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. 2008 May;26(5):561-9.
doi: 10.1038/nbt1402. Epub 2008 Apr 27.

A combinatorial library of lipid-like materials for delivery of RNAi therapeutics

Akin Akinc  1 , Andreas Zumbuehl, Michael Goldberg, Elizaveta S Leshchiner, Valentina Busini, Naushad Hossain, Sergio A Bacallado, David N Nguyen, Jason Fuller, Rene Alvarez, Anna Borodovsky, Todd Borland, Rainer Constien, Antonin de Fougerolles, J Robert Dorkin, K Narayanannair Jayaprakash, Muthusamy Jayaraman, Matthias John, Victor Koteliansky, Muthiah Manoharan, Lubomir Nechev, June Qin, Timothy Racie, Denitza Raitcheva, Kallanthottathil G Rajeev, Dinah W Y Sah, Jürgen Soutschek, Ivanka Toudjarska, Hans-Peter Vornlocher, Tracy S Zimmermann, Robert Langer, Daniel G Anderson
Affiliations

A combinatorial library of lipid-like materials for delivery of RNAi therapeutics

Akin Akinc et al. Nat Biotechnol. 2008 May.

Abstract

The safe and effective delivery of RNA interference (RNAi) therapeutics remains an important challenge for clinical development. The diversity of current delivery materials remains limited, in part because of their slow, multi-step syntheses. Here we describe a new class of lipid-like delivery molecules, termed lipidoids, as delivery agents for RNAi therapeutics. Chemical methods were developed to allow the rapid synthesis of a large library of over 1,200 structurally diverse lipidoids. From this library, we identified lipidoids that facilitate high levels of specific silencing of endogenous gene transcripts when formulated with either double-stranded small interfering RNA (siRNA) or single-stranded antisense 2'-O-methyl (2'-OMe) oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids were evaluated in three animal models: mice, rats and nonhuman primates. The studies reported here suggest that these materials may have broad utility for both local and systemic delivery of RNA therapeutics.

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Figures

Figure 1
Figure 1
Synthesis of lipidoids. (a) Alkyl-acrylate, alkyl-acrylamide and amino molecules were used to synthesize a combinatorial library of lipidoids. (b) Synthesis occurs through the conjugate addition of amines to an acrylate or acrylamide. Depending on the number of addition sites in the amino monomer, lipidoids can be formed with anywhere from 1 to 7 tails. Amino groups in the lipidoid can be quaternized by treatment with methyl iodide. For ease of nomenclature, lipidoids are named as follows: (amine number)(acrylate or acrylamide name)-(number of tails)(“+” if quaternized).
Figure 2
Figure 2
In vitro screening of lipidoids for siRNA delivery. (a) HeLa cells expressing both firefly and Renilla luciferase were treated with firefly luciferase targeting siRNA-lipidoid complexes. The average percent reduction in firefly luciferase activity after treatment with siRNA-lipidoid complexes at a 5:1 (wt/wt) ratio in quadruplicate is shown. For ease of analysis, data are grouped as follows: no test, 0–20% knockdown, 20–40%, 40–60%, 60–80%, 80–100%. (b) Optimized in vitro knockdown by lipidoids in HeLa cells. Lipidoids were optimized for delivery using four lipidoid/siRNA ratios: 5:1, 10:1, 15:1 and 20:1. Materials were tested in quadruplicate. Data are presented for the optimal siRNA/lipidoid wt/wt ratio for each lipidoid, including s.d. This data set includes only lipidoids with no significant cytoxicity, as assessed by no significant change in Renilla luciferase expression relative to untreated cells. (c–e) Dose response of silencing in HeLa (c), HepG2 (d) and primary macrophage cultures (e). Data were generated in quadruplicate, as a function of siRNA molarity, at ratio of 5:1 wt/wt. s.d. is shown. Day 5 GFP-expressing bone marrow-derived macrophage cultures were incubated with siRNA-lipidoid complexes composed of the indicated lipidoids or commercial transfection reagents (Lipofectamine 2000 and Lipofectamine RNAiMAX) and siGFP or siCD45 for 6 h. GFP expression was quantified by flow cytometry. Silencing is expressed as the percentage of untreated cultures performed in parallel.
Figure 3
Figure 3
In vivo delivery of siRNA to liver in rodents. (a) Mice (n = 3) received two daily i.v. injections of different lipidoid formulations of siRNA at a dose of 2.5 mg/kg. Factor VII protein levels were quantified 24 h after the second administration. (b) Simultaneous silencing of two genes in vivo in mice. Mice (n = 3) received a single i.v. bolus injection of a 98N12-5(1)-formulated 1:1 (wt/wt) mixture of siFVII and siApoB at 10, 6 or 4 mg/kg (5, 3 or 2 mg/kg of each siRNA). For comparison, control animals received PBS or lipidoid-formulated siFVII alone or lipidoid-formulated siApoB alone at 5, 3 or 2 mg/kg. Forty-eight hours after administration animals were killed and livers were harvested. Liver mRNA levels of Factor VII or ApoB (normalized to GAPDH) were determined by branched DNA assay. (c–e) Rats (n = 4) were injected with lipidoid-formulated siRNA at 1.25, 2.5, 5 and 10 mg/kg. Animals were bled and killed 48 h after administration; shown are liver mRNA levels (c), serum Factor VII protein levels (d) and prothrombin time (e). (f) Durability of silencing in rats. Rats (n = 5) received a single i.v. administration of lipidoid-formulated siRNA at 5 mg/kg. Animals were bled at various time points after administration and serum Factor VII protein levels were quantified. Data points represent group mean ± s.d. Data points marked with asterisks are statistically significant relative to control treated groups (*, P < 0.05; **, P < 0.005; ***, P < 0.001; t-test, single-tailed).
Figure 4
Figure 4
In vivo delivery of siRNA to lung and peritoneal macrophages and delivery of anti-miRs to liver. (a) Inhibition of RSV/A2 in BALB/c mouse lungs. Mice (n = 5) were intranasally administered naked siRNA or lipidoid-siRNA (siRSV or mm-siRSV) formulation at a dose of 2 mg/kg. Lungs were harvested at day 4 post-infection and assayed by immunostaining plaque assay. (b) Inhibition of CD45 protein in thioglycollate elicited mouse peritoneal macrophages. Mice (n = 4) were injected with thioglycollate (i.p.) 3 d before treatment with 10 mg/kg of lipodoid-formulated siCD45 or siGFP administered via i.p. injection. We analyzed 4 d post administration CD45 expression on macrophages by flow cytometry. Macrophage cells were gated and median fluorescence intensity of the CD45 staining is plotted. MFI, mean fluorescence intensity. (c) Lipidoid delivery of anti-miR122 in vivo. Nuclease protection assay and miRNA detection. Liver RNA samples of three representative animals per treatment group are shown. The U6 signal serves as RNA input control. Animals treated with antagomir122 show a marked decrease of miR-122 signal (lanes 7–9). Even further reduction of miR-122 signal is observed for lipidoid-formulated anti-miR122–treated animals (lanes 10–12). The mismatched control antagomir and anti-miR had little effect on miR-122 signals (lanes 4–6 and 13–15). (d) Derepression of miR-122 target genes after miR-122 inhibition in mice. Mice (n = 6) received i.v. injections of either lipidoid-anti-miR122 (black bars) or lipidoid-mm-anti-miR122 (gray bars) at a daily dose of 5 mg/kg for 3 consecutive days. Twenty-four hours after the last injection, expression levels of seven reported miR-122 target genes were analyzed. Data points represent group mean ± s.d. Data points marked with asterisks are statistically significant relative to control treated groups (* P < 0.05, ** P < 0.005, *** P < 0.001; t-test, single-tailed).
Figure 5
Figure 5
In vivo delivery in primates. (a) Extent and duration of serum ApoB-100 protein reduction in cynomolgus monkeys after single bolus i.v. administration of lipidoid-formulated siRNA For all groups except saline control; n = 6 for data points up to and including 2 d and n = 3 for data points after 2 d. For saline control, n = 4 for data points up to and including 2 d, and n = 2 for data points beyond 2 d. Data points represent group mean ± s.d. No error bars shown for saline group where n = 2. (b–d) Animals (n = 3) were treated with either formulated control siRNA at 2.5 mg/kg, formulated Apob targeting siRNA at 2.5 mg/kg, or formulated Apob targeting siRNA at 6.25 mg/kg. (b) Liver Apob mRNA levels normalized to GAPDH mRNA 48 h after administration. (c) Serum ApoB-100 protein reduction at 12, 24 and 48 h after administration as percentage of predose levels. (d) LDL-C and high-density lipoprotein (HDL) cholesterol levels at 48 h after administration, normalized to predose levels. Data points represent group mean ± s.d. Data points marked with asterisks are statistically significant relative to control treated groups (* P < 0.05, ** P < 0.005, *** P < 0.001; t-test, single-tailed).
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