Structure Tuning of Cationic Oligospermine–siRNA Conjugates for Carrier-Free Gene Silencing

Oligospermine–siRNA conjugates are able to induce efficient luciferase gene silencing upon carrier-free transfection. These conjugates are readily accessible by a versatile automated chemistry that we developed using a DMT-spermine phosphoramidite reagent. In this article, we used this chemistry to study a wide range of structural modifications of the oligospermine–siRNA conjugates, i.e., variation of conjugate positions and introduction of chemical modifications to increase nuclease resistance. At first we examined gene silencing activity of a series of siRNA–tris­(spermine) conjugates with and without chemical modifications in standard carrier assisted conditions. The three spermine units attached at one of the two ends of the sense strand or at the 3′-end of the antisense strand are compatible with gene silencing activity whereas attachment of spermine units at the 5′-end of the antisense strand abolished the activity. 2′-<i>O-</i>Methylated nucleotides introduced in the sense strand are compatible while not in the antisense strand. Thiophosphate links could be used without activity loss at the 3′-end of both strands and at the 5′-end of the sense strand to conjugate oligospermine. Consequently a series of oligospermine–siRNA conjugates containing 15 to 45 spermines units in various configurations were chosen, prepared, and examined in carrier-free conditions. Attachment of 30 spermine units singly at the 5′-end of the sense strand provides the most potent carrier-free siRNA. Longevity of luciferase gene silencing was studied using oligospermine–siRNA conjugates. Five day long efficiency with more than 80% gene expression knockdown was observed upon transfection without vector. Oligospermine–siRNA conjugates targeting cell-constitutive natural lamin A/C gene were prepared. Efficient gene silencing was observed upon carrier-free transfection of siRNA conjugates containing 20 or 30 spermine residues grafted at the 5′-end of the sense strand

Conjugating Phosphospermines to siRNAs for Improved Stability in Serum, Intracellular Delivery and RNAi-Mediated Gene Silencing

siRNAs are usually formulated with cationic polymers or lipids to form supramolecular particles capable of binding and crossing the negatively charged cell membrane. However, particles hardly diffuse through tissues when administered <i>in vivo</i>. We therefore are developing cationic siRNAs, composed of an antisense sequence annealed to an oligophosphospermine-conjugated sense strand. Cationic siRNAs have been previously shown to display gene silencing activity in human cell line (Nothisen et al. <i>J. Am. Chem. Soc.</i> <b>2009</b>). We have improved the synthesis, purification and characterization of oligospermine-oligoribonucleotide conjugates which provide cationic siRNAs with enhanced biological activity. We show data supporting their carrier-free intracellular delivery in a molecular, soluble state. Additional results on the relationship between global charge, uptake and silencing activity confirm the requirement for an overall positive charge of the conjugated siRNA in order to enter cells. Importantly, conjugated siRNAs made of natural phosphodiester nucleotides are protected from nuclease degradation by the oligophosphospermine moiety, operate through the RNAi mechanism and mediate specific gene silencing at submicromolar concentration in the presence of serum