We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.

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Background DNA vaccines offer unique potential for generating protective and therapeutic immunity against infectious and malignant diseases. Unfortunately, rapid degradation and poor cellular uptake has significantly limited the efficacy of 'naked' plasmid DNA vaccines. We have previously described stabilized plasmid lipid particles (SPLP) as effective nonviral gene delivery vehicles for the transfection of tumours at distal sites following intravenous administration. Based on their low toxicity and favourable transfection profile following systemic administration, we investigate SPLP as gene delivery vehicles for the generation of a systemically administered genetic vaccine.

Although it is well documented that the immunological activity of cytosine-guanine (CpG) motifs is abrogated by 5' methylation of the cytosine residue, encapsulation within stabilized lipid nanoparticles endows these methylated cytosine-guanine- (mCpG-) containing oligonucleotides (ODNs) with potent immunostimulatory activity in murine animal models. Surprisingly, not only do liposomal nanoparticulate (LN) mCpG ODN possess immunostimulatory activity, their potency is found to be equivalent and often greater than the equivalent unmethylated form, as judged by a number of ex vivo innate and adaptive immune parameters and anti-tumor efficacy in murine models. Preliminary data indicate that both methylated and unmethylated CpG ODN act through a common receptor signaling pathway, specifically via toll-like receptor (TLR) 9, based on observations of up-regulated TLR9 expression, induction of nitric oxide and dependence on endosomal maturation. This is confirmed in TLR9 knockout animals which show no immunostimulatory activity following treatment with LN-mCpG ODN. These data, therefore, indicate that the mCpG DNA is fully competent to interact with TLR9 to initiate potent immune responses. Furthermore, this work implicates an as yet unidentified mechanism upstream of TLR9 which regulates the relative activities of free methylated versus unmethylated CpG ODN that is effectively bypassed by particulate delivery of CpG ODN.