Stable nucleic acid lipid particles (SNALPs) are microscopic particles approximately 120 nanometers in diameter, smaller than the wavelengths of visible light. They have been used to deliver siRNAs therapeutically to mammals in vivo. In SNALPs, the siRNA is surrounded by a lipid bilayer containing a mixture of cationic and fusogenic lipids, coated with diffusible polyethylene glycol.
Introduction
RNA interference(RNAi) is a process that occurs naturally within the cytoplasm inhibiting gene expression at specific sequences. Regulation of gene expression through RNAi is possible by introducing small interfering RNAs(siRNAs), which effectively silence expression of a targeted gene. RNAi activates the RNA-induced silencing complex(RISC) containing siRNA, siRNA derived from cleaved dsRNA. The siRNA guides the RISC complex to a specific sequence on the mRNA that is cleaved by RISC and, consequently, silences those genes.
However, without modifications to the RNA backbone or inclusion of inverted bases at either end, siRNA instability in the plasma makes it extremely difficult to apply this technique in vivo. Pattern recognition receptors(PRRs), which can be grouped as endocytic PRRs or signaling PRRs, are expressed in all cells of the innate immune system. Signaling PRRs, in particular, include Toll-like receptors(TLRs) and are involved primarily with identifying pathogen-associated molecular patterns(PAMPs). For example, TLRs can recognize specific regions conserved in various pathogens, recognition stimulating an immune response with potentially devastating effects to the organism. In particular, TLR 3 recognizes both dsRNA characteristic of viral replication and siRNA, which is also double-stranded. In addition to this instability, another limitation of siRNA therapy concerns the inability to target a tissue with any specificity.
SNALPs, though, may provide the stability and specificity required for this mode of RNAi therapy to be effective. Consisting of a lipid bilayer, SNALPs are able to provide stability to siRNAs by protecting them from nucleases within the plasma that would degrade them. In addition, delivery of siRNAs is subject to endosomal trafficking, potentially exposing them to TLR3 and TLR7, and can lead to activation of interferons and proinflammatory cytokines. However, SNALPs allow siRNA uptake into the endosome without activating Toll-like receptors and consequently stimulating an impeding immune response, thus enabling siRNA escape from the endosome.
Development of SNALP delivery of siRNA
Downregulation of gene expression via siRNA has been an important research tool in in vitro studies. Susceptibility of siRNAs to nuclease degradation, though, makes use of them in vivo problematic. In 2005, researchers working with hepatitis B virus(HBV) in rodents, determined that certain modifications of the siRNA prevented degradation by nucleases within the plasma and lead to increased gene silencing compared to unmodified siRNA. Modifications to the sense and antisense strands were made differentially. With respect to both sense and antisense strands, 2'-OH was substituted with 2'-fluoro at all pyrimidine positions. In addition, sense strands were modified at all purine positions with deoxyribose, antisense strands modified with 2'-O-methyl at the same positions. The 5' and 3' ends of the sense strand were capped with abasic inverted repeats, while a phosphorothioate linkage was incorporated at the 3' end of the antisense strand.
Although this research demonstrated a potential RNAi therapy using modified siRNA, the 90% reduction in HBV DNA in rodents resulted from a 30 mg/kg dosage with frequent administration. Because this is not a viable dosing regime, this same group looked at the effects of encapsulating the siRNA in a PEGylated lipid bilayer, or SNALP. Specifically, the lipid bilayer facilitates uptake into the cell and subsequent release from the endosome, the PEGylated outer layer providing stability during formulation due to the resulting hydrophilicity of the exterior. According to this 2005 study, researchers obtained 90% reduction in HBV DNA with a 3 mg/kg/day dose of siRNA for three days, a dose substantially lower than the earlier study. In addition, in contrast to unmodified or modified and non-encapsulated siRNA, administration of SNALP-delivered siRNA resulted in no detectable levels of interferons, such as IFN-a, or inflammatory cytokines associated with immunostimulation. Even so, researchers acknowledged that more work was necessary in order to reach a feasible dose and dosing regime.