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In vivo delivery of lipophilic siRNA

RNA interference (RNAi) is a conserved biological process for specific silencing of gene expression. Small interfering RNAs (siRNAs) are important tools for the control of gene expression, such as post-transcriptional gene silencing in mammals, including humans. However, to function as desired, they need to be taken up by cells or tissues and delivered to selected targets. For in vivo applications of siRNAs, delivery across plasma membranes remains the most significant obstacle.

Synthetic oligonucleotides can be modified via conjugation to lipophilic functional groups to allow efficient uptake of siRNAs by cells and tissues. Lipophilic groups are often attached to the 3′-end of the sense strand via oligomethylene linkers of various lengths. Alternatively, various commercially available lipophilic phosphoramidites also allow conjugation to oligonucleotides.

Covalent siRNA cholesterol conjugates are known to increase import into cellular where they induce RNAi resulting in the silencing of endogenous genes in vivo. Wolfram et al. in 2007 reported the preparation of a variety of lipophilic carrier molecules useful for optimized cell delivery. Long-chain fatty acid conjugates are helpful for cell delivery. However, for all these lipophilic molecules, efficient and selective cellular uptake depends on their interaction with lipoprotein particles, lipoprotein receptors, and transmembrane proteins. As Wolfrum et al. reported, the efficiency of siRNA delivery into cells depends on the combined length of the linker and the lipophilic group.

Figure 1:  Lipophilic siRNA conjugate structure. siRNAs when conjugated to different lipids have different in vivo activities. The desired lipophile (L) is conjugated to the 3’-end of the sense strand of the desired siRNA, here via a trans-4-hydroxyprolinol linker (Modified after Wolfrum et al. 2007).


 

Figure 2:  Structures of lipophilic siRNAs with various lipids conjugated to the core structure of the siRNA conjugate.

Reference

Petrova NS, Chernikov IV, Meschaninova MI, Dovydenko IS, Venyaminova AG, Zenkova MA, Vlassov VV, Chernolovskaya EL. Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group. Nucleic Acids Res. 2012 Mar;40(5):2330-44. doi: 10.1093/nar/gkr1002. [PMC]

Wolfrum, C., Shi, S., Jayaprakash, K. et al. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat Biotechnol 25, 1149–1157 (2007). [link]


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