Adele Alagia

siRNA and RNAi optimization

The discovery and examination of the posttranscriptional gene regulatory mechanism known as RNA interference (RNAi) contributed to the identification of small interfering RNA (siRNA) and the comprehension of its enormous potential for clinical purposes. Theoretically, the ability of specific target gene downregulation makes the RNAi pathway an appealing solution for several diseases. Despite numerous hurdles resulting from the inherent properties of siRNA molecule and proper delivery to the target tissue, more than 50 RNA‐based drugs are currently under clinical testing. In this work, we analyze the recent literature in the optimization of siRNA molecules. In detail, we focused on describing the most recent advances of siRNA field aimed at optimize siRNA pharmacokinetic properties. Special attention has been given in describing the impact of RNA modifications in the potential off‐target effects (OTEs) such as saturation of the RNAi machinery, passenger strand‐mediated silencing, immunostimulation, and miRNA‐like OTEs as well as to recent developments on the delivery issue. The novel delivery systems and modified siRNA provide significant steps toward the development of reliable siRNA molecules for therapeutic use. WIREs RNA 2016, 7:316–329. doi: 10.1002/wrna.1337

Cationic vesicles based on non-ionic surfactant and synthetic aminolipids mediate delivery of antisense oligonucleotides into mammalian cells

A formulation based on a synthetic aminolipid containing a double-tailed with two saturated alkyl chains along with a non-ionic surfactant polysorbate-80 has been used to form lipoplexes with an antisense oligonucleotide capable of inhibiting the expression of Renilla luciferase mRNA. The resultant lipoplexes were characterized in terms of morphology, Zeta potential, average size, stability and electrophoretic shift assay. The lipoplexes did not show any cytotoxicity in cell culture up to 150 mM concentration. The gene inhibition studies demonstrated that synthetic cationic vesicles based on non-ionic surfactant and the appropriate aminolipid play an important role in enhancing cellular uptake of antisense oligonucleotides obtaining promising results and efficiencies comparable to commercially available cationic lipids in cultured mammalian cells. Based on these results, this amino lipid moiety could be considered as starting point for the synthesis of novel cationic lipids to obtain potential non-viral carriers for antisense and RNA interference therapies.

RNA/aTNA Chimeras: RNAi Effects and Nucleases Resistance of Single and Double Stranded RNAs

The RNA interference pathway (RNAi) is a specific and powerful biological process, triggered by small non-coding RNA molecules and involved in gene expression regulation. In this work, we explored the possibility of increasing the biological stability of these RNA molecules by replacing their natural ribose ring with an acyclic l-threoninol backbone. In particular, this modification has been incorporated at certain positions of the oligonucleotide strands and its effects on the biological properties of the siRNA have been evaluated. In vitro cellular RNAi assays have demonstrated that the l-threoninol backbone is well tolerated by the RNAi machinery in both double and single-stranded fashion, with activities significantly higher than those evinced by the unmodified RNAs and comparable to the well-known phosphorothioate modification. Additionally, this modification conferred extremely strong resistance to serum and 3′/5′-exonucleases. In view of these results, we applied this modification to the knockdown of a therapeutically relevant human gene such as apolipoprotein B (ApoB). Further studies on the activation of the innate immune system showed that l-threoninol-modified RNAs are slightly less stimulatory than unmodified RNAs.

Functionalization of the 3′-Ends of DNA and RNA Strands with N-ethyl-N-coupled Nucleosides: A Promising Approach To Avoid 3′-Exonuclease-Catalyzed Hydrolysis of Therapeutic Oligonucleotides

The development of nucleic acid derivatives to generate novel medical treatments has become increasingly popular, but the high vulnerability of oligonucleotides to nucleases limits their practical use. We explored the possibility of increasing the stability against 3′‐exonucleases by replacing the two 3′‐terminal nucleotides by N‐ethyl‐N‐coupled nucleosides. Molecular dynamics simulations of 3′‐N‐ethyl‐N‐modified DNA:Klenow fragment complexes suggested that this kind of alteration has negative effects on the correct positioning of the adjacent scissile phosphodiester bond at the active site of the enzyme, and accordingly was expected to protect the oligonucleotide from degradation. We verified that these modifications conferred complete resistance to 3′‐exonucleases. Furthermore, cellular RNAi experiments with 3′‐N‐ethyl‐N‐modified siRNAs showed that these modifications were compatible with the RNAi machinery. Overall, our experimental and theoretical studies strongly suggest that these modified oligonucleotides could be valuable for therapeutic applications.

Covalent Strategies for Targeting Messenger and Non-Coding RNAs: An Updated Review on siRNA, miRNA and antimiR Conjugates

Oligonucleotide-based therapy has become an alternative to classical approaches in the search of novel therapeutics involving gene-related diseases. Several mechanisms have been described in which demonstrate the pivotal role of oligonucleotide for modulating gene expression. Antisense oligonucleotides (ASOs) and more recently siRNAs and miRNAs have made important contributions either in reducing aberrant protein levels by sequence-specific targeting messenger RNAs (mRNAs) or restoring the anomalous levels of non-coding RNAs (ncRNAs) that are involved in a good number of diseases including cancer. In addition to formulation approaches which have contributed to accelerate the presence of ASOs, siRNAs and miRNAs in clinical trials; the covalent linkage between non-viral vectors and nucleic acids has also added value and opened new perspectives to the development of promising nucleic acid-based therapeutics. This review article is mainly focused on the strategies carried out for covalently modifying siRNA and miRNA molecules. Examples involving cell-penetrating peptides (CPPs), carbohydrates, polymers, lipids and aptamers are discussed for the synthesis of siRNA conjugates whereas in the case of miRNA-based drugs, this review article makes special emphasis in using antagomiRs, locked nucleic acids (LNAs), peptide nucleic acids (PNAs) as well as nanoparticles. The biomedical applications of siRNA and miRNA conjugates are also discussed.

Cationic nioplexes-in-polysaccharide-based hydrogels as versatile biodegradable hybrid materials to deliver nucleic acids

Two polysaccharide-based hydrogels made of only κ-carrageenan (4%; w/v) or of a mixture of methylcellulose:κ-carrageenan (2%; w/v) were used to encapsulate cationic nioplexes. These vesicular particles were made of a synthetic aminolipid and polysorbate-80 (Tween-80), as a non-ionic surfactant agent. According to oscillatory rheological measurements, the presence of nioplexes did not compromise the mechanical integrity of the gels. In vitro niosomal release experiments demonstrated the liberation of nioplexes up to 24 h, and the curves were fitted according to Higuchi, Korsmeyer-Peppas and Weibull equation models, which indicated Fickian-diffusion controlled mechanisms. Besides nioplexes, cervical cancer cells were also entrapped within the biohydrogels. Cell release confirmed that these materials did not affect the cell viability, allowing cells to spread and proliferate after 24 h. The applicability of these biocompatible hydrogels was also extended to gene delivery. In this regard, the best silencing activities were found when cationic niosomes were complexed with antisense oligonucleotides in KC hydrogels. Nioplexes were able to release through the hydrogel and promoted silencing of luciferase expression in the presence of serum without using commercially available cationic lipids. Overall, the formation of such hybrid materials by integrating cationic nioplexes within biodegradable hydrogels provides a new perspective for the delivery of macromolecular therapeutics.

Challenges and Opportunities for Oligonucleotide-Based Therapeutics by Antisense and RNA Interference Mechanisms

Oligonucleotide-based therapeutics may be one of the most promising approaches for the treatment of diseases. Although significant progress has been made in developing these agents as drugs, several hurdles remain to be overcome. One of the most promising approaches to overcome these difficulties is the preparation of modified oligonucleotides designed to increase cellular uptake and/or increase stability to nucleases. Herein, we report the developments done by our group in the synthesis of modified oligonucleotides directed to the generation of active compounds for gene inhibition. Specifically we will report the synthesis of novel nuclease-resistant oligonucleotides such as North bicyclo[3.1.0]hexane pseudosugars or N-coupled dinucleotide units. Also, the design of several siRNA conjugates carrying cell-penetrating peptides, lipids, intercalating agents, and carbohydrates will be described. Some of these novel derivatives show clear improvements in their biological and inhibitory properties.

siRNA Modified with 2′-Deoxy-2′-C-methylpyrimidine Nucleosides

(2′S)‐2′‐Deoxy‐2′‐C‐methyluridine and (2′R)‐2′‐deoxy‐2′‐C‐methyluridine were incorporated in the 3′‐overhang region of the sense and antisense strands and in positions 2 and 5 of the seed region of siRNA duplexes directed against Renilla luciferase, whereas (2′S)‐2′‐deoxy‐2′‐C‐methylcytidine was incorporated in the 6‐position of the seed region of the same constructions. A dual luciferase reporter assay in transfected HeLa cells was used as a model system to measure the IC50 values of 24 different modified duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3′‐overhang region by (2′S)‐ or (2′R)‐2′‐deoxy‐2′‐C‐methyluridine, reducing IC50 to half of the value observed for the natural control. The selectivity of the modified siRNA was measured, it being found that modifications in positions 5 and 6 of the seed region had a positive effect on the ON/OFF activity.

The impact of an extended nucleobase-2′-deoxyribose linker in the biophysical and biological properties of oligonucleotides

Interest in artificial DNA mimetics has been triggered by the widespread applications of nucleic acids as they are useful tools for modulation of the biophysical and biological properties of oligonucleotides. In this article, we describe the synthesis and properties of a novel thymine derivative (T*) containing an extended linker between the thymine nucleobase and the 2′-deoxyribose moiety. The modified 2′-deoxyribosyl derivative was prepared via coupling of a functionalized nucleobase to the amino group of 1-aminomethyl-2-deoxyribose, which was synthesized starting from an easily accessible cyano sugar available on a large-scale. Corresponding phosphoramidite and succinyl derivatives have also been incorporated into oligonucleotides at predetermined sites and defined internucleotidic motifs using the solid-phase synthesis approach. This derivative pairs equally well with adenine and guanine and it can be safely introduced at the 3′-end of the siRNAs to generate potent inhibitors of gene expression by the RNA interference mechanism.

Cellular uptake studies of antisense oligonucleotides using G-quadruplex-nanostructures. The effect of cationic residue on the biophysical and biological properties

Oligonucleotides carrying cationic peptides have been used to improve hybridization and cellular uptake of oligonucleotides. In this study, a series of four cationic amino acid derivatives were covalently linked at the 3′-termini with the objective of modifying the Tetrahymena telomeric repeat sequence d(TGGGGT). This led to the synthesis of a small cationic G-quadruplex-forming oligonucleotide series containing lysine, ornithine, homo-arginine and arginine moieties. The preparation of the appropriate solid-supports afforded the synthesis of 3′-lysine and 3′-ornithine oligonucleotides which were converted to homo-arginine and arginine conjugates through post-synthetic modifications. Circular dichroism and thermal denaturation experiments confirmed that the presence of the four cationic residues did not affect the formation of stable parallel G-quadruplex structures. Afterwards, a phosphorothioate oligonucleotide targeting Renilla luciferase mRNA was prepared at the 5′-termini of this telomeric sequence which afforded a series of phosphorothioate oligonucleotide/[d(TG4T)]4 hybrid constructs modified with lysine, ornithine, homo-arginine and arginine units after incubation. In addition, the phosphodiester/phosphorothioate mixed backbone contributed to the degradation of the G-quadruplex moiety by exonucleases liberating the antisense sequence. Cell culture analysis of gene expression showed that the formation of self-assembled G-quadruplex nanostructures did not disrupt the antisense mechanism and therefore were able to induce luciferase gene inhibition in mammalian cells without using cationic lipids. Flow cytometry analyses confirmed that fluorescently labelled antisense G4-quadruplex nanostructures were efficiently taken up by HeLa cells. These results suggest that G-quadruplex nanostructures may be used to improve cellular uptake of therapeutic oligonucleotides.