciferase was used as a model system in vitro.
Successful siRNA delivery was observed within a very narrow window of conditions, and only with the 25,000 branched PEI at an N:P ratio of 6:1 and 8:1 and with 200 nM siRNA. While the zeta potential and size of PEI:siRNA complexes correlated to transfection efficacy in some cases, complex stability may also affect transfection efficacy.
The ability of PEI to transfer functionally active siRNA to cells in culture is surprisingly dependent on its biophysical and structural characteristics when compared to its relative success and ease of use for DNA delivery.
Journal Article
Glucosylceramide synthase inhibition alleviates aberrations in synucleinopathy models
Mutations in the glucocerebrosidase gene (GBA) confer a heightened risk of developing Parkinson’s disease (PD) and other synucleinopathies, resulting in a lower age of onset and exacerbating disease progression. However, the precise mechanisms by which mutations in GBA increase PD risk and accelerate its progression remain unclear. Here, we investigated the merits of glucosylceramide synthase (GCS) inhibition as a potential treatment for synucleinopathies. Two murine models of synucleinopathy (a Gaucher-related synucleinopathy model, GbaD409V/D409V
and a A53T–α-synuclein overexpressing model harboring wild-type alleles of GBA, A53T–SNCA mouse model) were exposed to a brain-penetrant GCS inhibitor, GZ667161. Treatment of GbaD409V/D409V
mice with the GCS inhibitor reduced levels of glucosylceramide and glucosylsphingosine in the central nervous system (CNS), demonstrating target engagement. Remarkably, treatment with GZ667161 slowed the accumulation of hippocampal aggregates of α-synuclein, ubiquitin, and tau, and improved the associated memory deficits. Similarly, prolonged treatment of A53T–SNCA mice with GZ667161 reduced membrane-associated α-synuclein in the CNS and ameliorated cognitive deficits. The data support the contention that prolonged antagonism of GCS in the CNS can affect α-synuclein processing and improve behavioral outcomes. Hence, inhibition of GCS represents a diseasemodifying therapeutic strategy for GBA-related synucleinopathies and conceivably for certain forms of sporadic disease.
Journal Article
Preclinical pharmacology of glucosylceramide synthase inhibitor venglustat in a GBA-related synucleinopathy model
Mutations in
GBA
, the gene encoding the lysosomal enzyme glucocerebrosidase (GCase), represent the greatest genetic risk factor for developing synucleinopathies including Parkinson’s disease (PD). Additionally, PD patients harboring a mutant
GBA
allele present with an earlier disease onset and an accelerated disease progression of both motor and non-motor symptoms. Preclinical studies in mouse models of synucleinopathy suggest that modulation of the sphingolipid metabolism pathway via inhibition of glucosylceramide synthase (GCS) using a CNS-penetrant small molecule may be a potential treatment for synucleinopathies. Here, we aim to alleviate the lipid storage burden by inhibiting the de novo synthesis of the primary glycosphingolipid substrate of GCase, glucosylceramide (GlcCer). We have previously shown that systemic GCS inhibition reduced GlcCer and glucosylsphingosine (GlcSph) accumulation, slowed α-synuclein buildup in the hippocampus, and improved cognitive deficits. Here, we studied the efficacy of a brain-penetrant clinical candidate GCS inhibitor, venglustat, in mouse models of
GBA
-related synucleinopathy, including a heterozygous
Gba
mouse model which more closely replicates the typical
GBA
-PD patient genotype. Collectively, these data support the rationale for modulation of GCase-related sphingolipid metabolism as a therapeutic strategy for treating
GBA-
related synucleinopathies.
Journal Article
Cross-species efficacy of AAV-mediated ARSA replacement for metachromatic leukodystrophy
Metachromatic leukodystrophy (MLD) is an autosomal recessive neurodegenerative disorder caused by mutations in the arylsulfatase A ( ARSA ) gene, resulting in lower sulfatase activity and the toxic accumulation of sulfatides in the central and peripheral nervous system. Children account for 70% of cases and become progressively disabled, with death occurring within 10 years of disease onset. Gene therapy approaches to restore ARSA expression via adeno-associated virus (AAV) vectors have been promising but hampered by limited brain biodistribution. We report the development of an engineered capsid, AAV.GMU01, demonstrating superior biodistribution and transgene expression in the central nervous system of nonhuman primates (NHPs). Next, we show that AAV.GMU01- ARSA –treated MLD mice exhibit persistent, normal levels of sulfatase activity and a concomitant reduction in toxic sulfatides. Treated mice also show a reduction in MLD-associated pathology and auditory dysfunction. Lastly, we demonstrate that treatment with AAV.GMU01- ARSA in NHPs is well tolerated and results in potentially therapeutic ARSA expression in the brain. In summary, we propose AAV.GMU01- ARSA –mediated gene replacement as a clinically viable approach to achieve broad and therapeutic levels of ARSA.
Journal Article
The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil function
Bacterial superantigens (SAgs) cause Vβ-dependent T-cell proliferation leading to immune dysregulation associated with the pathogenesis of life-threatening infections such as toxic shock syndrome, and necrotizing pneumonia. Previously, we demonstrated that staphylococcal enterotoxin-like toxin X (SElX) from Staphylococcus aureus is a classical superantigen that exhibits T-cell activation in a Vβ-specific manner, and contributes to the pathogenesis of necrotizing pneumonia. Here, we discovered that SElX can also bind to neutrophils from human and other mammalian species and disrupt IgG-mediated phagocytosis. Site-directed mutagenesis of the conserved sialic acid-binding motif of SElX abolished neutrophil binding and phagocytic killing, and revealed multiple glycosylated neutrophil receptors for SElX binding. Furthermore, the neutrophil binding-deficient mutant of SElX retained its capacity for T-cell activation demonstrating that SElX exhibits mechanistically independent activities on distinct cell populations associated with acquired and innate immunity, respectively. Finally, we demonstrated that the neutrophil-binding activity rather than superantigenicity is responsible for the SElX-dependent virulence observed in a necrotizing pneumonia rabbit model of infection. Taken together, we report the first example of a SAg, that can manipulate both the innate and adaptive arms of the human immune system during S. aureus pathogenesis.
Journal Article
CNS-targeted gene therapy improves survival and motor function in a mouse model of spinal muscular atrophy
Spinal muscular atrophy (SMA) is a neuromuscular disease caused by a deficiency of survival motor neuron (SMN) due to mutations in the SMN1 gene. In this study, an adeno-associated virus (AAV) vector expressing human SMN (AAV8-hSMN) was injected at birth into the CNS of mice modeling SMA. Western blot analysis showed that these injections resulted in widespread expression of SMN throughout the spinal cord, and this translated into robust improvement in skeletal muscle physiology, including increased myofiber size and improved neuromuscular junction architecture. Treated mice also displayed substantial improvements on behavioral tests of muscle strength, coordination, and locomotion, indicating that the neuromuscular junction was functional. Treatment with AAV8-hSMN increased the median life span of mice with SMA-like disease to 50 days compared with 15 days for untreated controls. Moreover, injecting mice with SMA-like disease with a human SMN-expressing self-complementary AAV vector - a vector that leads to earlier onset of gene expression compared with standard AAV vectors - led to improved efficacy of gene therapy, including a substantial extension in median survival to 157 days. These data indicate that CNS-directed, AAV-mediated SMN augmentation is highly efficacious in addressing both neuronal and muscular pathologies in a severe mouse model of SMA.
Journal Article
Reduction in Emotional and Uncontrolled Eating Scores with Yoga Compared to Stretching: A Randomized Trial
Lowering stress through yoga may help individuals make better food choices and manage their weight.
To compare the effectiveness of yoga versus stretching for reducing stress levels, improving eating behaviors, and enhancing weight loss.
Randomized controlled 3-month trial with an additional 3-month follow-up.
Midwestern United States.
One hundred and sixty-two participants (146 females, mean age 43.7 years, BMI 34.0 kg/m2).
Participants were randomized to either yoga or stretching (control) intervention and asked to complete exercises 3 times a week for 3 months.
Perceived Stress Scale and Three Factor Eating Questionnaire Revised were completed, and weight was measured at baseline, 3, and 6 months.
Perceived stress decreased significantly in both groups at 3 months, and in just the yoga group at 6 months. There were no group differences at 6 months (-2.8 ± 6.6 yoga vs. -3.0 ± 6.4 control, P = .53). Emotional eating (EE) and uncontrolled eating (UE) were lower in the intervention group at 6 months (-16.0 ± 16.7 yoga vs. -3.3 ± 23.9 control, P = .04 and -11.8 ± 11.9 yoga vs. -3.6 ± 9.9 control, P = .01), with no difference at 3 months. Both EE (P = .026) and UE scores (P = .006) were lower for subjects who completed the program through 6 months. Changes in EE and UE were correlated with changes in perceived stress (r = 0.37, P < .01, df = 64 and r = 0.57, P < .001, df = 64, respectively) and cognitive restraint was negatively correlated with changes in perceived stress (r = -0.32, P < .01, df = 64) at 3 months.
Yoga may improve indicators of emotional eating and uncontrolled eating, which were correlated with a decrease in levels of perceived stress.
Eating behaviors; Obesity; Weight loss; Yoga, Stress.
Journal Article