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Cited by 206RNA Interference and Gene DeliveryNanoparticle-Based Drug DeliveryAdvancements in Transdermal Drug Delivery

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Inside out: optimization of lipid nanoparticle formulations for exterior complexation and in vivo delivery of saRNA

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Blakney, Anna K., McKay, Paul F., Yus, Bárbara Ibarzo, Aldon, Yoann, Shattock, Robin J.·2019·Gene Therapy
DOI 10.1038/s41434-019-0095-2PMC PMC6760535
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Abstract

Self-amplifying RNA (saRNA) is a promising biotherapeutic tool that has been used as a vaccine against both infectious diseases and cancer. saRNA has been shown to induce protein expression for up to 60 days and elicit immune responses with lower dosing than messenger RNA (mRNA). Because saRNA is a large (~9500 nt), negatively charged molecule, it requires a delivery vehicle for efficient cellular uptake and degradation protection. Lipid nanoparticles (LNPs) have been widely used for RNA formulations, where the prevailing paradigm is to encapsulate RNA within the particle, including the first FDA-approved small-interfering siRNA therapy. Here, we compared LNP formulations with cationic and ionizable lipids with saRNA either on the interior or exterior of the particle. We show that LNPs formulated with cationic lipids protect saRNA from RNAse degradation, even when it is adsorbed to the surface. Furthermore, cationic LNPs deliver saRNA equivalently to particles formulated with saRNA encapsulated in an ionizable lipid particle, both in vitro and in vivo. Finally, we show that cationic and ionizable LNP formulations induce equivalent antibodies against HIV-1 Env gp140 as a model antigen. These studies establish formulating saRNA on the surface of cationic LNPs as an alternative to the paradigm of encapsulating RNA.

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Crossref · 10.1038/s41434-019-0095-2

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Figures

Fig. 1 Characterization of saRNA lipid nanoparticle formulations. a Schematic of saRNA formulated on the interior or exterior of the lipid nanoparticles, with ionizable (C12-200) or cationic (DDA, DOTAP) complexing lipids. b Particle diameter (in nm) as determined by Nanoparticle tracking analysis (NTA) (bar graph) and their related polydispersity index (empty circles). c Surface charge of the LNPs as determined by zeta potential analysis measured on by the Zetasizer instrument. Bars represent means ± standard deviations for n = 3 for particle size and surface charge data
Fig. 1 Characterization of saRNA lipid nanoparticle formulations. a Schematic of saRNA formulated on the interior or exterior of the lipid nanoparticles, with ionizable (C12-200) or cationic (DDA, DOTAP) complexing lipids. b Particle diameter (in nm) as determined by Nanoparticle tracking analysis (NTA) (bar graph) and their related polydispersity index (empty circles). c Surface charge of the LNPs as determined by zeta potential analysis measured on by the Zetasizer instrument. Bars represent means ± standard deviations for n = 3 for particle size and surface charge dataCC BY · PubMed Central
Fig. 2 Transmission electron micrographs of LNP formulations. LNPs with saRNA on the interior (a–c) or exterior (d–f) of the particle, with C12-200 (a, d), DDA (b, e), and DOTAP (c, f) as the complexing lipid. Scale bar = 100 nm
Fig. 2 Transmission electron micrographs of LNP formulations. LNPs with saRNA on the interior (a–c) or exterior (d–f) of the particle, with C12-200 (a, d), DDA (b, e), and DOTAP (c, f) as the complexing lipid. Scale bar = 100 nmCC BY · PubMed Central
Fig. 3 Transfection efficiency of fLuc-encoding saRNA delivered on the interior or exterior of LNPs. saRNA was transfected under standard conditions without FCS ((−) FCS), in the presence of FCS ((+) 50% FCS), or in the presence of exogenous RNAse ((+) RNAse). Luciferase expression was evaluated 24 h after transfection and is expressed as relative light units (RLU). Bars represent means ± standard deviations for n = 3
Fig. 3 Transfection efficiency of fLuc-encoding saRNA delivered on the interior or exterior of LNPs. saRNA was transfected under standard conditions without FCS ((−) FCS), in the presence of FCS ((+) 50% FCS), or in the presence of exogenous RNAse ((+) RNAse). Luciferase expression was evaluated 24 h after transfection and is expressed as relative light units (RLU). Bars represent means ± standard deviations for n = 3CC BY · PubMed Central
Fig. 4 Effect of interior or exterior complexation on the protection of saRNA from RNAse degradation. a Formulations were subjected to RNAse, then purified by phenol–chloroform extraction, and analyzed by gel electrophoresis. Bars represent means ± standard deviations of the intensity of the nondegraded RNA band normalized to an equivalent sample that was not subjected to RNAse treatment for n = 2 samples. b Representative image of denaturing gel electrophoresis (Int: interior, Ext: exterior)
Fig. 4 Effect of interior or exterior complexation on the protection of saRNA from RNAse degradation. a Formulations were subjected to RNAse, then purified by phenol–chloroform extraction, and analyzed by gel electrophoresis. Bars represent means ± standard deviations of the intensity of the nondegraded RNA band normalized to an equivalent sample that was not subjected to RNAse treatment for n = 2 samples. b Representative image of denaturing gel electrophoresis (Int: interior, Ext: exterior)CC BY · PubMed Central
Fig. 5 In vivo luciferase expression of saRNA complexed to the interior or exterior of LNPs. a In vivo imaging (IVIS) visualization of mice injected intramuscularly with 5 μg of fLuc saRNA per leg and imaged 7 days after injection. b Quantification of IVIS luciferase expression with a line at the mean ± standard deviation for n = 5 mice (n = 10 legs) per group. Units are expressed as relative light units (RLU). Asterisk indicates significance applying an unpaired t-test with *p < 0.05 (p-values indicated); n.s., nonsignificant
Fig. 5 In vivo luciferase expression of saRNA complexed to the interior or exterior of LNPs. a In vivo imaging (IVIS) visualization of mice injected intramuscularly with 5 μg of fLuc saRNA per leg and imaged 7 days after injection. b Quantification of IVIS luciferase expression with a line at the mean ± standard deviation for n = 5 mice (n = 10 legs) per group. Units are expressed as relative light units (RLU). Asterisk indicates significance applying an unpaired t-test with *p < 0.05 (p-values indicated); n.s., nonsignificantCC BY · PubMed Central
Fig. 6 Antibody titers after immunization with HIV-1 Env gp140 saRNA complexed to the interior or exterior of LNPs. Bars represent means HIV-1 Env gp140-specific serum IgG antibody titer ± SEM, as determined by ELISA for n = 5 mice at each time point
Fig. 6 Antibody titers after immunization with HIV-1 Env gp140 saRNA complexed to the interior or exterior of LNPs. Bars represent means HIV-1 Env gp140-specific serum IgG antibody titer ± SEM, as determined by ELISA for n = 5 mice at each time pointCC BY · PubMed Central

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  1. Love KT, Mahon KP, Levins CG, Whitehead KA, Querbes W, Dorkin JR, et al. Lipid-like materials for low-dose, in vivo gene silencing. Proc Natl Acad Sci USA. 2010;107:1864–9. PubMed PMID: PMC2804742 · DOI · Paper page

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Metadata

Authors
Blakney, A. K., McKay, P. F., Yus, B. I., Aldon, Y., & Shattock, R. J.
Affiliations
Imperial College London (GB)
Corresponding author
Shattock, Robin J.
ORCID
Venue
Gene Therapy
Publisher
Springer Science and Business Media LLC
Published
2019-07-12
Volume
26
Issue
9
Pages
363-372
ISSN
  • 0969-7128 (ISSN-L)
  • 1476-5462
DOI
10.1038/s41434-019-0095-2
PMC
PMC6760535
OpenAlex
W2961919374
Cited by
206
References
37
Citations by year
2026: 19 · 2025: 25 · 2024: 30 · 2023: 38 · 2022: 37 · 2021: 32 · 2020: 23 · 2019: 2
FWCI
7.77
Type
journal article
Language
en
Open access
Licence
Locations
  • Gene Therapy (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion)
  • Spiral (Imperial College London) (submittedVersion, cc-by)
Funders
  • Whitaker Foundation
  • European Commission (794059, 681137)
  • RCUK | Engineering and Physical Sciences Research Council (EP/R013764/1)
  • EC | Horizon 2020 Framework Programme (681137)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmaceutical Science › Advancements in Transdermal Drug Delivery
Keywords
self-amplifying RNA, lipid nanoparticles, cationic lipids, ionizable lipids, surface complexation, vaccine adjuvants, RNA encapsulation
MeSH terms
Quaternary Ammonium Compounds, Animals, Cations, Fatty Acids, Monounsaturated, Female, Humans, Lipids, Mice, Inbred BALB C, Particle Size, Ribonucleases, RNA, Messenger, Transfection, HIV Antibodies, RNA Stability, Luciferases, Firefly, Mice, Nanoparticles, env Gene Products, Human Immunodeficiency Virus, HEK293 Cells

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