Exploring the Emergence of RNA Nucleosides and Nucleotides on the Early Earth

Understanding how life began is one of the most fascinating problems to solve. By approaching this enigma from a chemistry perspective, the goal is to define what series of chemical reactions could lead to the synthesis of nucleotides, amino acids, lipids, and other cellular components from simple feedstocks under prebiotically plausible conditions. It is well established that evolution of life involved RNA which plays central roles in both inheritance and catalysis. In this review, we present historically important and recently published articles aimed at understanding the emergence of RNA nucleosides and nucleotides on the early Earth

Docosanoic acid conjugation to siRNA enables functional and safe delivery to skeletal and cardiac muscles

Oligonucleotide therapeutics hold promise for the treatment of muscle- and heart-related diseases. However, oligonucleotide delivery across the continuous endothelium of muscle tissue is challenging. Here, we demonstrate that docosanoic acid (DCA) conjugation of small interfering RNAs (siRNAs) enables efficient (~5% of injected dose), sustainable ( \u3e 1 month), and non-toxic (no cytokine induction at 100 mg/kg) gene silencing in both skeletal and cardiac muscles after systemic injection. When designed to target myostatin (muscle growth regulation gene), siRNAs induced ~55% silencing in various muscle tissues and 80% silencing in heart, translating into a ~50% increase in muscle volume within 1 week. Our study identifies compounds for RNAi-based modulation of gene expression in skeletal and cardiac muscles, paving the way for both functional genomics studies and therapeutic gene modulation in muscle and heart

The chemical structure and phosphorothioate content of hydrophobically modified siRNAs impact extrahepatic distribution and efficacy

Small interfering RNAs (siRNAs) have revolutionized the treatment of liver diseases. However, robust siRNA delivery to other tissues represents a major technological need. Conjugating lipids (e.g. docosanoic acid, DCA) to siRNA supports extrahepatic delivery, but tissue accumulation and gene silencing efficacy are lower than that achieved in liver by clinical-stage compounds. The chemical structure of conjugated siRNA may significantly impact invivo efficacy, particularly in tissues with lower compound accumulation. Here, we report the first systematic evaluation of the impact of siRNA scaffold-i.e. structure, phosphorothioate (PS) content, linker composition-on DCA-conjugated siRNA delivery and efficacy in vivo. We found that structural asymmetry (e.g. 5- or 2-nt overhang) has no impact on accumulation, but is a principal factor for enhancing activity in extrahepatic tissues. Similarly, linker chemistry (cleavable versus stable) altered activity, but not accumulation. In contrast, increasing PS content enhanced accumulation of asymmetric compounds, but negatively impacted efficacy. Our findings suggest that siRNA tissue accumulation does not fully define efficacy, and that the impact of siRNA chemical structure on activity is driven by intracellular re-distribution and endosomal escape. Fine-tuning siRNA chemical structure for optimal extrahepatic efficacy is a critical next step for the progression of therapeutic RNAi applications beyond liver

Diverse lipid conjugates for functional extra-hepatic siRNA delivery in vivo

Small interfering RNA (siRNA)-based therapies are proving to be efficient for treating liver-associated disorders. However, extra-hepatic delivery remains challenging, limiting therapeutic siRNA utility. We synthesized a panel of fifteen lipid-conjugated siRNAs and systematically evaluated the impact of conjugate on siRNA tissue distribution and efficacy. Generally, conjugate hydrophobicity defines the degree of clearance and the liver-to-kidney distribution profile. In addition to primary clearance tissues, several conjugates achieve significant siRNA accumulation in muscle, lung, heart, adrenal glands and fat. Oligonucleotide distribution to extra-hepatic tissues with some conjugates was significantly higher than with cholesterol, a well studied conjugate, suggesting that altering conjugate structure can enhance extra-hepatic delivery. These conjugated siRNAs enable functional gene silencing in lung, muscle, fat, heart and adrenal gland. Required levels for productive silencing vary (5-200 mug/g) per tissue, suggesting that the chemical nature of conjugates impacts tissue-dependent cellular/intracellular trafficking mechanisms. The collection of conjugated siRNA described here enables functional gene modulation in vivo in several extra-hepatic tissues opening these tissues for gene expression modulation. A systemic evaluation of a panel of conjugated siRNA, as reported here, has not previously been investigated and shows that chemical engineering of lipid siRNAs is essential to advance the RNA therapeutic field

Hydrophobically Modified let-7b miRNA Enhances Biodistribution to NSCLC and Downregulates HMGA2 In Vivo

MicroRNAs (miRNAs) have increasingly been shown to be involved in human cancer, and interest has grown about the potential use of miRNAs for cancer therapy. miRNA levels are known to be altered in cancer cells, including in non-small cell lung cancer (NSCLC), a subtype of lung cancer that is the most prevalent form of cancer worldwide and that lacks effective therapies. The let-7 miRNA is involved in the regulation of oncogene expression in cells and directly represses cancer growth in the lung. let-7 is therefore a potential molecular target for tumor therapy. However, applications of RNA interference for cancer research have been limited by a lack of simple and efficient methods to deliver oligonucleotides (ONs) to cancer cells. In this study, we have used in vitro and in vivo approaches to show that HCC827 cells internalize hydrophobically modified let-7b miRNAs (hmiRNAs) added directly to the culture medium without the need for lipid formulation. We identified functional let-7b hmiRNAs targeting the HMGA2 mRNA, one of the let-7 target genes upregulated in NSCLC, and show that direct uptake in HCC827 cells induced potent and specific gene silencing in vitro and in vivo. Thus, hmiRNAs constitute a novel class of ONs that enable functional studies of genes involved in cancer biology and are potentially therapeutic molecules

Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways

Efficient delivery of therapeutic RNA beyond the liver is the fundamental obstacle preventing its clinical utility. Lipid conjugation increases plasma half-life and enhances tissue accumulation and cellular uptake of small interfering RNAs (siRNAs). However, the mechanism relating lipid hydrophobicity, structure, and siRNA pharmacokinetics is unclear. Here, using a diverse panel of biologically occurring lipids, we show that lipid conjugation directly modulates siRNA hydrophobicity. When administered in vivo, highly hydrophobic lipid-siRNAs preferentially and spontaneously associate with circulating low-density lipoprotein (LDL), while less lipophilic lipid-siRNAs bind to high-density lipoprotein (HDL). Lipid-siRNAs are targeted to lipoprotein receptor-enriched tissues, eliciting significant mRNA silencing in liver (65%), adrenal gland (37%), ovary (35%), and kidney (78%). Interestingly, siRNA internalization may not be completely driven by lipoprotein endocytosis, but the extent of siRNA phosphorothioate modifications may also be a factor. Although biomimetic lipoprotein nanoparticles have been explored for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation

Synthesis and evaluation of 2’-O-modified oligoribonucleotides bearing acetalester or alkyldithiomethyl biolabile groups in a siRNA prodrug-like approach

Les ARN interférents sont de puissants outils thérapeutiques et biologiques pour la mise en silence de l'expression des gènes. Afin d'améliorer leur stabilité enzymatique, leur biodistribution et leur pénétration cellulaire, nous proposons de développer une approche prodrogue d'ARN interférent. Ce manuscrit rapporte la synthèse et l'évaluation de pro-ARN masqués temporairement par des groupements biolabiles susceptibles d'être hydrolysés dans les cellules afin de libérer l'ARN naturel actif. Deux types de modifications sont présentés : des groupes acétalesters enlevés par des carboxyestérases et des groupes alkyldithiométhyles sensibles à un environnement réducteur. Dans une première partie, une nouvelle méthode de synthèse de pro-ARN partiellement modifiés en position 2' par des groupements acétalesters est décrite. Plusieurs groupements variant par leur caractère lipophile ou cationique sont évalués. Des résultats prometteurs d'études physico-chimiques, de stabilité enzymatique, de pénétration cellulaire et d'inhibition de gènes mettent en valeur l'intérêt d'utiliser certains pro-ARN modifiés en tant qu'outils thérapeutiques. Une deuxième partie présente une voie de synthèse originale de pro-ARN modifiés en position 2' par des groupements alkyldithiométyles. Les propriétés physico-chimiques, la stabilité enzymatique et le démasquage de ces pro-ARN sont décrits. Parallèlement, l'étude d'une réaction d'échange thiol-disulfure permettant l'incorporation de liens disulfures intrabrin au sein de duplex d'ARN et de constructions tige-boucles est détaillée dans ce manuscrit.SiRNA are powerful therapeutic and biological tools for gene silencing. In the aim of improving their stability, their biodistribution and their cellular delivery, we propose to develop a siRNA prodrug-like approach.This manuscript reports the synthesis and the study of pro-RNA temporarily masked by biolabile groups which could be hydrolyzed inside cells in order to release the active unmodified RNA. Two types of modifications are presented: acetalester groups removed by carboxyesterases and alkyldithiomethyl groups cleaved in a reducing environment within cells.In a first part, a new synthesis strategy of partially modified 2'-O-acetalester pro-RNA is described. Several acetalester groups varying in their lipophilicity and their charge are evaluated. Promising results obtained in physical-chemical studies, enzymatic stability and gene inhibition highlight the use of these modified pro-RNA as therapeutic drugs. A second part introduces an original approach for the synthesis of 2'-O-alkyldithiomethyl pro-RNA. The physical-chemical properties, the enzymatic stability and the unmasking of this pro-RNA are described.Moreover, the study of a thiol-disulfide exchange reaction allowing the incorporation of intrastrand disulfide bond into secondary structure duplex and hairpin is reported in this manuscript

Gene therapy with AR isoform 2 rescues spinal and bulbar muscular atrophy phenotype by modulating AR transcriptional activity

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, adult-onset neuromuscular condition caused by an abnormal polyglutamine (polyQ) tract expansion in androgen receptor (AR) protein. SBMA is a disease with high unmet clinical need. Recent studies have shown that mutant AR-altered transcriptional activity is key to disease pathogenesis. Restoring the transcriptional dysregulation without affecting other AR critical functions holds great promise for the treatment of SBMA and other AR-related conditions; however, how this targeted approach can be achieved and translated into a clinical application remains to be understood. Here, we characterized the role of AR isoform 2, a naturally occurring variant encoding a truncated AR lacking the polyQ-harboring domain, as a regulatory switch of AR genomic functions in androgen-responsive tissues. Delivery of this isoform using a recombinant adeno-associated virus vector type 9 resulted in amelioration of the disease phenotype in SBMA mice by restoring polyQ AR-dysregulated transcriptional activity

Therapeutic Oligonucleotides: An Outlook on Chemical Strategies to Improve Endosomal Trafficking

The potential of oligonucleotide therapeutics is undeniable as more than 15 drugs have been approved to treat various diseases in the liver, central nervous system (CNS), and muscles. However, achieving effective delivery of oligonucleotide therapeutics to specific tissues still remains a major challenge, limiting their widespread use. Chemical modifications play a crucial role to overcome biological barriers to enable efficient oligonucleotide delivery to the tissues/cells of interest. They provide oligonucleotide metabolic stability and confer favourable pharmacokinetic/pharmacodynamic properties. This review focuses on the various chemical approaches implicated in mitigating the delivery problem of oligonucleotides and their limitations. It highlights the importance of linkers in designing oligonucleotide conjugates and discusses their potential role in escaping the endosomal barrier, a bottleneck in the development of oligonucleotide therapeutics

A versatile post-synthetic method on a solid support for the synthesis of RNA containing reduction-responsive modifications

International audienceAn original post-synthetic method on a solid support was developed to introduce various disulfide bond containing groups at the 2'-OH of oligoribonucleotides (RNAs). It is based on a thiol disulfide exchange reaction between several readily accessible alkyldisulfanyl-pyridine derivatives and 2'-O-acetylthiomethyl RNA in the presence of butylamine. By this strategy, diverse 2'-O-alkyldithiomethyl RNAs were obtained. These modifications provided high nuclease resistance to RNA and were easily removed with glutathione treatment, thus featuring a potential use for siRNA prodrugs