Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo

Abstract

Special (lipid) delivery: The role of the ionizable lipid pK(a) in the in vivo delivery of siRNA by lipid nanoparticles has been studied with a large number of head group modifications to the lipids. A tight correlation between the lipid pK(a) value and silencing of the mouse FVII gene (FVII ED(50) ) was found, with an optimal pK(a) range of 6.2-6.5. The most potent cationic lipid from this study has ED(50) levels around 0.005 mg kg(-1) in mice and less than 0.03 mg kg(-1) in non-human primates.

Figure 1

2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA, 1). No difference in activity between enantiomers (4, 5) or the racemic mixture was observed (Table 1).

Figure 2

Determination of pK_a by in situ TNS fluorescence titration. The four amino lipids shown are examples illustrating the full pK_a range evaluated. Sigmoidal best-fit analyses were applied with pK_a defined as the pH at half-maximal fluorescence intensity. A) 14, pK_a=4.17; B) 52, pK_a=5.73; C) 19, pK_a=6.95; D) 48, pK_a=8.12 (see Supporting Information page 40 for details).

Figure 3

Plot of in vivo hepatic gene silencing activity vs. pK_a in mice. The 56 amino lipids were formulated in LNPs and subjected to an ED₅₀ analysis (described in Supporting Information, page 39) and plotted against their pK_a. For 15 lipids the ED₅₀ dose was not achieved, these are indicated by the open diamonds representing the highest dose tested for that lipid. For the remaining lipids (closed diamonds), a polynomial best-fit curve highlights the most active compounds, which exhibit an optimal pK_a between 6.2 and 6.5. Each data point is derived from a four-dose response curve with groups of n=4 mice per dose.

Figure 4

(6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (16, Table 1).

Figure 5

Plot of hepatic gene silencing activity in mice, pK_a and calculated protonated/unprotonated amino lipid (% mole fractions) for the set of novel lipids between pK_a 4 and 8.5. Each FVII ED₅₀ data point (blue) is derived from a four-dose response (n=4 mice per dose level). Open diamonds represent lipids for which the ED₅₀ was not achieved and are shown at the highest dose tested. The mole % of each amino lipid estimated to carry a positive charge in blood, assuming pH 7.4, is shown in red and the mole % amino lipid estimated to be uncharged in acidified endosomes at pH 5.5 is shown in green.

Figure 6

Lipid mixing supports pK_a value as the dominant factor in determining in vivo hepatic gene-silencing activity for a given set of amino lipids. Mixtures of amino lipids 15, 16, and 17 were made to incrementally shift the average surface pK_a value of the resulting LNP between 5.64 and 6.93. Numbers in parenthesis indicate the amount of each lipid in the mixture to make up the 40 mole % amino lipid component of the LNP. Bars represent group mean (n=4)±s.d.

Figure 7

Efficacy of siRNA-LNP containing amino lipid 16 in mice and non-human primates. An optimized formulation composition containing 50 mole % of 16 was employed in this study (see text and Supporting Information page 41 for details). Inset: residual FVII activity in mouse serum relative to a saline control group with a median effective siRNA dose (ED₅₀) of about 0.005 mg kg⁻¹; data points represent group mean (n=5)±s.d. Cynomolgus monkeys were administered siRNA targeting the TTR gene at doses of 0.03, 0.1 and 0.3 mg kg⁻¹ (bar graph). Data represent group mean (n=3)±s.d., expressed as TTR mRNA relative to GAPDH mRNA levels determined in liver samples, with the ED₅₀ estimated to be <0.03 mg kg⁻¹.