posted on 2016-02-20, 04:58authored byClément Paris, Valérie Moreau, Gaëlle Deglane, Loukmane Karim, Bernard Couturier, Marie-Elise Bonnet, Valérie Kedinger, Mélanie Messmer, Anne-Laure Bolcato-Bellemin, Jean-Paul Behr, Patrick Erbacher, Nathalie Lenne-Samuel
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 in vivo. 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. J. Am. Chem. Soc.2009). 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.