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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 -
EMBO Molecular Medicine Jun 2024Polycystic kidney disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations,...
Polycystic kidney disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations, including the Warburg effect and glutaminolysis, sustained in vitro by the enzyme asparagine synthetase (ASNS). Here, we used antisense oligonucleotides (ASO) against Asns in orthologous and slowly progressive PKD murine models and show that treatment leads to a drastic reduction of total kidney volume (measured by MRI) and a prominent rescue of renal function in the mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven by the amino acid response (AAR) branch of the integrated stress response (ISR). Metabolic profiling of PKD or control kidneys treated with Asns-ASO or Scr-ASO revealed major changes in the mutants, several of which are rescued by Asns silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine synthesis and proliferation in cystic epithelia. Notably, while several metabolic pathways were completely corrected by Asns-ASO, glycolysis was only partially restored. Accordingly, combining the glycolytic inhibitor 2DG with Asns-ASO further improved efficacy. Our studies identify a new therapeutic target and novel metabolic vulnerabilities in PKD.
Topics: Polycystic Kidney Diseases; Animals; Aspartate-Ammonia Ligase; Mice; Disease Models, Animal; Oligonucleotides, Antisense; Disease Progression; Humans; Kidney
PubMed: 38684863
DOI: 10.1038/s44321-024-00071-9 -
Frontiers in Bioscience (Landmark... Mar 2024The endogenous metabolism of polyunsaturated fatty acids is regulated by the fatty acid desaturase (FADS) gene cluster and is strongly associated with diseases such as...
BACKGROUND
The endogenous metabolism of polyunsaturated fatty acids is regulated by the fatty acid desaturase (FADS) gene cluster and is strongly associated with diseases such as atherosclerosis, dyslipidemia, and type 2 diabetes. However, the association between FADS and atherosclerosis remains a subject of debate.
METHODS
In this study, we specifically investigated the physiological role of Δ-5 fatty acid desaturase (FADS1) in aortic and peripheral vessel (namely, the femoral artery) atherosclerosis by targeting the selective knockdown of hepatic in apolipoprotein E-null (ApoE-/-) mice with antisense oligonucleotides (ASOs).
RESULTS
Knockdown of hepatic in ApoE-/- mice exacerbated aortic atherosclerosis and non-alcoholic fatty liver disease (NAFLD), resulting in weight loss. Upregulation of FADS1 mRNA expression in more severe atherosclerosis vascular tissues potentially caused the upregulation of angiopoietin-like 4 expression.
CONCLUSIONS
Our study demonstrated that knockdown of hepatic in ApoE-/- mice aggravates spontaneous atherosclerosis and NAFLD but does not affect peripheral atherosclerosis (femoral artery) induced by vascular cuff combined with tandem stenosis.
Topics: Animals; Fatty Acid Desaturases; Delta-5 Fatty Acid Desaturase; Atherosclerosis; Liver; Apolipoproteins E; Mice; Gene Knockdown Techniques; Male; Non-alcoholic Fatty Liver Disease; Mice, Inbred C57BL; Mice, Knockout; Oligonucleotides, Antisense
PubMed: 38682200
DOI: 10.31083/j.fbl2904131 -
Nucleic Acids Research Jun 2024MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that...
MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that members of the same miRNA family with high sequence similarity might have different roles and that this functional divergence might be rooted in their precursors' sequence. Current knock-down strategies such as antisense oligonucleotides (ASOs) or miRNA sponges cannot distinguish between identical or near identical miRNAs originating from different precursors to allow exploring unique functions of these miRNAs. We here develop a novel strategy based on short 2'-OMe/LNA-modified oligonucleotides to selectively target specific precursor molecules and ablate the production of individual members of miRNA families in vitro and in vivo. Leveraging the highly conserved Xenopus miR-181a family as proof-of-concept, we demonstrate that 2'-OMe/LNA-ASOs targeting the apical region of pre-miRNAs achieve precursor-selective inhibition of mature miRNA-5p production. Furthermore, we extend the applicability of our approach to the human miR-16 family, illustrating its universality in targeting precursors generating identical miRNAs. Overall, our strategy enables efficient manipulation of miRNA expression, offering a powerful tool to dissect the functions of identical or highly similar miRNAs derived from different precursors within miRNA families.
Topics: MicroRNAs; Animals; Humans; Oligonucleotides; Oligonucleotides, Antisense; RNA Precursors; Xenopus
PubMed: 38676942
DOI: 10.1093/nar/gkae284 -
International Journal of Molecular... Apr 2024Aryl hydrocarbon receptor (AHR), a transcription factor activated by many natural and synthetic ligands, represents an important mediator of the interplay between the... (Review)
Review
Aryl hydrocarbon receptor (AHR), a transcription factor activated by many natural and synthetic ligands, represents an important mediator of the interplay between the environment and the host's immune responses. In a healthy gut, AHR activation promotes tolerogenic signals, which help maintain mucosal homeostasis. AHR expression is defective in the inflamed gut of patients with inflammatory bowel diseases (IBD), where decreased AHR signaling is supposed to contribute to amplifying the gut tissue's destructive immune-inflammatory responses. We here review the evidence supporting the role of AHR in controlling the "physiological" intestinal inflammation and summarize the data about the therapeutic effects of AHR activators, both in preclinical mouse models of colitis and in patients with IBD.
Topics: Receptors, Aryl Hydrocarbon; Animals; Humans; Signal Transduction; Inflammatory Bowel Diseases; Inflammation; Colitis; Intestinal Mucosa; Mice
PubMed: 38674118
DOI: 10.3390/ijms25084527 -
Biomedicines Apr 2024Casimersen (AMONDYS 45) is an antisense oligonucleotide of the phosphorodiamidate morpholino oligomer subclass developed by Sarepta therapeutics. It was approved by the... (Review)
Review
Casimersen (AMONDYS 45) is an antisense oligonucleotide of the phosphorodiamidate morpholino oligomer subclass developed by Sarepta therapeutics. It was approved by the Food and Drug Administration (FDA) in February 2021 to treat Duchenne muscular dystrophy (DMD) in patients whose gene mutation is amenable to exon 45 skipping. Administered intravenously, casimersen binds to the pre-mRNA of the gene to skip a mutated region of an exon, thereby producing an internally truncated yet functional dystrophin protein in DMD patients. This is essential in maintaining the structure of a myocyte membrane. While casimersen is currently continuing in phase III of clinical trials in various countries, it was granted approval by the FDA under the accelerated approval program due to its observed increase in dystrophin production. This article discusses the pathophysiology of DMD, summarizes available treatments thus far, and provides a full drug review of casimersen (AMONDYS 45).
PubMed: 38672266
DOI: 10.3390/biomedicines12040912 -
Clinical Therapeutics Apr 2024Since 2014, several clinical studies focusing on centronuclear myopathies have been conducted, including a prospective natural history study, a gene transfer clinical...
BACKGROUND
Since 2014, several clinical studies focusing on centronuclear myopathies have been conducted, including a prospective natural history study, a gene transfer clinical trial and a clinical trial using an antisense oligonucleotide. Dedicated patient organizations have played an important role in this process. The experience of members of these organizations, either as a study participant, parent or as a patient organization member communicating with the sponsors are potentially very informative for future trial design.
METHODS
We investigated the burden of and the lessons learned from the first natural history studies and clinical trials from a patient perspective using a qualitative approach. We arranged 4 focus groups with a total of 37 participants from 3 large international patient organizations: ZNM-ZusammenStark!, the Myotubular Trust, and the MTM-CNM Family Connection. 4 themes, based on a systematic literature search were discussed: Expectations and preparation, Clinical study participation, Communication and Recommendations for future clinical trials. The focus group recordings were transcribed, anonymized, and uploaded to Atlas-ti version 8.1 software. The data were analyzed using a thematic content analysis.
RESULTS
Overall, participants were realistic in their expectations, hoping for small improvements of function and quality of life. The realization that trial participation does not equate to a treatment was challenging. Participating in a clinical study had a huge impact on many aspects of daily life, both for patients and their immediate families. First-hand insights into the burden of the design and its possible effect on performance were provided, resulting in numerous compelling recommendations for future clinical studies. Furthermore, participants stressed the importance of clear communication, which was considered to be especially vital in cases of severe adverse events. Finally, while patients were understanding of the importance of adhering to the regulations of good clinical practice, they indicated that they would strongly appreciate a greater understanding and/or acknowledgment of the patient perspective and a reflection of this perspective in future clinical trial design.
CONCLUSION
The acknowledgment and inclusion of patients' perspectives and efficient and effective communication is expected to improve patient recruitment and retention in future clinical studies, as well as more accurate assessment of the patient performance related to suitable planning of the study visits.
PubMed: 38670885
DOI: 10.1016/j.clinthera.2024.03.008 -
Cancer Genomics & Proteomics Apr 2024Epithelial ovarian cancer (EOC) is associated with a dismal prognosis due to development of resistance to chemotherapy and metastasis in the peritoneal cavity and... (Review)
Review
Epithelial ovarian cancer (EOC) is associated with a dismal prognosis due to development of resistance to chemotherapy and metastasis in the peritoneal cavity and distant organs. In order to identify new targets and treatment modalities we searched the literature for up- and and down-regulated circRNAs with efficacy in preclinical EOC-related in vivo systems. Our search yielded circRNAs falling into the following categories: cisplatin and paclitaxel resistance, transmembrane receptors, secreted factors, transcription factors, RNA splicing and processing factors, RAS pathway-related components, proteolysis and cell-cycle regulation, signaling-related proteins, and circRNAs regulating proteins in additional categories. These findings can be potentially translated by validation and manipulation of the corresponding targets, inhibition of circRNAs with antisense oligonucleotides (ASO), small interfering RNAs (siRNA) or small hairpin RNA (shRNA) or by reconstituting their activity.
Topics: Humans; RNA, Circular; Carcinoma, Ovarian Epithelial; Female; Ovarian Neoplasms; Animals; Gene Expression Regulation, Neoplastic; Molecular Targeted Therapy; Drug Resistance, Neoplasm; RNA
PubMed: 38670587
DOI: 10.21873/cgp.20442 -
Lakartidningen Apr 2024We present a patient with familial amyotrophic lateral sclerosis caused by an aggressive A4S mutation in the SOD1 gene. In 2020, the patient was enrolled in the VALOR...
We present a patient with familial amyotrophic lateral sclerosis caused by an aggressive A4S mutation in the SOD1 gene. In 2020, the patient was enrolled in the VALOR SOD1 gene therapy phase-3 trial. At screening, the ALSFRS-R score was 41 (48 is normal) and the level of CSF-neurofilament L (an indicator of ongoing neuronal damage) was 11 000 ng/L (ref <650 ng/L). In the four years following enrollment, the patient received monthly intrathecal treatment with tofersen, an antisense oligonucleotide compound that inhibits SOD1 protein expression and hence lowers the synthesis of toxic SOD1 protein species. Side effects have been minimal and mostly attributed to the spinal taps. The patient remains ambulatory with an active social lifestyle. The ALSFRS-R score has in the past 18 months stabilized around 35-37, CSF-NfL is 1 290 ng/L and plasma-NfL is 12 (reference <13). This is the first documented arresting intervention in a patient with ALS in Sweden.
Topics: Humans; Amyotrophic Lateral Sclerosis; Superoxide Dismutase-1; Genetic Therapy; Disease Progression; Male; Middle Aged; Mutation; Oligonucleotides, Antisense; Oligonucleotides
PubMed: 38666665
DOI: No ID Found