Influence of Cationic Lipid Composition on Gene Silencing Properties of Lipid Nanoparticle Formulations of siRNA in Antigen-Presenting Cells

Abstract

Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases.

Human macrophage and dendritic cell-specific silencing of high-mobility group protein B1 ameliorates sepsis in a humanized mouse model

“…D-glucan-encapsulated siRNA particles were reported as efficient oral-delivery vehicles that silenced genes in mouse macrophages in vitro and in vivo (25). An ionizable cationic lipid called DLinKC2DMA, which was originally shown to deliver siRNA to liver cells (26), was also reported to deliver siRNA to mouse macrophages and DCs (27). Similarly, a lipidoid nanoparticle called C12-200, also developed originally for liver-specific siRNA delivery (28), was recently shown to target monocytes, and silencing CCR2 with this reagent was shown to reduce a number of inflammatory conditions in mice (18).…”

The Niemann-Pick C1 Inhibitor NP3.47 Enhances Gene Silencing Potency of Lipid Nanoparticles Containing siRNA

“…22 The third area of interest concerns whether enhanced gene silencing potency observed in vitro in the presence of NP3.47 can be extended to in vivo situations. As noted elsewhere the gene silencing potency of LNP-siRNA systems for hepatocyte targets is extremely high, requiring doses of as little as 10 μg/kg body weight to achieve 50% gene silencing; 3,4 however, for other tissues dose levels of 1 mg/kg body weight or more are required, [23][24][25] limiting clinical applications. The addition of agents such as NP3.47 to enhance potency would appear an attractive option, however the problems associated with the likely toxicities of In this regard we have recently shown 26 that a hydrophobic prodrug derivative of dexamethasone, when associated with LNP systems is a 20-fold or more potent for suppressing immunostimulatory effects of encapsulated RNA or DNA as compared with free dexamethasone.…”

“…This is attributed to the avid accumulation of LNP systems by cells of the immune system such as macrophages and dendritic cells. 23 Thus, for specific tissues that avidly accumulate LNP systems, such as cells of the immune system, inclusion of a hydrophobic prodrug version of NP3.47 in the LNP may prove useful for increasing gene silencing potencies of LNPsiRNA systems. In summary, the results presented here show that the NPC1 inhibitor NP3.47 leads to enhanced gene silencing capabilities for LNP-siRNA systems in vitro.…”

The Role of micro RNAs in Breast Cancer Metastasis: Preclinical Validation and Potential Therapeutic Targets

“…Lipid Nanoparticles (LNP) are small lipoplexes that are broadly used for cell culture transfections. In the meanwhile, they were also tested for their suitability as in vivo transfectants (189,190). However, those compounds strongly accumulate in the liver and other filtering organs and are rather cell-type unspecific which makes them likely to cause side effects and toxicity issues (191).…”