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JCI Insight May 2024Diabetes increases the risk of both cardiovascular disease and kidney disease. Notably, most of the excess cardiovascular risk in people with diabetes is in those with...
Diabetes increases the risk of both cardiovascular disease and kidney disease. Notably, most of the excess cardiovascular risk in people with diabetes is in those with kidney disease. Apolipoprotein C3 (APOC3) is a key regulator of plasma triglycerides, and it has recently been suggested to play a role in both type 1 diabetes-accelerated atherosclerosis and kidney disease progression. To investigate if APOC3 plays a role in kidney disease in people with type 2 diabetes, we analyzed plasma levels of APOC3 from the Veterans Affairs Diabetes Trial. Elevated baseline APOC3 levels predicted a greater loss of renal function. To mechanistically test if APOC3 plays a role in diabetic kidney disease and associated atherosclerosis, we treated black and tan, brachyury, WT and leptin-deficient (OB; diabetic) mice, a model of type 2 diabetes, with an antisense oligonucleotide (ASO) to APOC3 or a control ASO, all in the setting of human-like dyslipidemia. Silencing APOC3 prevented diabetes-augmented albuminuria, renal glomerular hypertrophy, monocyte recruitment, and macrophage accumulation, partly driven by reduced ICAM1 expression. Furthermore, reduced levels of APOC3 suppressed atherosclerosis associated with diabetes. This suggests that targeting APOC3 might benefit both diabetes-accelerated atherosclerosis and kidney disease.
Topics: Apolipoprotein C-III; Diabetic Nephropathies; Diabetes Mellitus, Type 2; Animals; Atherosclerosis; Mice; Humans; Male; Female; Middle Aged; Aged; Oligonucleotides, Antisense; Disease Models, Animal
PubMed: 38743496
DOI: 10.1172/jci.insight.177268 -
Nucleic Acids Research Jun 2024Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited...
Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution.
Topics: Animals; Acetylgalactosamine; RNA, Small Interfering; Administration, Oral; Mice; Rats; RNA Interference; Male; Biological Availability; Humans; Rats, Sprague-Dawley; Macaca fascicularis; Liver; Macaca mulatta
PubMed: 38742636
DOI: 10.1093/nar/gkae350 -
BMC Oral Health May 2024Patients who suffer from myofascial orofacial pain could affect their quality of life deeply. The pathogenesis of pain is still unclear. Our objective was to assess...
Patients who suffer from myofascial orofacial pain could affect their quality of life deeply. The pathogenesis of pain is still unclear. Our objective was to assess Whether Voltage-gated calcium channel αδ-1(Cavα2δ-1) is related to myofascial orofacial pain. Rats were divided into the masseter tendon ligation group and the sham group. Compared with the sham group, the mechanical pain threshold of the masseter tendon ligation group was reduced on the 4th, 7th, 10th and 14th day after operation(P < 0.05). On the 14th day after operation, Cavα2δ-1 mRNA expression levels in trigeminal ganglion (TG) and the trigeminal spinal subnucleus caudalis and C1-C2 spinal cervical dorsal horn (Vc/C) of the masseter tendon ligation group were increased (P=0.021, P=0.012). Rats were divided into three groups. On the 4th day after ligating the superficial tendon of the left masseter muscle of the rats, 10 ul Cavα2δ-1 antisense oligonucleotide, 10 ul Cavα2δ-1 mismatched oligonucleotides and 10 ul normal saline was separately injected into the left masseter muscle of rats in Cavα2δ-1 antisense oligonucleotide group, Cavα2δ-1 mismatched oligonucleotides group and normal saline control group twice a day for 4 days. The mechanical pain threshold of the Cavα2δ-1 antisense oligonucleotides group was higher than Cavα2δ-1 mismatched oligonucleotides group on the 7th and 10th day after operation (P < 0.01). After PC12 cells were treated with lipopolysaccharide, Cavα2δ-1 mRNA expression level increased (P < 0.001). Cavα2δ-1 may be involved in the occurrence and development in myofascial orofacial pain.
Topics: Animals; Rats; Masseter Muscle; Male; Rats, Sprague-Dawley; Calcium Channels; Trigeminal Ganglion; Pain Threshold; Facial Pain; Spinal Cord Dorsal Horn; Oligonucleotides, Antisense; Myofascial Pain Syndromes; RNA, Messenger; Calcium Channels, L-Type
PubMed: 38735923
DOI: 10.1186/s12903-024-04338-y -
International Journal of Molecular... Apr 2024Antisense oligonucleotides (ASOs) are short oligodeoxynucleotides designed to bind to specific regions of target mRNA. ASOs can modulate pre-mRNA splicing, increase... (Review)
Review
Antisense oligonucleotides (ASOs) are short oligodeoxynucleotides designed to bind to specific regions of target mRNA. ASOs can modulate pre-mRNA splicing, increase levels of functional proteins, and decrease levels of toxic proteins. ASOs are being developed for the treatment of motor neuron diseases (MNDs), including spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA). The biggest success has been the ASO known as nusinersen, the first effective therapy for SMA, able to improve symptoms and slow disease progression. Another success is tofersen, an ASO designed to treat ALS patients with gene mutations. Both ASOs have been approved by the FDA and EMA. On the other hand, ASO treatment in ALS patients with the gene mutation did not show any improvement in disease progression. The aim of this review is to provide an up-to-date overview of ASO research in MNDs, from preclinical studies to clinical trials and, where available, regulatory approval. We highlight the successes and failures, underline the strengths and limitations of the current ASO research, and suggest possible approaches that could lead to more effective treatments.
Topics: Humans; Oligonucleotides, Antisense; Motor Neuron Disease; Animals; Muscular Atrophy, Spinal; Amyotrophic Lateral Sclerosis
PubMed: 38732027
DOI: 10.3390/ijms25094809 -
Nature Communications May 2024Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs)....
Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is hampered by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically inactive CRISPR-RfxCas13d RNA-targeting system (dCas13d/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against endogenous splicing factors. SpliceRUSH, a high-throughput screening method, was developed to map SREs in any gene of interest using a lentivirus gRNA library that tiles the genetic region, including distal intronic sequences. When applied to SMN2, a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identifies not only known SREs but also a previously unknown distal intronic SRE, which can be targeted to alter exon 7 splicing using either dCas13d/gRNA or ASOs. This technology enables a deeper understanding of splicing regulation with applications for RNA-based drug discovery.
Topics: Humans; RNA Splicing; CRISPR-Cas Systems; Survival of Motor Neuron 2 Protein; RNA, Guide, CRISPR-Cas Systems; Introns; Exons; HEK293 Cells; Oligonucleotides, Antisense; Muscular Atrophy, Spinal; Regulatory Sequences, Nucleic Acid; RNA Precursors
PubMed: 38714659
DOI: 10.1038/s41467-024-47140-8 -
Molecular Therapy. Nucleic Acids Jun 2024Recent studies have highlighted the effectiveness of using antisense oligonucleotides (ASOs) for cellular RNA regulation, including targets that are considered...
Recent studies have highlighted the effectiveness of using antisense oligonucleotides (ASOs) for cellular RNA regulation, including targets that are considered undruggable; however, manually designing optimal ASO sequences can be labor intensive and time consuming, which potentially limits their broader application. To address this challenge, we introduce a platform, the ASOptimizer, a deep-learning-based framework that efficiently designs ASOs at a low cost. This platform not only selects the most efficient mRNA target sites but also optimizes the chemical modifications for enhanced performance. Indoleamine 2,3-dioxygenase 1 (IDO1) promotes cancer survival by depleting tryptophan and producing kynurenine, leading to immunosuppression through the aryl-hydrocarbon receptor (Ahr) pathway within the tumor microenvironment. We used ASOptimizer to identify ASOs that target IDO1 mRNA as potential cancer therapeutics. Our methodology consists of two stages: sequence engineering and chemical engineering. During the sequence-engineering stage, we optimized and predicted ASO sequences that could target IDO1 mRNA efficiently. In the chemical-engineering stage, we further refined these ASOs to enhance their inhibitory activity while reducing their potential cytotoxicity. In conclusion, our research demonstrates the potential of ASOptimizer for identifying ASOs with improved efficacy and safety.
PubMed: 38706632
DOI: 10.1016/j.omtn.2024.102186 -
Molecular Metabolism Jun 2024The contribution of the mitochondrial electron transfer system to insulin secretion involves more than just energy provision. We identified a small RNA fragment...
OBJECTIVE
The contribution of the mitochondrial electron transfer system to insulin secretion involves more than just energy provision. We identified a small RNA fragment (mt-tRF-Leu) derived from the cleavage of a mitochondrially-encoded tRNA that is conserved between mice and humans. The role of mitochondrially-encoded tRNA-derived fragments remains unknown. This study aimed to characterize the impact of mt-tRF-Leu, on mitochondrial metabolism and pancreatic islet functions.
METHODS
We used antisense oligonucleotides to reduce mt-tRF-Leu levels in primary rat and human islet cells, as well as in insulin-secreting cell lines. We performed a joint transcriptome and proteome analysis upon mt-tRF-Leu inhibition. Additionally, we employed pull-down assays followed by mass spectrometry to identify direct interactors of the fragment. Finally, we characterized the impact of mt-tRF-Leu silencing on the coupling between mitochondrial metabolism and insulin secretion using high-resolution respirometry and insulin secretion assays.
RESULTS
Our study unveils a modulation of mt-tRF-Leu levels in pancreatic islets in different Type 2 diabetes models and in response to changes in nutritional status. The level of the fragment is finely tuned by the mechanistic target of rapamycin complex 1. Located within mitochondria, mt-tRF-Leu interacts with core subunits and assembly factors of respiratory complexes of the electron transfer system. Silencing of mt-tRF-Leu in islet cells limits the inner mitochondrial membrane potential and impairs mitochondrial oxidative phosphorylation, predominantly by affecting the Succinate (via Complex II)-linked electron transfer pathway. Lowering mt-tRF-Leu impairs insulin secretion of rat and human pancreatic β-cells.
CONCLUSIONS
Our findings indicate that mt-tRF-Leu interacts with electron transfer system complexes and is a pivotal regulator of mitochondrial oxidative phosphorylation and its coupling to insulin secretion.
Topics: Animals; Rats; Humans; Mitochondria; Insulin Secretion; Insulin-Secreting Cells; RNA, Transfer; Male; Insulin; Islets of Langerhans; Diabetes Mellitus, Type 2; RNA, Mitochondrial; Mice; Rats, Wistar; Electron Transport
PubMed: 38704026
DOI: 10.1016/j.molmet.2024.101955 -
Acta Naturae 2024Unproductive splicing is a mechanism of post-transcriptional gene expression control in which premature stop codons are inserted into protein-coding transcripts as a...
Unproductive splicing is a mechanism of post-transcriptional gene expression control in which premature stop codons are inserted into protein-coding transcripts as a result of regulated alternative splicing, leading to their degradation via the nonsense-mediated decay pathway. This mechanism is especially characteristic of RNA-binding proteins, which regulate each other's expression levels and those of other genes in multiple auto- and cross-regulatory loops. Deregulation of unproductive splicing is a cause of serious human diseases, including cancers, and is increasingly being considered as a prominent therapeutic target. This review discusses the types of unproductive splicing events, the mechanisms of auto- and cross-regulation, nonsense-mediated decay escape, and problems in identifying unproductive splice isoforms. It also provides examples of deregulation of unproductive splicing in human diseases and discusses therapeutic strategies for its correction using antisense oligonucleotides and small molecules.
PubMed: 38698955
DOI: 10.32607/actanaturae.27337 -
Nature Communications May 2024Mouse models of autosomal dominant polycystic kidney disease (ADPKD) show that intact primary cilia are required for cyst growth following the inactivation of...
Mouse models of autosomal dominant polycystic kidney disease (ADPKD) show that intact primary cilia are required for cyst growth following the inactivation of polycystin-1. The signaling pathways underlying this process, termed cilia-dependent cyst activation (CDCA), remain unknown. Using translating ribosome affinity purification RNASeq on mouse kidneys with polycystin-1 and cilia inactivation before cyst formation, we identify the differential 'CDCA pattern' translatome specifically dysregulated in kidney tubule cells destined to form cysts. From this, Glis2 emerges as a candidate functional effector of polycystin signaling and CDCA. In vitro changes in Glis2 expression mirror the polycystin- and cilia-dependent changes observed in kidney tissue, validating Glis2 as a cell culture-based indicator of polycystin function related to cyst formation. Inactivation of Glis2 suppresses polycystic kidney disease in mouse models of ADPKD, and pharmacological targeting of Glis2 with antisense oligonucleotides slows disease progression. Glis2 transcript and protein is a functional target of CDCA and a potential therapeutic target for treating ADPKD.
Topics: Animals; Humans; Male; Mice; Cilia; Disease Models, Animal; Kidney; Mice, Inbred C57BL; Mice, Knockout; Oligonucleotides, Antisense; Polycystic Kidney Diseases; Polycystic Kidney, Autosomal Dominant; Signal Transduction; TRPP Cation Channels
PubMed: 38693102
DOI: 10.1038/s41467-024-48025-6 -
Bio-protocol Apr 2024The field of oligonucleotide therapeutics is rapidly advancing, particularly for combating orphan diseases and cancer. However, the intrinsic instability of...
The field of oligonucleotide therapeutics is rapidly advancing, particularly for combating orphan diseases and cancer. However, the intrinsic instability of oligonucleotides, especially RNA, poses a substantial challenge in the face of the harsh conditions encountered intracellularly and in circulation. Therefore, evaluating the stability of oligos in serum is of great significance when developing oligonucleotide therapeutics. This protocol outlines a dependable and reproducible method for preparing oligonucleotide duplexes, coupled with confirmation by gel electrophoresis. Subsequently, the protocol defines a mechanism to assess the stability of the oligo duplexes in serum. This protocol seeks to establish a standardized reference for researchers, enabling them to compare the impact of various modifications on oligo stability and assess the degradation kinetics effectively. Key features • Adaptable for use with small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASOs), and other unmodified and modified oligonucleotides. • Does not necessitate any Biological Safety Level clearance and offers a rapid, cost-effective, and entirely in vitro procedure. • Allows researchers to evaluate multiple modification patterns that, when coupled with targeting activity, allow for selecting the best modification pattern prior to in vivo analysis.
PubMed: 38686344
DOI: 10.21769/BioProtoc.4975