Alpha-l-Locked nucleic acid-modified antisense oligonucleotides induce efficient splice modulation in vitro

Alpha-l-Locked nucleic acid (α-l-LNA) is a stereoisomeric analogue of locked nucleic acid (LNA), which possesses excellent biophysical properties and also exhibits high target binding affinity to complementary oligonucleotide sequences and resistance to nuclease degradations. Therefore, α-l-LNA nucleotides could be utilised to develop stable antisense oligonucleotides (AO), which can be truncated without compromising the integrity and efficacy of the AO. In this study, we explored the potential of α-l-LNA nucleotides-modified antisense oligonucleotides to modulate splicing by inducing Dmd exon-23 skipping in mdx mouse myoblasts in vitro. For this purpose, we have synthesised and systematically evaluated the efficacy of α-l-LNA-modified 2′-O-methyl phosphorothioate (2′-OMePS) AOs of three different sizes including 20mer, 18mer and 16mer AOs in parallel to fully-modified 2′-OMePS control AOs. Our results demonstrated that the 18mer and 16mer truncated AO variants showed slightly better exon-skipping efficacy when compared with the fully-23 modified 2′-OMePS control AOs, in addition to showing low cytotoxicity. As there was no previous report on using α-l-LNA-modified AOs in splice modulation, we firmly believe that this initial study could be beneficial to further explore and expand the scope of α-l-LNA-modified AO therapeutic molecules

Antisense oligonucleotides targeting angiogenic factors as potential cancer therapeutics

Cancer is one of the leading causes of death worldwide and conventional cancer therapies such as surgery, chemotherapy and radiotherapy do not address the underlying molecular pathologies, leading to inadequate treatment and tumour recurrence. Angiogenic factors, such as EGF, PDGF, bFGF, TGF-β, TGF-α, VEGF, Endoglin and Angiopoietins play important roles in regulating tumour development and metastasis, and serve as potential targets for developing cancer therapeutics. Nucleic acid-based therapeutic strategies have received significant attention in the last two decades, and antisense oligonucleotide-mediated intervention is a prominent therapeutic approach for targeted manipulation of gene expression. Clinical benefits of antisense oligonucleotides have been recognised by the US Food and Drug Administration, with full or conditional approval of Vitravene, Kynamro, Exondys51 and Spinraza. Herein, we review the scope of antisense oligonucleotides that target angiogenic factors towards tackling solid cancers

Antisense oligonucleotide-mediated splice switching: Potential therapeutic approach for cancer mitigation

Splicing is an essential process wherein precursor messenger RNA (pre-mRNA) is reshaped into mature mRNA. In alternative splicing, exons of any pre-mRNA get rearranged to form mRNA variants and subsequently protein isoforms, which are distinct both by structure and function. On the other hand, aberrant splicing is the cause of many disorders, including cancer. In the past few decades, developments in the understanding of the underlying biological basis for cancer progression and therapeutic resistance have identified many oncogenes as well as carcinogenic splice variants of essential genes. These transcripts are involved in various cellular processes, such as apoptosis, cell signaling and proliferation. Strategies to inhibit these carcinogenic isoforms at the mRNA level are promising. Antisense oligonucleotides (AOs) have been developed to inhibit the production of alternatively spliced carcinogenic isoforms through splice modulation or mRNA degradation. AOs can also be used to induce splice switching, where the expression of an oncogenic protein can be inhibited by the induction of a premature stop codon. In general, AOs are modified chemically to increase their stability and binding affinity. One of the major concerns with AOs is efficient delivery. Strategies for the delivery of AOs are constantly being evolved to facilitate the entry of AOs into cells. In this review, the different chemical modifications employed and delivery strategies applied are discussed. In addition to that various AOs in clinical trials and their efficacy are discussed herein with a focus on six distinct studies that use AO-mediated exon skipping as a therapeutic strategy to combat cancer

Phosphorothioate modification improves exon-skipping of antisense oligonucleotides based on sulfonyl phosphoramidates in mdx mouse myotubes

2′-O-Methyl (2′-OMe) antisense oligonucleotides (AOs) possessing a various number of 4-(trimethylammonio)butylsulfonyl or tosyl phosphoramidates (N+ and Ts-modifications, respectively) instead of a native phosphodiester linkage were designed to skip exon-23 in dystrophin pre-mRNA transcript in mdx mice myotubes. AOs bearing several zwitterionic N+ modifications in the sequence had remarkably increased thermal stability towards complementary mRNA in comparison with 2′-OMe-RNAs having negatively charged Ts and phosphorothioate (PS) linkages. However, only Ts-modified AOs exhibited a similar level of exon skipping in comparison with fully modified PS-containing 2′-OMe-RNA, whereas the exon skipping induced by N+ modified AOs was much lower with no exon-skipping detected for AOs having seven N+ modifications. The level of exon-skipping was improved once Ts and especially N+ moieties were used in combination with PS-modification, most likely through improved cellular and nuclear uptake of AOs. These results provide new insights on expanding the design of novel chemically modified AOs based on phosphate modifications

A preliminary study on the screening of emerging drug resistance among the caries pathogens isolated from carious dentine

Background: Dental caries being the commonest unmet public health problem indicates its need to urge the dentists to overcome this problem globally. Caries exhibit in different types and is found to be associated with co-aggregation property of microbial flora with other oral hygienic factors. In spite of the surgical removals, excavations and administration of antimicrobials for carious dentine, there seems to be repeated infection and chronic prevalence of caries. A complete understanding of microbial etiology and prevention of emerging drug-resistant strains will aid in the eradication of this chronic dentine problem condition from the oral cavity. Aim: This study is aimed to isolate the predominant bacterial pathogens associated with caries and to screen for the emergence of drug resistance among the isolated caries pathogens. Materials and Methods: Carious dentine specimens were collected from 75 endodontic patients and the samples were processed microbiologically to isolate the caries pathogens. Identification of the strains was done by standard biochemical characterization studies. Statistical analysis of the isolates was done by Pearson Chi-square test and Fisher′s exact test. The predominant isolates were subjected to antimicrobial sensitivity test using Kirby Bauer′s method. The results were recorded and analyzed for drug resistance. Results: Carious dentine samples yielded a high percentage of Lactobacillus sp., and Candida albicans from different type of caries. Among the study population, dentinal caries was the most predominant type affecting most males with other associated risk factors. Nearly 47.3% of the isolated Lactobacillus sp. and 55.5% of the yeast C. albicans were screened to show resistance against the antimicrobials used for the study. Conclusion: This study concludes by stating that Lactobacillus sp., and C. albicans are mostly involved in the caries etiology and show resistance to the commonest antimicrobial agent. This implicates the need for periodical antimicrobial susceptibility examination of the caries pathogens that will aid to prevent the emergence of resistance property among the dentinal pathogenic organisms

Oligonucleotides containing 2′-O-methyl-5-(1-phenyl-1,2,3-triazol-4-yl)uridines demonstrate increased affinity for RNA and induce exon-skipping in vitro

The nucleotide monomer containing the 1-phenyl-1,2,3-triazole group attached to the 5-position of 2′-O-methyluridine is hereby presented together with a derivative further substituted with a p-sulfonamide group on the phenyl ring. Both were conveniently synthesised, and synergistic effect of the modifications were demonstrated when introduced into oligonucleotides and hybridised to complementary RNA. The combination of stacking of the phenyltriazoles and the conformational steering from the 2′-OMe group gave thermally very stable duplexes. Exon skipping in the distrophin transcript using 20-mer 2′-OMePS sequences with two phenyltriazoles introduced in different positions with and without the sulfonamide demonstrated efficient exon skipping but at the same level as the 2′-OMePS reference ASO

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Not AvailableMicroplitis maculipennis Szépligeti is an important parasitoid of castor semilooper Acanthodelta janata (L.) (Lepidoptera), a major pest of castor (Ricinus communis L.). Microplitis Förster shares remarkable morphological resemblance with moderately diverse genus Snellenius Westwood. In this study, molecular characterization of M. maculipennis was done using Cytochrome Oxidase I (COI) to confirm its generic placement in the respective genus. The Bayesian Inference (BI) and Maximum Likelihood (ML) phylogenetic analysis performed with a total of 354 published BOLD database sequences (after pre-processing of a total of 2257 COI sequences) of Microplitis and Snellenius species, representing 129 named species and 226 species determined only to genus raises doubts on the retention of both these genera separately. Our studies reveal that COI gene could not discriminate Microplitis and Snellenius species clearly.Not Availabl

Antibacterial effect of squid ink on ESBL producing strains of <i style="mso-bidi-font-style:normal">Escherichia coli</i> and <i style="mso-bidi-font-style:normal">Klebsiella pneumoniae</i>.

338-343Present study consist a novel therapeutics from natural sources to prevent the emergence and proliferation of resistant microbial populations that can make a significant impact in treating clinically challenging microbial infections. Squid ink has proved to play various primary roles in the world of alternative medicine and has widest range of therapeutic applications. Present study is designed to report the antibacterial effect of the squid ink against the ESBL producing strains of <i style="mso-bidi-font-style: normal">E.coli and K.pneumoniae. ESBL strains are isolated from patients with typical urinary tract infections. They have been identified microbiologically and characterized by double disc synergy test and plasmid profiles. Active metabolite of squid ink was extracted using solvents and was checked for its antibacterial activity by agar well diffusion method. MIC value is determined by microbroth dilution method. Results conclude that the hexane extract of the squid ink scored high antibacterial activity against the ESBL producing strains of E.coli and K.pneumoniae. Present study suggests that squid ink is an enigmatic pigment of therapeutic value in near future for treatment of dreadful infections caused by the ESBL strains