scholarly journals Antisense oligonucleotides targeting translation inhibitory elements in 5′ UTRs can selectively increase protein levels

2017 ◽  
Vol 45 (16) ◽  
pp. 9528-9546 ◽  
Author(s):  
Xue-hai Liang ◽  
Hong Sun ◽  
Wen Shen ◽  
Shiyu Wang ◽  
Joyee Yao ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Protein Levels

scholarly journals MicroRNA-143 Regulates Adipocyte Differentiation

2004 ◽  
Vol 279 (50) ◽  
pp. 52361-52365 ◽  
Author(s):  
Christine Esau ◽  
Xiaolin Kang ◽  
Eigen Peralta ◽  
Elaine Hanson ◽  
Eric G. Marcusson ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Target Gene ◽  
Adipocyte Differentiation ◽  
Biological Function ◽  
Protein Translation ◽  
Functional Assay ◽  
Expression Data ◽  
Mirna Microarray ◽  
Protein Levels ◽  
Human Mirnas

MicroRNAs (miRNAs) are endogenously expressed 20-24 nucleotide RNAs thought to repress protein translation through binding to a target mRNA (1-3). Only a few of the more than 250 predicted human miRNAs have been assigned any biological function. In an effort to uncover miRNAs important during adipocyte differentiation, antisense oligonucleotides (ASOs) targeting 86 human miRNAs were transfected into cultured human pre-adipocytes, and their ability to modulate adipocyte differentiation was evaluated. Expression of 254 miRNAs in differentiating adipocytes was also examined on a miRNA microarray. Here we report that the combination of expression data and functional assay results identified a role for miR-143 in adipocyte differentiation. miR-143 levels increased in differentiating adipocytes, and inhibition of miR-143 effectively inhibited adipocyte differentiation. In addition, protein levels of the proposed miR-143 target ERK5 (4) were higher in ASO-treated adipocytes. These results demonstrate that miR-143 is involved in adipocyte differentiation and may act through target gene ERK5.

Pharmacological Characterization and Structure Activity Relationship of FXI Antisense Oligonucleotides in Cynomolgus Monkeys.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2101-2101
Author(s):  
Robert MacLeod ◽  
Jeff Crosby ◽  
Chenguang Zhao ◽  
Dacao Gao ◽  
Chris May ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Anticoagulant Activity ◽  
Bleeding Risk ◽  
Intrinsic Pathway ◽  
Antithrombotic Agents ◽  
Pharmacological Characterization ◽  
Protein Levels ◽  
Intravascular Thrombosis ◽  
Single Chemical

Abstract Abstract 2101 Poster Board II-78 The absence of abnormal bleeding associated with congenital deficiencies of the intrinsic coagulation pathway factor, FXI, suggests that this pathway is not important for normal blood coagulation in vivo. However, recent work in mice and higher species demonstrates that the intrinsic pathway is an important contributor to pathologic intravascular thrombosis, suggesting that targeting this pathway may yield effective antithrombotic agents with high safety. We have previously demonstrated that second generation antisense oligonucleotides (ASOs) targeting the intrinsic pathway member FXI were potent antithrombotic agents and that this activity was achieved in several mouse models of thrombosis without any increase in bleeding risk (Zhang et al. ASH 2008). Here we characterize the pharmacological activity, chemical SAR and safety profile of FXI antisense oligonucleotides administered subcutaneously in Cynomologus monkeys. Our first monkey study was designed to address the PK/PD kinetics of FXI ASOs from a single chemical series (20 mers, 5-10-5 MOE Gapmers). Two dose escalation regimes were evaluated (1) 5mg/kg (3wks), 10mg/kg (3 wks) followed by 25 mg/kg (6wks) and (2) 5mg/kg (3wks) followed by 10mg/kg (9 wks). The subsequent study was designed to evaluate chemical SAR around the active ASOs identified in the study 1. FXI antisense oligonucleotides FXI-AS1, FXI-AS2 demonstrated dose and time dependent pharmacologic activity, including, decreased FXI mRNA in liver (up to 90%), decreased FXI protein levels and FXI activity measured in plasma, with a maximal inhibition of >80% observed at 25mg/kg on both schedules, prolonged activated partial thromboplastin times (aPTT), maximal aPTT ratio of 2.O, but no change in prothrombin time (PT) as expected. Similar to previous studies in mouse the anticoagulant activity observed from FXI depletion in monkeys was not associated with increased bleeding risk as assessed in a skin bleeding time test. Additionally, FXI ASOs had no deleterious effects on organ weights, platelets, or on measures of liver and kidney function. The second study confirmed the effects of FXI-AS1 and FXI-AS2 and demonstrated that shorter ASOs with decreased gap size were equally potent. These results further support the development of FXI ASOs as human therapeutics for the treatment of coagulation related disorders with the potential for improved safety profiles. Disclosures: MacLeod: Isis Pharmaceuticals: Employment. Crosby:Isis Pharmaceuticals, Inc.: Employment. Zhao:Isis Pharmaceuticals, Inc.: Employment. Gao:Isis Pharmaceuticals, Inc.: Employment. May:Isis Pharmaceuticals, Inc.: Employment. Zhang:Isis Pharmaceuticals, Inc.: Consultancy. Lowenberg:Isis Pharmaceuticals, Inc.: Consultancy. Levi:Isis Pharmaceuticals, Inc.: Consultancy. Monia:Isis Pharmaceuticals, Inc.: Employment.

scholarly journals Antisense oligonucleotide-based therapeutic strategy for progranulin-deficient frontotemporal dementia

2022 ◽  
Author(s):  
Geetika Aggarwal ◽  
Subhashis Banerjee ◽  
Spencer A. Jones ◽  
Monica D. Pavlack ◽  
Yousri Benchaar ◽  
...  
Keyword(s):  
Binding Site ◽  
Frontotemporal Dementia ◽  
Antisense Oligonucleotide ◽  
Antisense Oligonucleotides ◽  
Therapeutic Strategy ◽  
Neurological Diseases ◽  
Therapeutic Modality ◽  
Loss Of Function ◽  
Common Cause ◽  
Protein Levels

Loss-of-function GRN mutations result in progranulin haploinsufficiency and are a common cause of frontotemporal dementia (FTD). Antisense oligonucleotides (ASOs) are emerging as a promising therapeutic modality for neurological diseases, but ASO-based strategies for increasing target protein levels are still relatively limited. Here, we report the use of ASOs to increase progranulin protein levels by targeting the miR-29b binding site in the 3′ UTR of the GRN mRNA, resulting in increased translation.

scholarly journals Reduction of integrin alpha 4 activity through splice modulating antisense oligonucleotides

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
May T. Aung-Htut ◽  
Iain Comerford ◽  
Russell Johnsen ◽  
Kerrie Foyle ◽  
Sue Fletcher ◽  
...  
Keyword(s):  
Protein Expression ◽  
Disease Progression ◽  
Antisense Oligonucleotides ◽  
Dermal Fibroblasts ◽  
Mrna Processing ◽  
Jurkat Cells ◽  
Experimental Autoimmune Encephalomyelitis Mouse ◽  
Protein Levels ◽  
Human T Lymphocyte ◽  
Regulate Protein

Abstract With recent approvals of antisense oligonucleotides as therapeutics, there is an increasing interest in expanding the application of these compounds to many other diseases. Our laboratory focuses on developing therapeutic splice modulating antisense oligonucleotides to treat diseases potentially amendable to intervention during pre-mRNA processing, and here we report the use of oligomers to down-regulate integrin alpha 4 protein levels. Over one hundred antisense oligonucleotides were designed to induce skipping of individual exons of the ITGA4 transcript and thereby reducing protein expression. Integrin alpha 4-mediated activities were evaluated in human dermal fibroblasts and Jurkat cells, an immortalised human T lymphocyte cell line. Peptide conjugated phosphorodiamidate morpholino antisense oligomers targeting ITGA4 were also assessed for their effect in delaying disease progression in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. With the promising results in ameliorating disease progression, we are optimistic that the candidate oligomer may also be applicable to many other diseases associated with integrin alpha 4 mediated inflammation. This highly specific strategy to down-regulate protein expression through interfering with normal exon selection during pre-mRNA processing should be applicable to many other gene targets that undergo splicing during expression.

scholarly journals Open reading frame correction using antisense oligonucleotides for the treatment of cystic fibrosis caused by CFTR-W1282X

2021 ◽  
Author(s):  
Wren E. Michaels ◽  
Cecilia Pena-Rasgado ◽  
Rusudan Kotaria ◽  
Robert J. Bridges ◽  
Michelle L. Hastings
Keyword(s):  
Cystic Fibrosis ◽  
Protein Function ◽  
Antisense Oligonucleotides ◽  
Gene Mutations ◽  
Severe Form ◽  
Open Reading Frame ◽  
Common Mutation ◽  
Reading Frame ◽  
Protein Levels ◽  
Cftr Protein

CFTR gene mutations that result in the introduction of premature termination codons (PTCs) are common in cystic fibrosis (CF). This mutation type causes a severe form of the disease, likely because of low CFTR mRNA expression as a result of nonsense mediated mRNA decay (NMD), as well as production of a non-functional, truncated CFTR protein. Current therapeutics for CF, which target residual protein function, are less effective in patients with these types of mutations, due in part to low CFTR protein levels. Splice-switching antisense oligonucleotides (ASOs) designed to induce skipping of exons in order to restore the mRNA open reading frame have shown therapeutic promise pre-clinically and clinically for a number of diseases. We hypothesized that ASO-mediated skipping of CFTR exon 23 would recover CFTR activity associated with terminating mutations in the exon, including CFTR p.W1282X, the 5th most common mutation in CF. Here, we show that CFTR lacking the amino acids encoding exon 23 is partially functional and responsive to corrector and modulator drugs currently in clinical use. ASO-induced exon 23 skipping rescued CFTR expression and chloride current in primary human bronchial epithelial cells isolated from homozygote CFTR-W1282X patients. These results support the use of ASOs in treating CF patients with CFTR class I mutations in exon 23 that result in unstable CFTR mRNA and truncations of the CFTR protein.

scholarly journals BACE1 Inhibition Using 2’-OMePS Steric Blocking Antisense Oligonucleotides

Genes ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 705
Author(s):  
Chakravarthy ◽  
Veedu
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Amyloid Precursor Protein ◽  
Antisense Oligonucleotides ◽  
Precursor Protein ◽  
Exon 2 ◽  
Protein Levels ◽  
Bace1 Expression ◽  
Beta Peptide ◽  
Bace1 Inhibition

Amyloid beta-peptide is produced by the cleavage of amyloid precursor protein by two secretases, a β-secretase, beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and a γ-secretase. It has been hypothesised that partial inhibition of BACE1 in individuals with a high risk of developing Alzheimer’s disease may be beneficial in preventing cognitive decline. In this study, we report the development of a novel antisense oligonucleotide (AO) that could efficiently downregulate the BACE1 transcript and partially inhibit BACE1 protein. We designed and synthesised a range of 2’-OMethyl-modified antisense oligonucleotides with a phosphorothioate backbone across various exons of the BACE1 transcript, of which AO2, targeting exon 2, efficiently downregulated BACE1 RNA expression by 90%. The sequence of AO2 was later synthesised with a phosphorodiamidate morpholino chemistry, which was found to be not as efficient at downregulating BACE1 expression as the 2’-OMethyl antisense oligonucleotides with a phosphorothioate backbone variant. AO2 also reduced BACE1 protein levels by 45%. In line with our results, we firmly believe that AO2 could be used as a potential preventative therapeutic strategy for Alzheimer’s disease.

scholarly journals Suppression of kinesin expression in cultured hippocampal neurons using antisense oligonucleotides.

1992 ◽  
Vol 117 (3) ◽  
pp. 595-606 ◽  
Author(s):  
A Ferreira ◽  
J Niclas ◽  
R D Vale ◽  
G Banker ◽  
K S Kosik
Keyword(s):  
Neurite Outgrowth ◽  
Cell Body ◽  
Heavy Chain ◽  
Antisense Oligonucleotides ◽  
Hippocampal Neurons ◽  
Full Recovery ◽  
Synapsin I ◽  
Neurite Length ◽  
Protein Levels ◽  
Cultured Hippocampal Neurons

Kinesin, a microtubule-based force-generating molecule, is thought to translocate organelles along microtubules. To examine the function of kinesin in neurons, we sought to suppress kinesin heavy chain (KHC) expression in cultured hippocampal neurons using antisense oligonucleotides and study the phenotype of these KHC "null" cells. Two different antisense oligonucleotides complementary to the KHC sequence reduced the protein levels of the heavy chain by greater than 95% within 24 h after application and produced identical phenotypes. After inhibition of KHC expression for 24 or 48 h, neurons extended an array of neurites often with one neurite longer than the others; however, the length of all these neurites was significantly reduced. Inhibition of KHC expression also altered the distribution of GAP-43 and synapsin I, two proteins thought to be transported in association with membranous organelles. These proteins, which are normally localized at the tips of growing neurites, were confined to the cell body in antisense-treated cells. Treatment of the cells with the corresponding sense oligonucleotides affected neither the distribution of GAP-43 and synapsin I, nor the length of neurites. A full recovery of neurite length occurred after removal of the antisense oligonucleotides from the medium. These data indicate that KHC plays a role in the anterograde translocation of vesicles containing GAP-43 and synapsin I. A deficiency in vesicle delivery may also explain the inhibition of neurite outgrowth. Despite the inhibition of KHC and the failure of GAP-43 and synapsin I to move out of the cell body, hippocampal neurons can extend processes and acquire as asymmetric morphology.

scholarly journals Role of Ras Isoforms in the Stimulated Proliferation of Human Renal Fibroblasts in Primary Culture

2000 ◽  
Vol 11 (9) ◽  
pp. 1600-1606 ◽  
Author(s):  
CLAIRE C. SHARPE ◽  
MARK E. C. DOCKRELL ◽  
MAZHAR I. NOOR ◽  
BRETT P. MONIA ◽  
BRUCE M. HENDRY
Keyword(s):  
Primary Culture ◽  
Reverse Transcription ◽  
Intracellular Signaling ◽  
Antisense Oligonucleotides ◽  
Primary Cultures ◽  
Fibroblast Cell ◽  
Ras Protein ◽  
Protein Levels ◽  
Reverse Transcription Pcr ◽  
Serum Stimulation

Abstract. The proliferation of renal fibroblasts is implicated in the pathophysiologic processes of renal fibrosis. Many of the growth factors involved in proliferation are known to activate intracellular signaling pathways that converge on Ras monomeric GTPases. Although three ras family genes exist, their functional specificity is not yet known. Using antisense oligonucleotides, a role for Kirsten (Ki)-Ras in the stimulated proliferation of a primate renal fibroblast cell line was previously demonstrated. This study examines Ras in primary cultures of adult human renal fibroblasts. Using reverse transcription-PCR, mRNA for Harvey (Ha)-ras, Ki(4B)-ras, and neural (N)-ras, but not Ki(4A)-ras, were detected. Antisense oligonucleotides targeting Ha-, Ki-, and N-ras mRNA, which were used for liposomal transfection at 100 to 200 nM, were demonstrated to be active and isoform-specific in quantitative reverse transcription-PCR assays. Cellular Ras protein levels, as estimated using isoform-specific monoclonal antibodies, indicated that Ki-Ras was the predominantly expressed isoform (>95% of total Ras protein) under both serum-containing and serum-free conditions, with N- and Ha-Ras being detected in small amounts. Consistent with this finding, the antisense oligonucleotide directed against Ki-Ras reduced total cellular Ras levels by >70%, whereas Ha-Ras, N-Ras, and control oligonucleotides had no significant effect. Proliferation of oligonucleotide-transfected cells was measured using epidermal growth factor (EGF) and serum stimulation. The Ki-Ras oligonucleotide at 100 nM reduced serum-stimulated proliferation by >50% and EGF-stimulated proliferation by 25%, compared with data obtained with the control oligonucleotide (P < 0.01). The N-Ras oligonucleotide was not active, compared with the control oligonucleotide. The Ha-Ras oligonucleotide was not significantly active at 100 nM but reduced serum-stimulated proliferation by 13% and EGF-stimulated growth by 40% at 200 nM (P < 0.01). These results demonstrate that Ki-Ras(4B) is the predominantly expressed Ras isoform in human renal fibroblasts in primary culture and is important for both serum- and EGF-stimulated proliferation. Ha-Ras appears to be expressed at low levels but may also play a distinct role in stimulated proliferation.

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