REVIEW | doi:10.20944/preprints201905.0239.v1
Subject: Medicine & Pharmacology, Gastroenterology Keywords: oligonucleotide therapeutics; RNA interference; antisense; aptamer; decoy; pancreatic cancer
Online: 20 May 2019 (10:12:46 CEST)
Although there is a several array of diagnostic and therapeutic choices for pancreatic cancer in recent years, a crucial medical approach for the refractory disease is still needed. Oligonucleotide therapeutics, such as those based on antisense RNAs, RNA interference, aptamers and decoys, are promising agents against pancreatic cancer because they identify a specific nucleotide sequence or protein and interfere with gene expression as molecular-targeted agents. Within just the past quarter-century, the diversity and feasibility of these drugs as diagnostic or therapeutic tools have dramatically increased. Actually, there have been several clinical and preclinical studies of oligonucleotides for patients with pancreatic cancer so far. To support the discovery of effective diagnostic or therapeutic options by using oligonucleotide-based strategies in the absence of satisfactory therapies for long-term survival and the rising trend of diseases, we summarize the current clinical trials of oligonucleotide therapeutics for pancreatic cancer patients with underlying preclinical or scientific data and focus on the possibility of oligonucleotides to target pancreatic cancer in clinical implications.
REVIEW | doi:10.20944/preprints202106.0683.v1
Subject: Medicine & Pharmacology, Allergology Keywords: Osteoarthritis; rheumatoid arthritis; synovitis; cartilage; bone; antisense; oligonucleotides; therapeutics.
Online: 28 June 2021 (16:01:16 CEST)
Osteoarthritis and rheumatoid arthritis are two of the most common chronic inflammatory joint diseases, for which there remains a great clinical need to develop safer and more efficacious pharmacological treatments. The pathology of both osteoarthritis and rheumatoid arthritis involves multiple tissues within the joint, including the synovial joint lining and the bone, as well as the articular cartilage in osteoarthritis. In this review, we discuss the potential for the development of oligonucleotide therapies for these disorders by examining the evidence that oligonucleotides can modulate the key cellular pathways that drive the pathology of the inflammatory diseased joint pathology as well as evidence in preclinical in vivo models that oligonucleotides can modify disease progression.
REVIEW | doi:10.20944/preprints201804.0322.v2
Subject: Biology, Other Keywords: antisense oligonucleotides; antisense therapy; DNA insecticides; RNAi; medicine; agriculture; forestry
Online: 31 May 2018 (12:11:59 CEST)
Antisense oligonucleotides (ASO), short single-stranded polymers based on DNA or RNA chemistries and synthesized in vitro, regulate gene expression by binding in a sequence-specific manner to an RNA target. The functional activity and selectivity in the action of ASOs largely depends on the combination of nitrogenous bases in a target sequence. This simple and natural property of nucleic acids provides an attractive route by which scientists can create different ASO-based techniques. Over the last 50 years, planned and realized applications in the field of antisense and nucleic acid nanotechnologies have produced astonishing results and posed new challenges for further developments, exemplifying the essence of the post-genomic era. Today the majority of ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake. This review critically analyzes some successful cases using the antisense approach in medicine to address severe diseases, such as Duchenne muscular dystrophy and spinal muscular atrophy, and suggests some prospective directions for future research. We also examine in detail the elaboration of unmodified insect-specific DNA insecticides and RNA preparations in the areas of agriculture and forestry, a relatively new branch of ASO that allows circumvention of the use of non-selective chemical insecticides. When considering the variety of successful ASO modifications with an efficient signal-to-noise ratio of action, coupled with the affordability of in vitro oligonucleotide synthesis and post-synthesis procedures, we predict that the next half-century will produce a fruitful yield of tools created from effective ASO-based end products.
CONCEPT PAPER | doi:10.20944/preprints202004.0412.v1
Subject: Life Sciences, Microbiology Keywords: coronavirus; COVID-19; antisense oligonucleotide; ASO; LNA GapmeR
Online: 23 April 2020 (11:30:39 CEST)
The severity of the global COVID-19 pandemic, with a high transmission rate, 2.6-4.7% lethality and a huge economic impact, poses an urgent need for efficient medical treatments and vaccines. Currently, there are only non-specific treatments to assist the patients in acute respiratory distress during the inflammatory step following the preliminary infection by SARS-CoV-2. Clinical trials of drug repurposing were quickly launched at the international level. Specific treatments such as the transfusion of plasma from patients who have recovered into infected patients or the use of specific inhibitors of the viral RNA-polymerase complex are promising strategies to block infection. To complete the therapeutic arsenal, we believe that the opportunity of targeting the SARS-CoV-2 genome by RNA therapy should be deeply investigated. In the present paper, we propose to design specific antisense oligonucleotides targeting transcripts encoding viral proteins associated to replication and transcription of SARS-CoV-2, aiming to block infection. We designed antisense oligonucleotides targeting the genomic 5’ untranslated region (5’-UTR), open reading frames 1a and 1b (ORF1a and ORF1b) governing expression of the replicase/transcriptase complex, and the gene N encoding the nucleoprotein that is genome-associated. To maximize the probability of efficiency, we predicted the antisense oligonucleotides by using two design methods: i) conventional antisense oligonucleotides with 100% phosphorothioate modifications (ASO); ii) antisense locked nucleic acids GapmeR. After binding the viral RNA target, the hetero-duplexes antisense oligonucleotide-RNA are cleaved by RNAse H1. Nine potent ASO candidates were found and we selected five of them targeting ORF1a (3), ORF1b (1) and N (1). Nine GapmeR candidates were predicted with excellent properties and we retained four of them targeting 5’-UTR (1), ORF1a (3), ORF1b (1) and N (1). The most potent GapmeR candidate targets the 5’-UTR, a key genomic domain with multiple functions in the viral cycle. By this open publication, we are pleased to share these in silico results with the scientific community in hopes of stimulating innovation in translational research in order to fight the unprecedented COVID-19 pandemic. These antisense oligonucleotide candidates should be now experimentally evaluated.
REVIEW | doi:10.20944/preprints202103.0050.v1
Subject: Life Sciences, Biochemistry Keywords: antisense; antibiotic resistance; RNase P; RNase H; nucleotide analogs
Online: 2 March 2021 (09:17:47 CET)
Antisense technologies consist of the utilization of oligonucleotides or oligonucleotide analogs to interfere with undesirable biological processes, commonly through inhibition of expression of selected genes. This field holds a lot of promise for the treatment of a very diverse group of diseases including viral and bacterial infections, genetic disorders, and cancer. To date, drugs approved for utilization in clinics or in clinical trials target diseases other than bacterial infections. Although several groups and companies are working on different strategies, the application of antisense technologies to prokaryotes still lags with respect to those that target other human diseases. In those cases where the focus is on bacterial pathogens, a subset of the research is dedicated to produce antisense compounds that silence or reduce expression of antibiotic resistance genes. Therefore, these compounds will be adjuvants administered with the antibiotic to which they reduce resistance levels. A varied group of oligonucleotide analogs like phosphorothioate or phosphorodiamidate morpholino residues, as well as peptide nucleic acids, locked nucleic acids and bridge nucleic acids, the latter two in gapmer configuration, have been utilized to reduce resistance levels. The major mechanisms of inhibition include eliciting cleavage of the target mRNA by the host’s RNase H or RNase P, and steric hindrance. The different approaches targeted resistance to β-lactams including carbapenems, aminoglycosides, chloramphenicol, macrolides, and fluoroquinolones.
ARTICLE | doi:10.20944/preprints202005.0432.v1
Subject: Medicine & Pharmacology, Nutrition Keywords: antisense; HIV-1 nef; stop codon readthrough; selenium; thioredoxin reductase
Online: 26 May 2020 (13:16:09 CEST)
The HIV-1 nef gene terminates in a 3’-UGA stop codon, which is highly conserved in the main group of HIV-1 subtypes, along with a downstream potential coding region that could extend the nef protein by 33 amino acids, if readthrough of the stop codon occurs. Antisense tethering interactions (ATIs) between a viral mRNA and a host selenoprotein mRNA are a potential viral strategy for the capture of a host selenocysteine insertion sequence (SECIS) element (Taylor et al, 2016) . This mRNA hijacking mechanism could enable the expression of virally encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine (SeC). Here we show that readthrough of the 3’-terminal UGA codon of nef occurs during translation of HIV-1 nef expression constructs in transfected cells. This was accomplished via fluorescence microscopy image analysis and flow cytometry of HEK 293 cells, transfected with engineered GFP reporter gene plasmid constructs, in which GFP can only be expressed by translational recoding of the UGA codon. SiRNA knockdown of thioredoxin reductase 1 (TR1) mRNA resulted in a 67% decrease in GFP expression, presumably due to reduced availability of the components involved in selenocysteine incorporation for the stop codon readthrough, thus supporting the proposed ATI. Addition of 20 nM sodium selenite to the media significantly enhanced stop codon readthrough in the pNefATI1 plasmid construct, by >100%, supporting the hypothesis that selenium is involved in the UGA readthrough mechanism.
ARTICLE | doi:10.20944/preprints202302.0033.v1
Subject: Medicine & Pharmacology, Pathology & Pathobiology Keywords: Neuromuscular disorder; NCALD; SMA; SMN2; Antisense oligonucleotide; Genetic modifier; Therapy; hiPSCs;
Online: 2 February 2023 (07:54:05 CET)
Here we investigated the longer-term effect of Ncald-ASOs by additional i.c.v. bolus injection at PND28. Two weeks after injection of 500 µg Ncald-ASO in wild-type mice, NCALD was significantly reduced in brain and spinal cord and well tolerated. Next, we performed a double-blinded preclinical study combining low-dose SMN-ASO (PND1) with 2x i.c.v. Ncald-ASO or CTRL-ASO (100 µg at PND2, 500 µg at PND28). Ncald-ASO re-injection significantly ameliorated electrophysiological defects and NMJ denervation at 2 months. Moreover, we developed and identified a nontoxic and highly efficient human NCALD-ASO that significantly reduced NCALD in hiPSCs-derived MNs. This improved both neuronal activity and growth cone maturation of SMA MNs, emphasizing the additional protective effect of NCALD-ASO treatment.
CONCEPT PAPER | doi:10.20944/preprints202212.0334.v1
Subject: Biology, Agricultural Sciences & Agronomy Keywords: essential oil plants; antisense oligoilators; secondary metabolites; oligonucleotide insecticides; green agriculture
Online: 19 December 2022 (09:48:52 CET)
Successful management of the synthesis of secondary metabolites of essential oil plants is the basis for the economic growth of the essential oil industry. Against the backdrop of a growing global population and a decrease in land available for cultivation, simple and effective ways to increase the content of certain components in essential oils are becoming increasingly important. Selection is no longer keeping pace with market needs, which stimulates the search for faster methods to control the biosynthesis of secondary metabolites. In this article, using the genus Lavandula as an example, we will consider the prospects for use of antisense oligonucleotides (ASO), oligoilators, to rapidly increase the concentration of valuable components in essential oil. This article discusses the use of unmodified ASOs as regulators of plant secondary metabolism to increase the synthesis of individual valuable components, presenting a completely new way to increase the yield of valuable substances based on unique nucleotide sequences. The proposed approach is effective, affordable, safe, and also significantly reduces the time needed to obtain plants that synthesize the required concentrations of target substances. Oligoilators can the used along with oligonucleotide insecticides in complex formulations used for green agriculture. Further investigation is needed to determine maximum economic efficiency of this approach.
ARTICLE | doi:10.20944/preprints202201.0173.v1
Subject: Medicine & Pharmacology, Behavioral Neuroscience Keywords: γ-synuclein; AAV vector; antisense oligonucleotide; cognitive dysfunction; dopamine; motor deficits
Online: 12 January 2022 (14:27:18 CET)
The synuclein family consists of α-, β-, and γ-Synuclein (α-Syn, β-Syn, and γ-Syn), expressed in the neurons and concentrated in synaptic terminals. While α-Syn is at the center of interest due to its implication in the pathogenesis of Parkinson’s disease (PD) and other synucleinopathies, limited information exists on the other members. The current study aimed at investigating the biological role of γ-Syn controlling the midbrain dopamine (DA) function. We generated two different mouse models with i) γ-Syn overexpression induced by an adeno-associated viral vector and ii) γ-Syn knockdown induced by a ligand-conjugated antisense oligonucleotide, to modify the endogenous γ-Syn transcription levels in midbrain DA neurons. The progressive overexpression of γ-Syn decreased DA neurotransmission in the nigrostriatal and mesocortical pathways. In parallel, mice evoked motor deficits in the rotarod and impaired cognitive performance as assessed by novel object recognition, passive avoidance, and Morris water maze tests. Conversely, acute γ-Syn knockdown selectively in DA neurons facilitated forebrain DA neurotransmission. Importantly, modifications in γ-Syn expression did not induce the loss of DA neurons or changes in α-Syn expression. Collectively, our data strongly suggest that DA re-lease/re-uptake processes in the nigrostriatal and mesocortical pathways are partially dependent on SNc/VTA γ-Syn transcription levels, and are linked to modulation of DA transporter function, similar to α-Syn.
REVIEW | doi:10.20944/preprints202112.0273.v1
Subject: Biology, Other Keywords: RNA therapeutics; cardiovascular disease; mRNA therapeutics; siRNA therapeutics; antisense oligonucleotide therapeutics
Online: 16 December 2021 (14:07:05 CET)
Abstract Purpose of review: RNA therapeutics are a new and rapidly expanding class of drugs to prevent or treat a wide spectrum of diseases. We discuss the defining characteristics of the diverse family of molecules under the RNA therapeutics umbrella. Recent findings:RNA therapeutics are designed to regulate gene expression in a transient manner. For example, depending upon the strategy employed, RNA therapies offer the versatility to replace, supplement, correct, suppress, or eliminate the expression of a targeted gene. RNA therapies include antisense nucleotides, microRNAs and small interfering RNAs, RNA aptamers, and messenger RNAs. Further, we discuss the mechanism(s) by which different RNA therapies either reduce or increase the expression of their targets. Summary: We review the RNA therapeutics approved (and those in trials) to treat cardiovascular indications. RNA-based therapeutics are a new, rapidly growing class of drugs that will offer new alternatives for an increasing array of cardiovascular conditions.
REVIEW | doi:10.20944/preprints201906.0032.v1
Subject: Life Sciences, Molecular Biology Keywords: nucleic acids analogs, antisense, CRISPR, antibiotic resistance, myotonic dystrophy, cholesterol, hematologic malignancy,
Online: 5 June 2019 (08:11:12 CEST)
Oligonucleotides are key compounds widely used for research, diagnostics, and therapeutics. The rapid increase in oligonucleotide-based applications, together with the progress in nucleic acids research, led to the design of nucleotide analogs that when being part of these oligomers enhance their efficiency, bioavailability, or stability. One of the most useful nucleotide analogs are the first-generation bridge nucleic acids (BNA), also known as locked nucleic acids (LNA), which were used in combination with ribonucleotides, deoxyribonucleotides, or other analogs to construct oligomers with diverse applications. However, there is still room to improve their efficiency, bioavailability, stability, and, importantly, toxicity. A second generation BNA, BNANC (2'-O,4'-aminoethylene bridged nucleic acid), has been recently made available. Oligomers containing these analogs not only showed less toxicity when compared to LNA-containing compounds but in some cases also exhibited higher specificity. Although there are still few applications where BNANC-containing compounds were researched, the results are very promising warranting more efforts in incorporating these analogs for other applications. Furthermore, newer BNA compounds will be introduced in the near future offering great hope to oligonucleotide-based fields of research and applications.
REVIEW | doi:10.20944/preprints202007.0744.v1
Subject: Life Sciences, Biochemistry Keywords: long non-coding RNA (lncRNA); RNA polymerase II (RNAPII) transcription; gene regulation; tandem transcriptional interference (tTI); antisense transcription; DNA processing
Online: 31 July 2020 (10:47:08 CEST)
RNA polymerase II (RNAPII) frequently transcribes non-protein coding DNA sequences in eukaryotic genomes into long non-coding RNA (lncRNA). Here, we focus on the impact of the act of lncRNA transcription on nearby functional DNA units. Distinct molecular mechanisms linked to the position of lncRNA relative to the coding gene illustrate how non-coding transcription controls gene expression. We review the biological significance of the act of lncRNA transcription on DNA processing, highlighting common themes, such as mediating cellular responses to environmental changes. This review presents the background in chromatin signaling to appreciate examples in different organisms where we can interpret functions of non-coding DNA through the act of RNAPII transcription.
ARTICLE | doi:10.20944/preprints201902.0172.v4
Subject: Life Sciences, Molecular Biology Keywords: RNA-dependent amplification of mammalian mRNA; physiologically occurring intracellular PCR, iPCR; RNA-dependent RNA polymerase, RdRp; chimeric RNA; sense-strand RNA; antisense-strand RNA
Online: 12 June 2019 (12:21:59 CEST)
The transfer of protein-encoding genetic information from DNA to RNA to protein, a process formalized as the “Central Dogma of Molecular Biology”, has undergone a significant evolution since its inception. It was amended to account for the information flow from RNA to DNA, the reverse transcription, and for the information transfer from RNA to RNA, the RNA-dependent RNA synthesis. These processes, both potentially leading to protein production, were initially described only in viral systems, and although RNA-dependent RNA polymerase activity was shown to be present, and RNA-dependent RNA synthesisfound to occur, in mammalian cells, its function was presumed to be restricted to regulatory. However, recent results, obtained with multiple mRNA species in several mammalian systems, strongly indicate the occurrence of protein-encoding RNA to RNA information transfer in mammalian cells. It can result in the rapid production of the extraordinary quantities of specific proteins as was seen in cases of terminal cellular differentiation and during cellular deposition of extracellular matrix molecules. A malfunction of this process may be involved in pathologies associated either with the deficiency of a protein normally produced by this mechanism or with the abnormal abundanceof a protein or of its C-terminal fragment. It seems to be responsible for some types of familial thalassemia and may underlie the overproduction of beta amyloid in sporadic Alzheimer’s disease. The aim of the present article is to systematize the current knowledge and understanding of this pathway. The outlined framework introduces unexpected features of the mRNA amplification such as its ability to generate polypeptides non-contiguously encoded in the genome, its second Tier, a physiologically occurring intracellular polymerase chain reaction, iPCR, a Two-Tier Paradox and RNA Dark Matter. RNA-dependent mRNA amplification represents a new mode of genomic protein-encoding information transfer in mammalian cells. Its potential physiological impact is substantial, it appears relevant to multiple pathologies and its understanding opens new venues of therapeutic interference, it suggests powerful novel bioengineering approaches and its further rigorous investigations are highly warranted.