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Nucleic Acids Research Jul 2023Although targeting TfR1 to deliver oligonucleotides to skeletal muscle has been demonstrated in rodents, effectiveness and pharmacokinetic/pharmacodynamic (PKPD)...
Although targeting TfR1 to deliver oligonucleotides to skeletal muscle has been demonstrated in rodents, effectiveness and pharmacokinetic/pharmacodynamic (PKPD) properties remained unknown in higher species. We developed antibody-oligonucleotide conjugates (AOCs) towards mice or monkeys utilizing anti-TfR1 monoclonal antibodies (αTfR1) conjugated to various classes of oligonucleotides (siRNA, ASOs and PMOs). αTfR1 AOCs delivered oligonucleotides to muscle tissue in both species. In mice, αTfR1 AOCs achieved a > 15-fold higher concentration to muscle tissue than unconjugated siRNA. A single dose of an αTfR1 conjugated to an siRNA against Ssb mRNA produced > 75% Ssb mRNA reduction in mice and monkeys, and mRNA silencing was greatest in skeletal and cardiac (striated) muscle with minimal to no activity in other major organs. In mice the EC50 for Ssb mRNA reduction in skeletal muscle was >75-fold less than in systemic tissues. Oligonucleotides conjugated to control antibodies or cholesterol produced no mRNA reduction or were 10-fold less potent, respectively. Tissue PKPD of AOCs demonstrated mRNA silencing activity primarily driven by receptor-mediated delivery in striated muscle for siRNA oligonucleotides. In mice, we show that AOC-mediated delivery is operable across various oligonucleotide modalities. AOC PKPD properties translated to higher species, providing promise for a new class of oligonucleotide therapeutics.
Topics: Mice; Animals; Oligonucleotides; Oligonucleotides, Antisense; Antibodies; RNA, Small Interfering; RNA, Messenger; Muscle, Skeletal
PubMed: 37224533
DOI: 10.1093/nar/gkad415 -
Proceedings of the National Academy of... Jul 2023Aberrant alternative splicing of mRNAs results in dysregulated gene expression in multiple neurological disorders. Here, we show that hundreds of mRNAs are incorrectly...
Aberrant alternative splicing of mRNAs results in dysregulated gene expression in multiple neurological disorders. Here, we show that hundreds of mRNAs are incorrectly expressed and spliced in white blood cells and brain tissues of individuals with fragile X syndrome (FXS). Surprisingly, the gene is transcribed in >70% of the FXS tissues. In all -expressing FXS tissues, RNA itself is mis-spliced in a CGG expansion-dependent manner to generate the little-known -217 RNA isoform, which is comprised of exon 1 and a pseudo-exon in intron 1. -217 is also expressed in FXS premutation carrier-derived skin fibroblasts and brain tissues. We show that in cells aberrantly expressing mis-spliced , antisense oligonucleotide (ASO) treatment reduces -217, rescues full-length RNA, and restores FMRP (Fragile X Messenger RibonucleoProtein) to normal levels. Notably, gene reactivation in transcriptionally silent FXS cells using 5-aza-2'-deoxycytidine (5-AzadC), which prevents DNA methylation, increases -217 RNA levels but not FMRP. ASO treatment of cells prior to 5-AzadC application rescues full-length expression and restores FMRP. These findings indicate that misregulated RNA-processing events in blood could serve as potent biomarkers for FXS and that in those individuals expressing , ASO treatment may offer a therapeutic approach to mitigate the disorder.
Topics: Humans; Fragile X Syndrome; Trinucleotide Repeat Expansion; Oligonucleotides, Antisense; Decitabine; Fragile X Mental Retardation Protein; Oligonucleotides; RNA
PubMed: 37364131
DOI: 10.1073/pnas.2302534120 -
Neurotherapeutics : the Journal of the... Jul 2022Despite extensive research, amyotrophic lateral sclerosis (ALS) remains a progressive and invariably fatal neurodegenerative disease. Limited knowledge of the underlying... (Randomized Controlled Trial)
Randomized Controlled Trial
Despite extensive research, amyotrophic lateral sclerosis (ALS) remains a progressive and invariably fatal neurodegenerative disease. Limited knowledge of the underlying causes of ALS has made it difficult to target upstream biological mechanisms of disease, and therapeutic interventions are usually administered relatively late in the course of disease. Genetic forms of ALS offer a unique opportunity for therapeutic development, as genetic associations may reveal potential insights into disease etiology. Genetic ALS may also be amenable to investigating earlier intervention given the possibility of identifying clinically presymptomatic, at-risk individuals with causative genetic variants. There is increasing evidence for a presymptomatic phase of ALS, with biomarker data from the Pre-Symptomatic Familial ALS (Pre-fALS) study showing that an elevation in blood neurofilament light chain (NfL) precedes phenoconversion to clinically manifest disease. Tofersen is an investigational antisense oligonucleotide designed to reduce synthesis of superoxide dismutase 1 (SOD1) protein through degradation of SOD1 mRNA. Informed by Pre-fALS and the tofersen clinical development program, the ATLAS study (NCT04856982) is designed to evaluate the impact of initiating tofersen in presymptomatic carriers of SOD1 variants associated with high or complete penetrance and rapid disease progression who also have biomarker evidence of disease activity (elevated plasma NfL). The ATLAS study will investigate whether tofersen can delay the emergence of clinically manifest ALS. To our knowledge, ATLAS is the first interventional trial in presymptomatic ALS and has the potential to yield important insights into the design and conduct of presymptomatic trials, identification, and monitoring of at-risk individuals, and future treatment paradigms in ALS.
Topics: Humans; Superoxide Dismutase-1; Amyotrophic Lateral Sclerosis; Superoxide Dismutase; Neurodegenerative Diseases; Oligonucleotides, Antisense; Biomarkers; RNA, Messenger; Mutation
PubMed: 35585374
DOI: 10.1007/s13311-022-01237-4 -
Drugs Apr 2024Eplontersen (Wainua™) is a ligand-conjugated antisense oligonucleotide directed to TTR, which is being developed by Ionis Pharmaceuticals and AstraZeneca for the... (Review)
Review
Eplontersen (Wainua™) is a ligand-conjugated antisense oligonucleotide directed to TTR, which is being developed by Ionis Pharmaceuticals and AstraZeneca for the treatment of TTR-mediated amyloidosis (ATTR). Eplontersen, which is targeted to the liver by a ligand containing three N-acetyl galactosamine residues, binds to wild-type and variant TTR mRNA, thus reducing the levels of circulating TTR protein and amyloid deposition. Subcutaneous eplontersen reduced serum TTR levels, inhibited neuropathy progression and improved health-related quality of life in patients with polyneuropathy of hereditary ATTR (ATTRv-PN; v for variant) in a phase III trial. Based on these results, eplontersen was approved in the USA for the treatment of ATTRv-PN on 21 December 2023 and is currently undergoing regulatory review for a similar indication in the EU, the UK, Switzerland and Canada. Eplontersen is also undergoing phase III development for ATTR cardiomyopathy. This article summarizes the milestones in the development of eplontersen leading to this first approval for ATTRv-PN.
Topics: Humans; Amyloid Neuropathies, Familial; Prealbumin; Drug Approval; Oligonucleotides; Oligonucleotides, Antisense; Quality of Life; Clinical Trials, Phase III as Topic
PubMed: 38413492
DOI: 10.1007/s40265-024-02008-5 -
Nucleic Acids Research Feb 2023Inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is...
Inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. Here, we show that the well-tolerated and orally bioavailable synthetic sphingolipid analog, SH-BC-893, increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance their activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold without toxicity. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts endolysosomal trafficking to improve the activity of oligonucleotides in extrahepatic tissues.
Topics: Animals; Mice; Oligonucleotides; Endosomes; Endocytosis; Biological Transport; Oligonucleotides, Antisense; RNA, Small Interfering
PubMed: 36727438
DOI: 10.1093/nar/gkad023 -
Cancer Research Mar 2022The M2 pyruvate kinase (PKM2) isoform is upregulated in most cancers and plays a crucial role in regulation of the Warburg effect, which is characterized by the...
UNLABELLED
The M2 pyruvate kinase (PKM2) isoform is upregulated in most cancers and plays a crucial role in regulation of the Warburg effect, which is characterized by the preference for aerobic glycolysis over oxidative phosphorylation for energy metabolism. PKM2 is an alternative-splice isoform of the PKM gene and is a potential therapeutic target. Antisense oligonucleotides (ASO) that switch PKM splicing from the cancer-associated PKM2 to the PKM1 isoform have been shown to induce apoptosis in cultured glioblastoma cells when delivered by lipofection. Here, we explore the potential of ASO-based PKM splice switching as a targeted therapy for liver cancer. A more potent lead constrained-ethyl (cEt)/DNA ASO induced PKM splice switching and inhibited the growth of cultured hepatocellular carcinoma (HCC) cells. This PKM isoform switch increased pyruvate-kinase activity and altered glucose metabolism. In an orthotopic HCC xenograft mouse model, the lead ASO and a second ASO targeting a nonoverlapping site inhibited tumor growth. Finally, in a genetic HCC mouse model, a surrogate mouse-specific ASO induced Pkm splice switching and inhibited tumorigenesis, without observable toxicity. These results lay the groundwork for a potential ASO-based splicing therapy for HCC.
SIGNIFICANCE
Antisense oligonucleotides are used to induce a change in PKM isoform usage in hepatocellular carcinoma, reversing the Warburg effect and inhibiting tumorigenesis.
Topics: Alternative Splicing; Animals; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Transformation, Neoplastic; Glycolysis; Humans; Liver Neoplasms; Mice; Oligonucleotides, Antisense; Protein Isoforms; Pyruvate Kinase
PubMed: 34921016
DOI: 10.1158/0008-5472.CAN-20-0948 -
RNA Biology 2022RNA therapeutics comprise a diverse group of oligonucleotide-based drugs such as antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and short hairpin...
RNA therapeutics comprise a diverse group of oligonucleotide-based drugs such as antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs) that can be designed to selectively interact with drug targets currently undruggable with small molecule-based drugs or monoclonal antibodies. Furthermore, RNA-based therapeutics have the potential to modulate entire disease pathways, and thereby represent a new modality with unprecedented potential for generating disease-modifying drugs for a wide variety of human diseases, including central nervous system (CNS) disorders. Here, we describe different strategies for delivering RNA drugs to the CNS and review recent advances in clinical development of ASO drugs and siRNA-based therapeutics for the treatment of neurological diseases and neuromuscular disorders. 2'-MOE: 2'--(2-methoxyethyl); 2'--Me: 2'--methyl; 2'-F: 2'-fluoro; AD: Alzheimer's disease; ALS: Amyotrophic lateral sclerosis; ALSFRS-R: Revised Amyotrophic Lateral Sclerosis Functional Rating Scale; ARC: Antibody siRNA Conjugate; AS: Angelman Syndrome; ASGRP: Asialoglycoprotein receptor; ASO: Antisense oligonucleotide; AxD: Alexander Disease; BBB: Blood brain barrier; Bp: Basepair; CNM: Centronuclear myopathies; CNS: Central Nervous System; CPP: Cell-penetrating Peptide; CSF: Cerebrospinal fluid; DMD: Duchenne muscular dystrophy; DNA: Deoxyribonucleic acid; FAP: Familial amyloid polyneuropathy; FALS: Familial amyotrophic lateral sclerosis; FDA: The United States Food and Drug Administration; GalNAc: N-acetylgalactosamine; GoF: Gain of function; hATTR: Hereditary transthyretin amyloidosis; HD: Huntington's disease; HRQOL: health-related quality of life; ICV: Intracerebroventricular; IT: Intrathecal; LNA: Locked nucleic acid; LoF: Loss of function; mRNA: Messenger RNA; MS: Multiple Sclerosis; MSA: Multiple System Atrophy; NBE: New Biological Entity; NCE: New Chemical Entity; NHP: Nonhuman primate; nt: Nucleotide; PD: Parkinson's disease; PNP: Polyneuropathy; PNS: Peripheral nervous system; PS: Phosphorothioate; RISC: RNA-Induced Silencing Complex; RNA: Ribonucleic acid; RNAi: RNA interference; s.c.: Subcutaneous; siRNA: Small interfering RNA; SMA: Spinal muscular atrophy; SMN: Survival motor neuron; TTR: Transthyretin.
Topics: Animals; Amyloid Neuropathies, Familial; Amyotrophic Lateral Sclerosis; Neuromuscular Diseases; Oligonucleotides, Antisense; Quality of Life; RNA, Messenger; RNA, Small Interfering; United States; Genetic Therapy; Humans; Disease Models, Animal
PubMed: 35482908
DOI: 10.1080/15476286.2022.2066334 -
Nature Medicine Jan 2022Expansions of a GC repeat in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating...
Expansions of a GC repeat in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating adult-onset neurodegenerative disorders. Using C9-ALS/FTD patient-derived cells and C9ORF72 BAC transgenic mice, we generated and optimized antisense oligonucleotides (ASOs) that selectively blunt expression of GC repeat-containing transcripts and effectively suppress tissue levels of poly(GP) dipeptides. ASOs with reduced phosphorothioate content showed improved tolerability without sacrificing efficacy. In a single patient harboring mutant C9ORF72 with the GC repeat expansion, repeated dosing by intrathecal delivery of the optimal ASO was well tolerated, leading to significant reductions in levels of cerebrospinal fluid poly(GP). This report provides insight into the effect of nucleic acid chemistry on toxicity and, to our knowledge, for the first time demonstrates the feasibility of clinical suppression of the C9ORF72 gene. Additional clinical trials will be required to demonstrate safety and efficacy of this therapy in patients with C9ORF72 gene mutations.
Topics: Animals; C9orf72 Protein; Fibroblasts; Humans; Mice; Mice, Transgenic; Mutation; Neurons; Oligonucleotides, Antisense
PubMed: 34949835
DOI: 10.1038/s41591-021-01557-6 -
Cell Jun 2022Spinal muscular atrophy (SMA) is a motor-neuron disease caused by mutations of the SMN1 gene. The human paralog SMN2, whose exon 7 (E7) is predominantly skipped, cannot...
Spinal muscular atrophy (SMA) is a motor-neuron disease caused by mutations of the SMN1 gene. The human paralog SMN2, whose exon 7 (E7) is predominantly skipped, cannot compensate for the lack of SMN1. Nusinersen is an antisense oligonucleotide (ASO) that upregulates E7 inclusion and SMN protein levels by displacing the splicing repressors hnRNPA1/A2 from their target site in intron 7. We show that by promoting transcriptional elongation, the histone deacetylase inhibitor VPA cooperates with a nusinersen-like ASO to promote E7 inclusion. Surprisingly, the ASO promotes the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to RNA polymerase II elongation that inhibits E7 inclusion. By removing the roadblock, VPA counteracts the chromatin effects of the ASO, resulting in higher E7 inclusion without large pleiotropic effects. Combined administration of the nusinersen-like ASO and VPA in SMA mice strongly synergizes SMN expression, growth, survival, and neuromuscular function.
Topics: Animals; Chromatin; Exons; Mice; Muscular Atrophy, Spinal; Oligonucleotides, Antisense; RNA Splicing
PubMed: 35688133
DOI: 10.1016/j.cell.2022.04.031 -
Nature Apr 2024Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions. TS type 1 (TS1) is...
Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.
Topics: Animals; Female; Humans; Male; Mice; Alternative Splicing; Autistic Disorder; Calcium; Calcium Channels, L-Type; Cell Movement; Dendrites; Exons; Long QT Syndrome; Neurons; Oligonucleotides, Antisense; Organoids; Prosencephalon; Syndactyly; Interneurons
PubMed: 38658687
DOI: 10.1038/s41586-024-07310-6