Efficient intracellular delivery is critical to the successful application of synthetic antisense oligonucleotides (ASOs) to modulate gene expression. The conjugation of cell-penetrating peptides ...(CPPs) to ASOs has been shown to significantly improve their intracellular delivery. It is important, however, that formation of the covalent linkage between the peptide and oligonucleotide is efficient and orthogonal, to ensure high yields and a homogeneous product. Described herein are efficient and facile methodologies for the conjugation of peptides to ASOs, and their subsequent labeling with various moieties such as fluorescent dyes for intracellular tracking studies.
Amyotrophic lateral sclerosis (ALS) is a progressive paralyzing disease characterized by tissue oxidative damage and motor neuron degeneration. This study investigated the in vivo effect of ...diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)), which is an orally bioavailable, blood-brain barrier-permeable complex. In vitro the compound inhibits the action of peroxynitrite on Cu,Zn-superoxide dismutase (SOD1) and subsequent nitration of cellular proteins. Oral treatment of transgenic SOD1G93A mice with CuII(atsm) at presymptomatic and symptomatic ages was performed. The mice were examined for improvement in lifespan and motor function, as well as histological and biochemical changes to key disease markers. Systemic treatment of SOD1G93A mice significantly delayed onset of paralysis and prolonged lifespan, even when administered to symptomatic animals. Consistent with the properties of this compound, treated mice had reduced protein nitration and carbonylation, as well as increased antioxidant activity in spinal cord. Treatment also significantly preserved motor neurons and attenuated astrocyte and microglial activation in mice. Furthermore, CuII(atsm) prevented the accumulation of abnormally phosphorylated and fragmented TAR DNA-binding protein-43 (TDP-43) in spinal cord, a protein pivotal to the development of ALS. CuII(atsm) therefore represents a potential new class of neuroprotective agents targeting multiple major disease pathways of motor neurons with therapeutic potential for ALS.
Background: CuII(atsm) (diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) was orally administrated to transgenic SOD1G93A mice.
Results: Treatment significantly prolonged lifespan with preservation of motor neurons. Reduced protein oxidation, attenuated astrocyte, and microglial activation also resulted from treatment.
Conclusion: CuII(atsm) is neuroprotective in this model even when treatment begins after the onset of disease symptoms.
Significance: The drug has therapeutic potential for amyotrophic lateral sclerosis.
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder caused by loss of motor neurons. ALS incidence is skewed towards males with typically earlier age of onset and limb ...site of onset. The androgen receptor (AR) is the major mediator of androgen effects in the body and is present extensively throughout the central nervous system, including motor neurons. Mutations in the AR gene lead to selective lower motor neuron degeneration in male spinal bulbar muscular atrophy (SBMA) patients, emphasising the importance of AR in maintaining motor neuron health and survival. To evaluate a potential role of AR in onset and progression of ALS, we generated SOD1
mice with either neural AR deletion or global human AR overexpression. Using a Cre-LoxP conditional gene knockout strategy, we report that neural deletion of AR has minimal impact on the disease course in SOD1
male mice. This outcome was potentially confounded by the metabolically disrupted Nestin-Cre phenotype, which likely conferred the profound lifespan extension observed in the SOD1
double transgenic male mice. In addition, overexpression of human AR produced no benefit to disease onset and progression in SOD1
mice. In conclusion, the disease course of SOD1
mice is independent of AR expression levels, implicating other mechanisms involved in mediating the sex differences in ALS. Our findings using Nestin-Cre mice, which show an inherent metabolic phenotype, led us to hypothesise that targeting hypermetabolism associated with ALS may be a more potent modulator of disease, than AR in this mouse model.
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Antisense oligonucleotides (ASOs) are an emerging class of gene-specific therapeutics for diseases associated with the central nervous system (CNS). However, ASO delivery across the ...blood–brain barrier (BBB) to their CNS target cells remains a major challenge. Since ASOs are mainly taken up into the brain capillary endothelial cells interface through endosomal routes, entrapment in the endosomal compartment is a major obstacle for efficient CNS delivery of ASOs. Therefore, we evaluated the effectiveness of a panel of cell-penetrating peptides (CPPs) bearing several endosomal escape domains for the intracellular delivery, endosomal release and antisense activity of FDA-approved Spinraza (Nusinersen), an ASO used to treat spinal muscular atrophy (SMA). We identified a CPP, HA2-ApoE(131–150), which, when conjugated to Nusinersen, showed efficient endosomal escape capability and significantly increased the level of full-length functional mRNA of the survival motor neuron 2 (SMN2) gene in SMA patient-derived fibroblasts. Treatment of SMN2 transgenic adult mice with this CPP-PMO conjugate resulted in a significant increase in the level of full-length SMN2 in the brain and spinal cord. This work provides proof-of-principle that integration of endosomal escape domains with CPPs enables higher cytosolic delivery of ASOs, and more importantly enhances the efficiency of BBB-permeability and CNS activity of systemically administered ASOs.
Abstract Spinal muscular atrophy results from diminished levels of survival motor neuron (SMN) protein in spinal motor neurons. Low levels of SMN also occur in models of amyotrophic lateral sclerosis ...(ALS) caused by mutant superoxide dismutase 1 (SOD1) and genetic reduction of SMN levels exacerbates the phenotype of transgenic SOD1G93A mice. Here, we demonstrate that SMN protein is significantly reduced in the spinal cords of patients with sporadic ALS. To test the potential of SMN as a modifier of ALS, we overexpressed SMN in 2 different strains of SOD1G93A mice. Neuronal overexpression of SMN significantly preserved locomotor function, rescued motor neurons, and attenuated astrogliosis in spinal cords of SOD1G93A mice. Despite this, survival was not prolonged, most likely resulting from SMN mislocalization and depletion of gems in motor neurons of symptomatic mice. Our results reveal that SMN upregulation slows locomotor deficit onset and motor neuron loss in this mouse model of ALS. However, disruption of SMN nuclear complexes by high levels of mutant SOD1, even in the presence of SMN overexpression, might limit its survival promoting effects in this specific mouse model. Studies in emerging mouse models of ALS are therefore warranted to further explore the potential of SMN as a modifier of ALS.
Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality worldwide, is characterised by the homozygous loss of the survival motor neuron 1 (SMN1) gene. The consequent degeneration ...of spinal motor neurons and progressive atrophy of voluntary muscle groups results in paralysis and eventually premature infantile death. Humans possess a second nearly identical copy of SMN1, known as SMN2. However, SMN2 produces only 10–20% functional SMN protein due to aberrant splicing of its pre-mRNA that leads to the exclusion of exon 7. This level of SMN is insufficient to rescue the phenotype. Recently developed splice-switching antisense oligonuclotides (SSO) have shown great promise in correcting the aberrant splicing of SMN2 towards producing functional SMN protein. Several FDA approved drugs are being repurposed for SMA treatment including valproic acid (VPA), a histone deacetylase inhibitor, which has been shown to increase overall SMN2 expression. In this study, we have characterised the effects of single and combined treatment of VPA and a SSO based on phosphorodiamidate morpholino oligomer (PMO) chemistry. We conjugated both VPA and PMO to a single cell-penetrating peptide (Apolipoprotein E (ApoE)) for their simultaneous intracellular delivery. Treatment of SMA Type I patient-derived fibroblasts with the conjugates showed no additive increase in the level of full-length SMN2 mRNA expression over both 4 and 16 h treatments indicating that conjugation of VPA to ApoE-PMO has limited benefit. However, treatment with a combination of VPA and ApoE-PMO induced more favourable splice switching activity than either agent alone, promoting exon 7 inclusion in SMN2 transcripts. Our results suggest that combination therapy of VPA and ApoE-PMO is superior in upregulating SMN2 production in vitro, as compared to singular treatment of each compound at both transcriptional and protein levels. This study provides the first indication of a novel dual therapy approach for the potential treatment of SMA.
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•Antisense oligonuclotides (ASO) and Valproate increased protein in Spinal Muscular Atrophy patient-derived fibroblast.•Conjugation of VPA to peptide-ASO for simultaneous intracellular delivery is not therapeutically favourable.•Combination therapy of VPA and peptide-ASO is superior compared to singular treatment of each compound.•Combination of VPA and peptide-ASO is not toxic at a therapeutically relevant concentrations.•This dual therapy approach represent a potential treatment for Spinal Muscular Atrophy patients.
Amyotrophic lateral sclerosis (ALS) is characterized by death and dysfunction of motor neurons that result in a rapidly progressing loss of motor function. While there are some data on alterations at ...the blood-brain barrier (BBB) in ALS and their potential impact on CNS trafficking of drugs, little is reported on the impact of this disease on the expression of drug-handling proteins in the small intestine and liver. This may impact the dosing of the many medicines that individuals with ALS are prescribed. In the present study, a proteomic evaluation was performed on small intestine and liver samples from postnatal day 120 SOD1
mice (a model of familial ALS that harbors a human mutant form of superoxide dismutase 1) and wild-type (WT) littermates (
= 7/genotype/sex). Untargeted, quantitative proteomics was undertaken using either label-based tandem mass tag (TMT) or label-free data-independent acquisition (DIA) acquisition strategies on high-resolution mass spectrometric instrumentation. Copper chaperone for superoxide dismutase (CCS) was significantly higher in SOD1
samples compared to the WT samples for both sexes and tissues, therefore representing a potential biomarker for ALS in this mouse model. Relative to WT mice, male SOD1
mice had significantly different proteins (
< 0.05, |fold-change|>1.2) in the small intestine (male 22, female 1) and liver (male 140, female 3). This included an up-regulation of intestinal transporters for dietary glucose solute carrier (SLC) SLC5A1 and cholesterol (Niemann-Pick c1-like 1), as well as for several drugs (e.g., SLC15A1), in the male SOD1
mice. There was both an up-regulation (e.g., SLCO2A1) and down-regulation (ammonium transporter rh type b) of transporters in the male SOD1
liver. In addition, there was both an up-regulation (e.g., phosphoenolpyruvate carboxykinase) and down-regulation (e.g., carboxylesterase 1) of metabolizing enzymes in the male SOD1
liver. This proteomic data set identified male-specific changes to key small intestinal and hepatic transporters and metabolizing enzymes that may have important implications for the bioavailability of nutrients and drugs in individuals with ALS.
Amyotrophic lateral sclerosis (ALS) is characterized by death and dysfunction of motor neurons that result in a rapidly progressing loss of motor function. While there are some data on alterations at ...the blood–brain barrier (BBB) in ALS and their potential impact on CNS trafficking of drugs, little is reported on the impact of this disease on the expression of drug-handling proteins in the small intestine and liver. This may impact the dosing of the many medicines that individuals with ALS are prescribed. In the present study, a proteomic evaluation was performed on small intestine and liver samples from postnatal day 120 SOD1G93A mice (a model of familial ALS that harbors a human mutant form of superoxide dismutase 1) and wild-type (WT) littermates (n = 7/genotype/sex). Untargeted, quantitative proteomics was undertaken using either label-based tandem mass tag (TMT) or label-free data-independent acquisition (DIA) acquisition strategies on high-resolution mass spectrometric instrumentation. Copper chaperone for superoxide dismutase (CCS) was significantly higher in SOD1G93A samples compared to the WT samples for both sexes and tissues, therefore representing a potential biomarker for ALS in this mouse model. Relative to WT mice, male SOD1G93A mice had significantly different proteins (P adj < 0.05, |fold-change|>1.2) in the small intestine (male 22, female 1) and liver (male 140, female 3). This included an up-regulation of intestinal transporters for dietary glucose solute carrier (SLC) SLC5A1 and cholesterol (Niemann-Pick c1-like 1), as well as for several drugs (e.g., SLC15A1), in the male SOD1G93A mice. There was both an up-regulation (e.g., SLCO2A1) and down-regulation (ammonium transporter rh type b) of transporters in the male SOD1G93A liver. In addition, there was both an up-regulation (e.g., phosphoenolpyruvate carboxykinase) and down-regulation (e.g., carboxylesterase 1) of metabolizing enzymes in the male SOD1G93A liver. This proteomic data set identified male-specific changes to key small intestinal and hepatic transporters and metabolizing enzymes that may have important implications for the bioavailability of nutrients and drugs in individuals with ALS.
Neuromuscular and neurodegenerative diseases are mostly modeled using genetically modified animals such as mice. However, animal models do not recapitulate all the phenotypes that are specific to ...human disease. This is mainly due to the genetic, anatomical and physiological difference in the neuromuscular systems of animals and humans. The emergence of direct and indirect human somatic cell reprogramming technologies may overcome this limitation because they enable the use of disease and patient-specific cellular models as enhanced platforms for drug discovery and autologous cell-based therapy. Induced pluripotent stem cells (iPSCs) and urine-derived stem cells (USCs) are increasingly employed to recapitulate the pathophysiology of various human diseases. Recent cell-based modeling approaches utilize highly complex differentiation systems that faithfully mimic human tissue- and organ-level dysfunctions. In this review, we discuss promising cellular models, such as USC- and iPSC-based approaches, that are currently being used to model human neuromuscular and neurodegenerative diseases.
Introduction
Pimavanserin is approved in the USA to treat hallucinations and delusions associated with Parkinson’s disease psychosis (PDP).
Objectives
We evaluated mortality in patients with PDP ...after initiation of pimavanserin or comparator atypical antipsychotics, overall, over time, and across subgroups.
Methods
A cohort of patients aged ≥65 years in the USA with PDP newly initiating pimavanserin or a comparator atypical antipsychotic (clozapine, quetiapine, risperidone, olanzapine, aripiprazole, brexpiprazole) was identified in 2016–2019 Medicare claims data. All-cause mortality in the propensity score–matched treatment groups was compared with hazard ratios (HRs) and 95% confidence intervals (CIs) estimated with Cox-proportional hazards models. Cumulative incidence curves and time period–specific models evaluated risk over time. Subgroup and sensitivity analyses were performed, including a sub-cohort of long-term care (LTC) or skilled nursing facility (SNF) residents.
Results
We identified 2892 pimavanserin initiators and 19,083 comparator initiators (overall 47% female, mean age = 80.9 years, LTC/SNF residents = 30%). Before matching, pimavanserin users had fewer severe comorbidities and more anti-Parkinson medication use than comparators. Matching resulted in 2891 patients in both groups, and all covariates were well balanced. In the matched cohort, the HR for mortality for pimavanserin versus comparator was 0.78 (95% CI 0.67–0.91), with the lowest time period–specific HRs in the first 180 days. Hazard ratios were similar across sensitivity analyses and subgroups. In LTC/SNF residents, the HR was 0.78 (95% CI 0.60–1.01).
Conclusion
The observed mortality rates were lower among patients treated with pimavanserin compared with those treated with other atypical antipsychotics.
Study registration
European Union Post-authorization Study (EU PAS) register number 46331.
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