Rational design of cationic lipids for siRNA delivery

Journal name:
Nature Biotechnology
Volume:
28,
Pages:
172–176
Year published:
DOI:
doi:10.1038/nbt.1602
Received
Accepted
Published online

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.

At a glance

Figures

  1. Proposed mechanism of action for membrane disruptive effects of cationic lipids and structural diagram of DLinDMA divided into headgroup, linker and hydrocarbon chain domains.
    Figure 1: Proposed mechanism of action for membrane disruptive effects of cationic lipids and structural diagram of DLinDMA divided into headgroup, linker and hydrocarbon chain domains.

    In isolation, cationic lipids and endosomal membrane anionic lipids such as phosphatidylserine adopt a cylindrical molecular shape, which is compatible with packing in a bilayer configuration. However, when cationic and anionic lipids are mixed together, they combine to form ion pairs where the cross-sectional area of the combined headgroup is less than that of the sum of individual headgroup areas in isolation. The ion pair therefore adopts a molecular 'cone' shape, which promotes the formation of inverted, nonbilayer phases such as the hexagonal HII phase illustrated. Inverted phases do not support bilayer structure and are associated with membrane fusion and membrane disruption9, 21.

  2. In vivo evaluation of novel cationic lipids.
    Figure 2: In vivo evaluation of novel cationic lipids.

    (a) Silencing activity of DLinDAP (), DLinDMA (), DLin-K-DMA () and DLin-KC2-DMA (•) screening formulations in the mouse Factor VII model. All LNP-siRNA systems were prepared using the preformed vesicle (PFV) method and were composed of ionizable cationic lipid, DSPC, cholesterol and PEG-lipid (40:10:40:10 mol/mol) with a Factor VII siRNA/total lipid ratio of ~0.05 (wt/wt). Data points are expressed as a percentage of PBS control animals and represent group mean (n = 5) ± s.d., and all formulations were compared within the same study. (b) Influence of headgroup extensions on the activity of DLin-K-DMA. DLin-K-DMA () had additional methylene groups added between the DMA headgroup and the ketal ring linker to generate DLin-KC2-DMA (•), DLin-KC3-DMA () and DLin-KC4-DMA (). The activity of PFV formulations of each lipid was assessed in the mouse Factor VII model. Data points are expressed as a percentage of PBS control animals and represent group mean (n = 4) ± s.d. (c) Chemical structures of novel cationic lipids.

  3. Efficacy of KC2-SNALP in rodents and nonhuman primates.
    Figure 3: Efficacy of KC2-SNALP in rodents and nonhuman primates.

    (a) Improved efficacy of KC2-SNALP relative to the initial screening formulation tested in mice. The in vivo efficacy of KC2-SNALP () was compared to that of the unoptimized DLin-KC2-DMA screening (that is, PFV) formulation (•) in the mouse Factor VII model. Data points are expressed as a percentage of PBS control animals and represent group mean (n = 5) ± s.d. (b) Efficacy of KC2-SNALP in nonhuman primates. Cynomolgus monkeys (n = 3 per group) received a total dose of either 0.03, 0.1, 0.3 or 1 mg/kg siTTR, or 1 mg/kg siApoB formulated in KC2-SNALP or PBS as 15-min intravenous infusions (5 ml/kg) through the cephalic vein. Animals were euthanized 48 h after administration. TTR mRNA levels relative to GAPDH mRNA levels were determined in liver samples. Data points represent group mean ± s.d. *, P < 0.05; **, P < 0.005.

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Author information

  1. These authors contributed equally to this work.

    • Sean C Semple &
    • Akin Akinc

Affiliations

  1. Tekmira Pharmaceuticals, Burnaby, British Columbia, Canada.

    • Sean C Semple,
    • Jianxin Chen,
    • Ammen P Sandhu,
    • Barbara L Mui,
    • Connie K Cho,
    • Derrick Stebbing,
    • Erin J Crosley,
    • Ed Yaworski,
    • Kieu Lam,
    • Lloyd B Jeffs,
    • Merete L Eisenhardt,
    • Sandra K Klimuk,
    • Ying K Tam,
    • Ian MacLachlan,
    • Thomas D Madden &
    • Michael J Hope
  2. Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA.

    • Akin Akinc,
    • Dinah W Y Sah,
    • J Robert Dorkin,
    • June Qin,
    • Kallanthottathil G Rajeev,
    • Lubomir Nechev,
    • Muthusamy Jayaraman,
    • Martin A Maier,
    • Michael J Weinstein,
    • Qingmin Chen,
    • Rene Alvarez,
    • Scott A Barros,
    • Soma De,
    • Todd Borland,
    • Verbena Kosovrasti,
    • William L Cantley,
    • Muthiah Manoharan,
    • Mark A Tracy &
    • Antonin de Fougerolles
  3. Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.

    • Ismail M Hafez,
    • Kim F Wong,
    • Mikameh Kazem &
    • Pieter R Cullis
  4. Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada.

    • Masuna Srinivasulu &
    • Marco A Ciufolini
  5. Present address: Alcana Technologies, Vancouver, British Columbia, Canada.

    • Jianxin Chen,
    • Barbara L Mui,
    • Ying K Tam,
    • Thomas D Madden &
    • Michael J Hope

Contributions

J.C., M.A.C., P.R.C., T.D.M., M.J.H. and K.F.W. designed and advised on novel lipids. J.C., K.F.W. and M.S. synthesized novel lipids. M.J.H., T.D.M., J.C., K.F.W., M.M., K.G.R., M.A.M., M.T. and M.J. analyzed and interpreted lipid data. T.D.M., M.J.H. and M.A.T. co-directed novel lipid synthesis and screening program. S.C.S. designed and directed rodent in vivo studies. S.C.S., S.K.K., B.L.M., K.L., M.L.E., M.K., A.P.S., Y.K.T., S.A.B., W.L.C., M.J.W. and E.J.C. generated rodent in vivo data, including Factor VII and tolerability analyses. L.N., V.K., T.B., R.A., Q.C. and D.W.Y.S. developed novel siRNAs targeting TTR. R.A. and A.A. designed and directed NHP in vivo studies. S.C.S., S.K.K., A.A., B.L.M., I.M., A.P.S., Y.K.T., R.A., T.B., D.W. Y. S., S.A.B., J.Q., J.R.D. and A.d.F. analyzed and interpreted in vivo data. B.L.M., K.L., A.P.S., S.K.K., S.C.S. and E.J.C. generated and characterized preformed vesicle formulations with novel lipids. D.S. and C.K.C. developed methods and designed and conducted HPLC lipid analyses of preformed vesicle formulations. E.Y. and L.B.J. prepared SNALP formulations. P.R.C. directed biophysical studies and advised on methods. A.P.S., I.M.H., S.D. and K.W. performed biophysical characterization studies (pKa, NMR, differential scanning calorimetric) of novel lipids and formulations. M.J.H., P.R.C., T.D.M., A.P.S., I.M.H. and K.F.W. analyzed biophysical data. S.C.S., M.J.H., A.A. and P.R.C. co-wrote the manuscript. T.D.M., M.M., M.A.M., M.A.T. and A.D.F. reviewed and edited the manuscript. S.C.S., M.J.H., A.A., P.R.C., I.M. and A.D.F. were responsible for approval of the final draft.

Competing financial interests

Authors are employees of Alnylam, Tekmira, or Alcana or receive funding from Alnylam.

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  1. Supplementary Text and Figures (348 KB)

    Supplementary Fig. 1, Supplementary Tables 1–4 and Supplementary Syntheses 1 and 2

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