Silencing Antibiotic Resistance with Antisense Oligonucleotides

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 targeting resistance to β-lactams include carbapenems, aminoglycosides, chloramphenicol, macrolides, and fluoroquinolones. The purpose of this short review is to summarize the attempts to develop antisense compounds that inhibit expression of resistance to antibiotics.

Design and In Vitro Evaluation of Splice-Switching Oligonucleotides Bearing Locked Nucleic Acids, Amido-Bridged Nucleic Acids, and Guanidine-Bridged Nucleic Acids

Our group previously developed a series of bridged nucleic acids (BNAs), including locked nucleic acids (LNAs), amido-bridged nucleic acids (AmNAs), and guanidine-bridged nucleic acids (GuNAs), to impart specific characteristics to oligonucleotides such as high-affinity binding and enhanced enzymatic resistance. In this study, we designed a series of LNA-, AmNA-, and GuNA-modified splice-switching oligonucleotides (SSOs) with different lengths and content modifications. We measured the melting temperature (Tm) of each designed SSO to investigate its binding affinity for RNA strands. We also investigated whether the single-stranded SSOs formed secondary structures using UV melting analysis without complementary RNA. As a result, the AmNA-modified SSOs showed almost the same Tm values as the LNA-modified SSOs, with decreased secondary structure formation in the former. In contrast, the GuNA-modified SSOs showed slightly lower Tm values than the LNA-modified SSOs, with no inhibition of secondary structures. We also evaluated the exon skipping activities of the BNAs in vitro at both the mRNA and protein expression levels. We found that both AmNA-modified SSOs and GuNA-modified SSOs showed higher exon skipping activities than LNA-modified SSOs but each class must be appropriately designed in terms of length and modification content.

Silencing Antibiotic Resistance with Antisense Oligonucleotides

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.

Development of aptamer-based inhibitors for CRISPR/Cas system

The occurrence of accidental mutations or deletions caused by genome editing with CRISPR/Cas9 system remains a critical unsolved problem of the technology. Blocking excess or prolonged Cas9 activity in cells is considered as one means of solving this problem. Here, we report the development of an inhibitory DNA aptamer against Cas9 by means of in vitro selection (systematic evolution of ligands by exponential enrichment) and subsequent screening with an in vitro cleavage assay. The inhibitory aptamer could bind to Cas9 at low nanomolar affinity and partially form a duplex with CRISPR RNA, contributing to its inhibitory activity. We also demonstrated that improving the inhibitory aptamer with locked nucleic acids efficiently suppressed Cas9-directed genome editing in cells and reduced off-target genome editing. The findings presented here might enable the development of safer and controllable genome editing for biomedical research and gene therapy.

A short de novo synthesis of nucleoside analogs

Nucleoside analogs are commonly used in the treatment of cancer and viral infections. Their syntheses benefit from decades of research but are often protracted, unamenable to diversification, and reliant on a limited pool of chiral carbohydrate starting materials. We present a process for rapidly constructing nucleoside analogs from simple achiral materials. Using only proline catalysis, heteroaryl-substituted acetaldehydes are fluorinated and then directly engaged in enantioselective aldol reactions in a one-pot reaction. A subsequent intramolecular fluoride displacement reaction provides a functionalized nucleoside analog. The versatility of this process is highlighted in multigram syntheses of d- or l-nucleoside analogs, locked nucleic acids, iminonucleosides, and C2′- and C4′-modified nucleoside analogs. This de novo synthesis creates opportunities for the preparation of diversity libraries and will support efforts in both drug discovery and development.

Think Different with RNA Therapy: Can Antisense Oligonucleotides Be Used to Inhibit Replication and Transcription of SARS-Cov-2?

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.

The Role of APAL/ST8SIA6-AS1 lncRNA in PLK1 Activation and Mitotic Catastrophe of Tumor Cells

Background Tumor growth can be addicted to vital oncogenes, but whether long noncoding RNAs (lncRNAs) are essential to cancer survival is largely uncharacterized. Methods We retrieved Gene Expression Omnibus datasets to identify lncRNA overexpression in 257 cancers vs 196 normal tissues and analyzed the association of ST8SIA6-AS1 (termed Aurora A/Polo-like-kinase 1 [PLK1]–associated lncRNA, APAL) with the clinical outcomes of multiple types of cancer from public RNA sequencing and microarray datasets as well as from in-house cancer cohorts. Loss- and gain-of-function experiments were performed to explore the role of APAL in cancers in vitro and in vivo. RNA pulldown and RNA immunoprecipitation were used to investigate APAL-interacting proteins. All statistical tests were two-sided. Results APAL is overexpressed in multiple human cancers associated with poor clinical outcome of patients. APAL knockdown causes mitotic catastrophe and massive apoptosis in human breast, lung, and pancreatic cancer cells. Overexpressing APAL accelerates cancer cell cycle progression, promotes proliferation, and inhibits chemotherapy-induced apoptosis. Mechanism studies show that APAL links up PLK1 and Aurora A to enhance Aurora A-mediated PLK1 phosphorylation. Notably, targeting APAL inhibits the growth of breast and lung cancer xenografts in vivo (MCF-7 xenografts: mean tumor weight, control = 0.18 g [SD = 0.03] vs APAL locked nucleic acids = 0.07 g [SD = 0.02], P < .001, n = 8 mice per group; A549 xenografts: mean tumor weight control = 0.36 g [SD = 0.10] vs APAL locked nucleic acids = 0.10 g [SD = 0.04], P < .001, n = 9 mice per group) and the survival of patient-derived breast cancer organoids in three-dimensional cultures. Conclusions Our data highlight the essential role of lncRNA in cancer cell survival and the potential of APAL as an attractive therapeutic target for a broad-spectrum of cancers.