Cholesterol Conjugated siRNA View image detail

Delivering RNAi: The Challenges and Strategies

Arguably the biggest challenge in translating the breakthrough science of RNAi into a broad new class of human therapeutics is delivering siRNAs to the appropriate tissues within the body. This is not an entirely new problem to drug development and similar obstacles apply to related oligonucleotide therapeutics (e.g. antisense technologies). In fact, considerable experience gained from years of developing nucleic acid delivery methods can now be applied to the development of RNAi therapeutics, while new, innovative technologies are being devised due to the immense interest in RNAi. It is expected that depending on the therapeutic application and target tissue, different delivery strategies will ultimately be used in the clinic.

An RNAi therapeutic has to cross the cellular membrane barrier

In an unmodified form, siRNAs are negatively charged molecules, and to reach their site of action (e.g. the cytoplasm), they must overcome the equally negatively charged outer cell plasma membrane and the “grease-like” barrier of the cell membrane. It is therefore surprising that efficient delivery of unformulated siRNAs can be obtained for a few tissues, particularly mucosal epithelia. Still, for most purposes, delivery formulations are required to ensure cellular entry. Cationic liposomes and polymers are some of the more popular methods to overcome electrostatic repulsion by binding and neutralizing the negative charge of a number of siRNAs at once. Following incorporation of the siRNAs, the liposomes can favorably interact with the plasma membrane and deliver their siRNA cargo into the cytoplasm to harness the endogenous RNAi machinery and effect gene silencing.

Conjugation approaches have also shown promise in facilitating cellular entry of siRNAs. This may involve conjugating siRNAs to small peptides that can penetrate cellular membranes, or to simple small molecules such as a cholesterol moiety. Cholesterol-conjugation of siRNAs allowed Alnylam scientists to demonstrate for the first time functional in vivo delivery of an siRNA following systemic administration in mice. Cholesterol-conjugation has the added benefit of functioning as a carrier for the siRNA in the blood thereby improving its pharmacokinetic properties. This is of particular importance for the systemic delivery of RNAi therapeutics.