Nonviral delivery of synthetic siRNAs in vivo

Abstract

Sequence-specific gene silencing using small interfering RNA (siRNA) is a Nobel prize-winning technology that is now being evaluated in clinical trials as a potentially novel therapeutic strategy. This article provides an overview of the major pharmaceutical challenges facing siRNA therapeutics, focusing on the delivery strategies for synthetic siRNA duplexes in vivo, as this remains one of the most important issues to be resolved. This article also highlights the importance of understanding the genocompatibility/toxicogenomics of siRNA delivery reagents in terms of their impact on gene-silencing activity and specificity. Collectively, this information is essential for the selection of optimally acting siRNA delivery system combinations for the many proposed applications of RNA interference.

Figure 1. The pharmaceutical challenges to effective siRNA delivery and activity in vivo.

The challenges for effective siRNA delivery and activity in vivo range from the effective empirical or in silico design and selection of an effective siRNA sequence to its chemical modification or formulation with delivery vectors to improve biological stability and pharmacokinetics (steps i–iv). Optimally designed and formulated siRNA should then efficiently enter target cells and escape endosomal and other intracellular compartments to become highly bioavailable inside the cells so as to exert sequence-specific gene-silencing activity with no or minimal effects on nontargeted genes or inadvertent stimulation of the immune system (steps v–x) (see text for detailed explanation).

Figure 2. A schematic depiction of the structural components required of a targeted cationic complex or nanoparticle for siRNA delivery in vivo.

Ordered assembly of the complex or nanoparticle (~100 nm in size) typically involves the use of a biocompatible/genocompatible lipid or polymer core that enters tissues and cells readily. The siRNA can be either entrapped within the core, adsorbed onto the surface via ionic interactions, or covalently attached through the sense strand to one of the surface components. The particle can then be modified to bear PEG polymer chains that improve the pharmacokinetic and biodistribution behavior by prolonging half-lives in vivo. PEGylation also serves to improve the biocompatibility of the particles and, because of shielding of the negative charges, to decrease nonspecific interactions with extracellular matrix components. Targeting to a specific tissue or cell type can be facilitated by addition of a receptor ligand or antibody that may be attached to the surface or conjugated to the PEG. Following uptake by endocytosis, intracellular bioavailability can be enhanced through endosomal escape of siRNA by including a fusogenic lipid or pH-sensitive peptide in the complex or nanoparticle (see text for specific examples).