Designing Chemically Modified Oligonucleotides for Targeted Gene Silencing

RNA interference (RNAi) is an ancient biological mechanism used to defend against external invasion It theoretically can silence any disease-related genes in a sequence-specific manner, making small interfering RNA (siRNA) a promising therapeutic modality After a two-decade journey from its discovery, two approvals of siRNA therapeutics, ONPATTRO® (patisiran) and GIVLAARI™ (givosiran), have been achieved by Alnylam Pharmaceuticals Reviewing the long-term pharmaceutical history of human beings, siRNA therapy currently has set up an extraordinary milestone, as it has already changed and will continue to change the treatment and management of human diseases It can be administered quarterly, even twice-yearly, to achieve therapeutic effects, which is not the case for small molecules and antibodies The drug development process was extremely hard, aiming to surmount complex obstacles, such as how to efficiently and safely deliver siRNAs to desired tissues and cells and how to enhance the performance of siRNAs with respect to their activity, stability, specificity and potential off-target effects In this review, the evolution of siRNA chemical modifications and their biomedical performance are comprehensively reviewed All clinically explored and commercialized siRNA delivery platforms, including the GalNAc (N-acetylgalactosamine)–siRNA conjugate, and their fundamental design principles are thoroughly discussed The latest progress in siRNA therapeutic development is also summarized This review provides a comprehensive view and roadmap for general readers working in the field

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Therapeutic siRNA: state of the art.

Genome editing offers promising solutions to genetic disorders by editing DNA sequences or modulating gene expression. The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) technology can be used to edit single or multiple genes in a wide variety of cell types and organisms in vitro and in vivo. Herein, we review the rapidly developing CRISPR/Cas9-based technologies for disease modeling and gene correction and recent progress toward Cas9/guide RNA (gRNA) delivery based on viral and nonviral vectors. We discuss the relative merits of delivering the genome editing elements in the form of DNA, mRNA, or protein, and the opportunities of combining viral delivery of a transgene encoding Cas9 with nonviral delivery of gRNA. We highlight the lessons learned from nonviral gene delivery in the past three decades and consider their applicability for CRISPR/Cas9 delivery. We also include a discussion of bioinformatics tools for gRNA design and chemical modifications...

CRISPR/Cas9-Based Genome Editing for Disease Modeling and Therapy: Challenges and Opportunities for Nonviral Delivery

Over the last decade, great enthusiasm has evolved for microRNA (miRNA) therapeutics. Part of the excitement stems from the fact that a miRNA often regulates numerous related mRNAs. As such, modulation of a single miRNA allows for parallel regulation of multiple genes involved in a particular disease. While many studies have shown therapeutic efficacy using miRNA inhibitors, efforts to restore or increase the function of a miRNA have been lagging behind. The miR-29 family has gained a lot of attention for its clear function in tissue fibrosis. This fibroblast-enriched miRNA family is downregulated in fibrotic diseases which induces a coordinate increase of many extracellular matrix genes. Here, we show that intravenous injection of synthetic RNA duplexes can increase miR-29 levels in vivo for several days. Moreover, therapeutic delivery of these miR-29 mimics during bleomycin-induced pulmonary fibrosis restores endogenous miR-29 function whereby decreasing collagen expression and blocking and reversing pulmonary fibrosis. Our data support the feasibility of using miRNA mimics to therapeutically increase miRNAs and indicate miR-29 to be a potent therapeutic miRNA for treating pulmonary fibrosis.

https://www.embopress.org/doi/pdf/10.15252/emmm.201303604

MicroRNA mimicry blocks pulmonary fibrosis

The present disclosure provides modified nucleosides, nucleotides, and nucleic acids, and methods of using them.

Modified nucleosides, nucleotides, and nucleic acids, and uses thereof

Considerable attention has focused on the use of alternatives to the native ribose and phosphate backbone of small interfering RNAs for therapeutic applications of the RNA interference pathway. In this synopsis, we highlight the less common chemical modifications, namely, those of the RNA nucleobases. Base modifications have the potential to lend insight into the mechanism of gene silencing and to lead to novel methods to overcome off-target effects that arise due to deleterious protein binding or mis-targeting of mRNA.

Chemical Modification of siRNA Bases to Probe and Enhance RNA Interference

Disclosed are compositions and methods related to modified nucleobases. Also disclosed are compositions and methods related to modified interfering RNAs. Also disclosed are compositions and methods related to modified guanine bases for controlling off-target effects in RNA interference.

Methods and compositions related to modified guanine bases for controlling off-target effects in rna interference

Small interfering double-stranded RNAs have been synthesized bearing one or more base modifications at nucleotide positions 4, 11, and/or 16 in the guide strand. The chemically modified base is an N(2)-alkyl-8-oxo-7,8-dihydroguanine (alkyl = propyl, benzyl) that can alternatively pair in a Watson-Crick sense opposite cytosine (C) or as a Hoogsteen pair opposite adenine (A). Cellular delivery with C opposite led to effective targeting of A-containing but not C-containing mRNA sequences in a dual luciferase assay with RNA interference levels that were generally as good as or better than unmodified sequences. The higher activity is ascribed to an inhibitory effect of the alkyl group projecting into the minor groove of double-stranded RNA preventing off-target binding to proteins such as PKR (RNA-activated protein kinase).

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8-Oxoguanosine switches modulate the activity of alkylated siRNAs by controlling steric effects in the major versus minor grooves.

N2-Alkyl analogues of 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG) were synthesized (alkyl = propyl, benzyl) via reductive amination of the protected OG nucleoside and incorporated into various positions of an RNA strand. Thermal stability studies of duplexes containing A or C opposite a single modified base revealed only moderate destabilization. Both OG as well as its N2-alkyl analogues can pair opposite A or C with nearly equal stability, potentially offering a new means of modulating RNA−protein interactions in the minor vs major grooves.

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Synthesis of N2-alkyl-8-oxo-7,8-dihydro-2'-deoxyguanosine derivatives and effects of these modifications on RNA duplex stability.

Exposure of cells to phenolic compounds through exogenous and endogenous sources can lead to deleterious effects via nucleobase modifications of DNA occurring under oxidative conditions. 2′-Deoxygu...

Arunkumar Kannan

Papers

Chemical Modification of siRNA Bases to Probe and Enhance RNA Interference

Methods and compositions related to modified guanine bases for controlling off-target effects in rna interference

8-Oxoguanosine switches modulate the activity of alkylated siRNAs by controlling steric effects in the major versus minor grooves.

Synthesis of N2-alkyl-8-oxo-7,8-dihydro-2'-deoxyguanosine derivatives and effects of these modifications on RNA duplex stability.

Copper/H2O2-Mediated Oxidation of 2′-Deoxyguanosine in the Presence of 2-Naphthol Leads to the Formation of Two Distinct Isomeric Adducts