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Cited by 103RNA Interference and Gene DeliveryNanoparticle-Based Drug DeliveryAdvancements in Transdermal Drug Delivery

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Efficient Delivery of Antisense Oligonucleotides Using Bioreducible Lipid Nanoparticles In Vitro and In Vivo

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Yang, Liu, Ma, Feihe, Liu, Fang, Chen, Jinjin, Zhao, Xuewei, Xu, Qiaobing·2020·Molecular Therapy - Nucleic Acids
DOI 10.1016/j.omtn.2020.01.018PMC PMC7036716
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Abstract

The efficient delivery of antisense oligonucleotides (ASOs) to the targeted cells and organs remains a challenge, in particular, in vivo . Here, we investigated the ability of a library of biodegradable lipid nanoparticles (LNPs) in delivering ASO to both cultured human cells and animal models. We first identified three top-performing lipids through in vitro screening using GFP-expressing HEK293 cells. Next, we explored these three candidates for delivering ASO to target proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA in mice. We found that lipid 306-O12B-3 showed efficiency with the median effective dose (ED 50 ) as low as 0.034 mg·kg -1 , which is a notable improvement over the efficiency reported in the literature. No liver or kidney toxicity was observed with a dose up to 5 mg·kg -1 of this ASO/LNP formulation. The biodegradable LNPs are efficient and safe in the delivery of ASO and pave the way for clinical translation.

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Graphical abstract CC BY-NC-ND · PubMed Central
Figure 1 Design of Bioreducible Lipids for Effective ASO Delivery (A) Schematic illustration of the mechanism of LNPs for ASO delivery both in vitro and in vivo. For in vitro study, cultured HEK cell lines stably expressing GFP were treated with the LNPs packing with ASO. The ASO sequences were able to target and silence GFP mRNA, followed by the fade in GFP intensity. On the other hand, the optimal lipids screened from in vitro study were applied to PCSK9 mRNA knockdown in vivo. BALB/c mice were intravenously administrated with ASO-loaded LNPs. Once the ASO/LNP complexes arrived at hepatocytes, the LNPs were destabilized and degraded at the reductive intracellular environments, triggering the release of ASO. The PCSK9 targeting ASO then specifically hybridized with mRNA by Watson-Crick base pairing, followed by the nonspecific activation of endonuclease RNase H, which cleaved the RNA strand only in the form of a DNA-RNA hybrid. This mechanism led to the degradation of mRNA and the halt of PCSK9 protein translation. (B) Synthetic route (I) and the chemical structure of lipid 306-O12B-3 (II). (C) The chemical structures of amine head (R) groups and acrylate tails (R′).
Figure 1 Design of Bioreducible Lipids for Effective ASO Delivery (A) Schematic illustration of the mechanism of LNPs for ASO delivery both in vitro and in vivo. For in vitro study, cultured HEK cell lines stably expressing GFP were treated with the LNPs packing with ASO. The ASO sequences were able to target and silence GFP mRNA, followed by the fade in GFP intensity. On the other hand, the optimal lipids screened from in vitro study were applied to PCSK9 mRNA knockdown in vivo. BALB/c mice were intravenously administrated with ASO-loaded LNPs. Once the ASO/LNP complexes arrived at hepatocytes, the LNPs were destabilized and degraded at the reductive intracellular environments, triggering the release of ASO. The PCSK9 targeting ASO then specifically hybridized with mRNA by Watson-Crick base pairing, followed by the nonspecific activation of endonuclease RNase H, which cleaved the RNA strand only in the form of a DNA-RNA hybrid. This mechanism led to the degradation of mRNA and the halt of PCSK9 protein translation. (B) Synthetic route (I) and the chemical structure of lipid 306-O12B-3 (II). (C) The chemical structures of amine head (R) groups and acrylate tails (R′).CC BY-NC-ND · PubMed Central
Figure 2 In vitro Screening of Lipids for ASO Delivery (A) HEK cells stably expressing GFP were treated with or without ASO/LNP complexes. ASO can specifically target GFP mRNA sequences (GFP-ASO). With fixed acrylate tail length at O16B, lipids of ten different heads in two to four tail numbers were screened. The LNPs were mixed with GFP-ASO at the ratio of 15/1 (w/w); *p < 0.01 versus all other samples. (B) The three tail numbers of lipids were further investigated in different tail lengths (O12B-O18B) with top-head groups of 113 and 306; *p < 0.01 versus all other samples. (C) Optimization of the N/P ratio of the three best-performing lipids 113-O14B-3, 113-O16B-3, and 306-O12B-3. The ASO/LNP complexes were prepared by mixing GFP-ASO with fixed concentration at 0.1 μg/mL, and differing LNP concentrations ranged from 0.2 μg/mL to 2 μg/mL; *p < 0.01 versus all other samples at the same lipid group. (D) The comparison of the ASO transfection efficiency mediated by bioreducible lipids or LPF 2000. The GFP-HEK cells were treated with ASO/LNP complexes with GFP-ASO concentration increasing from 0.3 to 30 nM, with the fixed N/P ratio of 15/1. (E) The cell viability of GFP-HEK cells treated with bioreducible LNPs or LPF 2000 in a dose-dependent manner; same conditions as (D). For all negative control groups, GFP-HEK cells were treated with 16.7 nM of formulated, scrambled GFP-ASO; naked GFP-ASO; or PBS; *p < 0.01 versus all other samples at the same concentration group. For all of the in vitro experiments, the LNPs were fabricated by directly dissolving in sodium acetate buffer (25 mM, pH 5.2), followed by a brief sonication and dialysis. The GFP intensity was then analyzed by flow cytometry after 16 h of postdelivery. Data are presented as mean ± SD (n ≥ 3).
Figure 2 In vitro Screening of Lipids for ASO Delivery (A) HEK cells stably expressing GFP were treated with or without ASO/LNP complexes. ASO can specifically target GFP mRNA sequences (GFP-ASO). With fixed acrylate tail length at O16B, lipids of ten different heads in two to four tail numbers were screened. The LNPs were mixed with GFP-ASO at the ratio of 15/1 (w/w); *p < 0.01 versus all other samples. (B) The three tail numbers of lipids were further investigated in different tail lengths (O12B-O18B) with top-head groups of 113 and 306; *p < 0.01 versus all other samples. (C) Optimization of the N/P ratio of the three best-performing lipids 113-O14B-3, 113-O16B-3, and 306-O12B-3. The ASO/LNP complexes were prepared by mixing GFP-ASO with fixed concentration at 0.1 μg/mL, and differing LNP concentrations ranged from 0.2 μg/mL to 2 μg/mL; *p < 0.01 versus all other samples at the same lipid group. (D) The comparison of the ASO transfection efficiency mediated by bioreducible lipids or LPF 2000. The GFP-HEK cells were treated with ASO/LNP complexes with GFP-ASO concentration increasing from 0.3 to 30 nM, with the fixed N/P ratio of 15/1. (E) The cell viability of GFP-HEK cells treated with bioreducible LNPs or LPF 2000 in a dose-dependent manner; same conditions as (D). For all negative control groups, GFP-HEK cells were treated with 16.7 nM of formulated, scrambled GFP-ASO; naked GFP-ASO; or PBS; *p < 0.01 versus all other samples at the same concentration group. For all of the in vitro experiments, the LNPs were fabricated by directly dissolving in sodium acetate buffer (25 mM, pH 5.2), followed by a brief sonication and dialysis. The GFP intensity was then analyzed by flow cytometry after 16 h of postdelivery. Data are presented as mean ± SD (n ≥ 3).CC BY-NC-ND · PubMed Central
Figure 3 Biodistribution of ASO/LNP Complexes in Major Organs (Brain, Heart, Lung, Liver, Spleen, and Kidney) The three best-performing lipids, 113-O14B-3, 113-O16B-3, and 306-O12B-3, chosen from the in vitro screening studies were formulated in the following formulation: lipids/cholesterol/DOPE/DSPE-PEG2000 at the weight ratio 16/4/1/1. The Alexa 750-labeled ASOs were mixed with formulated LNPs at the weight ratio of 1/15, followed by the intravenous injection to mice. The results showed that all of the ASO-loaded LNPs exhibited most fluorescence signals in the liver, indicating the complexes mainly accumulating in this organ. Also, the ASO-Alexa 750s delivered by 306-O12B-3 exhibited the strongest fluorescence intensity in the liver.
Figure 3 Biodistribution of ASO/LNP Complexes in Major Organs (Brain, Heart, Lung, Liver, Spleen, and Kidney) The three best-performing lipids, 113-O14B-3, 113-O16B-3, and 306-O12B-3, chosen from the in vitro screening studies were formulated in the following formulation: lipids/cholesterol/DOPE/DSPE-PEG2000 at the weight ratio 16/4/1/1. The Alexa 750-labeled ASOs were mixed with formulated LNPs at the weight ratio of 1/15, followed by the intravenous injection to mice. The results showed that all of the ASO-loaded LNPs exhibited most fluorescence signals in the liver, indicating the complexes mainly accumulating in this organ. Also, the ASO-Alexa 750s delivered by 306-O12B-3 exhibited the strongest fluorescence intensity in the liver.CC BY-NC-ND · PubMed Central
Figure 4 In Vivo Evaluation of the Three Best-Performing Lipids (113-O14B-3, 113-O16B-3, and 306-O12B-3) for PCSK9-ASO Delivery The “#” represents the ED50 (mg·kg-1) for different lipids in PCSK9-ASO delivery in vivo. All of the LNPs were composed of cationic lipid, cholesterol, DOPE, and DSPE-PEG2000 (16/4/1/1 w/w) with a PCSK9-ASO-to-lipid ratio of 1/15 (w/w). Animals were treated with single-dose intravenous administration at varying ASO doses (mg·kg-1). Mice were sacrificed after 72 h of postinjection. PCSK9 mRNA levels relative to β-actin mRNA levels were determined in liver samples. Data points are expressed as a percentage of saline control animals and represent group mean ± SD (n = 3).
Figure 4 In Vivo Evaluation of the Three Best-Performing Lipids (113-O14B-3, 113-O16B-3, and 306-O12B-3) for PCSK9-ASO Delivery The “#” represents the ED50 (mg·kg-1) for different lipids in PCSK9-ASO delivery in vivo. All of the LNPs were composed of cationic lipid, cholesterol, DOPE, and DSPE-PEG2000 (16/4/1/1 w/w) with a PCSK9-ASO-to-lipid ratio of 1/15 (w/w). Animals were treated with single-dose intravenous administration at varying ASO doses (mg·kg-1). Mice were sacrificed after 72 h of postinjection. PCSK9 mRNA levels relative to β-actin mRNA levels were determined in liver samples. Data points are expressed as a percentage of saline control animals and represent group mean ± SD (n = 3).CC BY-NC-ND · PubMed Central
Figure 5 Improved Efficacy of the Optimized Formulation for the Lipid 306-O12B-3 Compared to the Initial Formulation in PCSK9-ASO Delivery In Vivo The “#” represents the ED50 (mg·kg-1) for different lipids in PCSK9-ASO delivery in vivo. (A) The in vivo PCSK9 mRNA silencing level mediated by the optimized formulation (cationic lipid/cholesterol/DOPE/DSPE-PEG2000 at the ratio of 16/4/4/1, w/w) was compared to that of the initial screening formulation (16/4/1/1, w/w). (B) The optimized formulation evaluated by the serum PCSK9 protein level. (C) The optimized formulation assessed by the serum total cholesterol level. The lipid and PCSK9-ASO were combined at a ratio of 15/1 (w/w); *p < 0.01 or **p < 0.01 versus all other samples at the same dose group. Animals were treated with single-dose intravenous administration at varying ASO dose levels. The control group was injected with saline. Mice were sacrificed after 72 h of postinjection. PCSK9 mRNA levels relative to β-actin mRNA levels were determined in liver samples. Data points are expressed as a percentage of saline control animals and represent group mean ± SD (n = 3).
Figure 5 Improved Efficacy of the Optimized Formulation for the Lipid 306-O12B-3 Compared to the Initial Formulation in PCSK9-ASO Delivery In Vivo The “#” represents the ED50 (mg·kg-1) for different lipids in PCSK9-ASO delivery in vivo. (A) The in vivo PCSK9 mRNA silencing level mediated by the optimized formulation (cationic lipid/cholesterol/DOPE/DSPE-PEG2000 at the ratio of 16/4/4/1, w/w) was compared to that of the initial screening formulation (16/4/1/1, w/w). (B) The optimized formulation evaluated by the serum PCSK9 protein level. (C) The optimized formulation assessed by the serum total cholesterol level. The lipid and PCSK9-ASO were combined at a ratio of 15/1 (w/w); *p < 0.01 or **p < 0.01 versus all other samples at the same dose group. Animals were treated with single-dose intravenous administration at varying ASO dose levels. The control group was injected with saline. Mice were sacrificed after 72 h of postinjection. PCSK9 mRNA levels relative to β-actin mRNA levels were determined in liver samples. Data points are expressed as a percentage of saline control animals and represent group mean ± SD (n = 3).CC BY-NC-ND · PubMed Central

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Metadata

Authors
Yang, L., Ma, F., Liu, F., Chen, J., Zhao, X., & Xu, Q.
Affiliations
Tufts University (US)
Corresponding author
Xu, Qiaobing
ORCID
Venue
Molecular Therapy - Nucleic Acids
Publisher
Elsevier BV
Published
2020-03
Volume
19
Pages
1357-1367
ISSN
2162-2531 (ISSN-L)
DOI
10.1016/j.omtn.2020.01.018
PMC
PMC7036716
OpenAlex
W3002543944
Cited by
103
References
42
Citations by year
2026: 14 · 2025: 16 · 2024: 21 · 2023: 18 · 2022: 15 · 2021: 12 · 2020: 7
FWCI
4.22
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Therapy — Nucleic Acids (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Institutes of Health (R01 EB027170-01, 1R21EB024041-01)
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
antisense oligonucleotides, lipid nanoparticles, biodegradable lipids, nucleic acid delivery, PCSK9, lipid screening, in vivo delivery, toxicity, gene silencing

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