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• W3002543944 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 (ED50) 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. 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 (ED50) 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. Antisense oligonucleotides (ASOs) are single-strand DNAs serving as effective drugs to reduce the messenger RNA level in cells. ASOs are usually 16–25 bases long and are designed to hybridize with mRNA through Watson-Crick base-pairing specificity.1Raouane M. Desmaële D. Urbinati G. Massaad-Massade L. Couvreur P. Lipid conjugated oligonucleotides: a useful strategy for delivery.Bioconjug. Chem. 2012; 23: 1091-1104Crossref PubMed Scopus (114) Google Scholar, 2Crooke S.T. Molecular Mechanisms of Antisense Oligonucleotides.Nucleic Acid Ther. 2017; 27: 70-77Crossref PubMed Scopus (188) Google Scholar, 3Shen X. Corey D.R. Chemistry, mechanism and clinical status of antisense oligonucleotides and duplex RNAs.Nucleic Acids Res. 2018; 46: 1584-1600Crossref PubMed Scopus (374) Google Scholar, 4Crooke S.T. Molecular mechanisms of action of antisense drugs.Biochim. Biophys. Acta. 1999; 1489: 31-44Crossref PubMed Scopus (350) Google Scholar This DNA-RNA hybrid recruits and activates the endonuclease RNase H, which cleaves the RNA strand, leading to the degradation of the mRNA and halting protein translation.5Nakamura H. Oda Y. Iwai S. Inoue H. Ohtsuka E. Kanaya S. Kimura S. Katsuda C. Katayanagi K. Morikawa K. et al.How does RNase H recognize a DNA.RNA hybrid?.Proc. Natl. Acad. Sci. USA. 1991; 88: 11535-11539Crossref PubMed Scopus (195) Google Scholar, 6Crooke S.T. Wang S. Vickers T.A. Shen W. Liang X.H. Cellular uptake and trafficking of antisense oligonucleotides.Nat. Biotechnol. 2017; 35: 230-237Crossref PubMed Scopus (316) Google Scholar, 7Lima W.F. Vickers T.A. Nichols J. Li C. Crooke S.T. Defining the factors that contribute to on-target specificity of antisense oligonucleotides.PLoS ONE. 2014; 9: e101752Crossref PubMed Scopus (35) Google Scholar Moreover, ASO can redirect the mRNA splicing machinery to include or delete specific exons by complementary binding to RNA sequences at the early transcriptional stage.8Juliano R.L. Ming X. Nakagawa O. The chemistry and biology of oligonucleotide conjugates.Acc. Chem. Res. 2012; 45: 1067-1076Crossref PubMed Scopus (97) Google Scholar,9Vickers T.A. Crooke S.T. The rates of the major steps in the molecular mechanism of RNase H1-dependent antisense oligonucleotide induced degradation of RNA.Nucleic Acids Res. 2015; 43: 8955-8963Crossref PubMed Scopus (43) Google Scholar ASO affiliating to mRNA can also sterically inhibit the formation of the ribosomal complex, resulting in the hindrance of protein translation.10Mercatante D.R. Kole R. Control of alternative splicing by antisense oligonucleotides as a potential chemotherapy: effects on gene expression.Biochim. Biophys. Acta. 2002; 1587: 126-132Crossref PubMed Scopus (38) Google Scholar,11Chery J. RNA therapeutics: RNAi and antisense mechanisms and clinical applications.Postdoc J. 2016; 4: 35-50Crossref PubMed Google Scholar The use of synthetic ASO to regulate gene expression has been developed for many years. Within the past few years, the US Food and Drug Administration (FDA) approved two ASO-mediated therapies for the treatment of Duchenne muscular dystrophy and spinal muscular atrophy, respectively.12Stein C.A. Eteplirsen Approved for Duchenne Muscular Dystrophy: The FDA Faces a Difficult Choice.Mol. Ther. 2016; 24: 1884-1885Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar,13US Food and Drug AdministrationFDA approves first drug for spinal muscular atrophy.Press Anouncement. 2016; (December 23, 2016)https://www.fda.gov/news-events/press-announcements/fda-approves-first-drug-spinal-muscular-atrophyGoogle Scholar However, optimization of ASO delivery is still urgently needed for the further advancement of ASO in the clinic. An optimal delivery system needs to be cell specific, controllable, and able to protect the nucleic acids from nuclease degradation.14Rinaldi C. Wood M.J.A. Antisense oligonucleotides: the next frontier for treatment of neurological disorders.Nat. Rev. Neurol. 2018; 14: 9-21Crossref PubMed Scopus (361) Google Scholar Substantial progress has been made in developing lipid-based and polymer-based nanocarriers to facilitate nucleic acid delivery.15Falzarano M.S. Passarelli C. Ferlini A. Nanoparticle delivery of antisense oligonucleotides and their application in the exon skipping strategy for Duchenne muscular dystrophy.Nucleic Acid Ther. 2014; 24: 87-100Crossref PubMed Scopus (37) Google Scholar, 16Akinc A. Zumbuehl A. Goldberg M. Leshchiner E.S. Busini V. Hossain N. Bacallado S.A. Nguyen D.N. Fuller J. Alvarez R. et al.A combinatorial library of lipid-like materials for delivery of RNAi therapeutics.Nat. Biotechnol. 2008; 26: 561-569Crossref PubMed Scopus (937) Google Scholar, 17Semple S.C. Akinc A. Chen J. Sandhu A.P. Mui B.L. Cho C.K. Sah D.W. Stebbing D. Crosley E.J. Yaworski E. et al.Rational design of cationic lipids for siRNA delivery.Nat. Biotechnol. 2010; 28: 172-176Crossref PubMed Scopus (1167) Google Scholar, 18Jayaraman M. Ansell S.M. Mui B.L. Tam Y.K. Chen J. Du X. Butler D. Eltepu L. Matsuda S. Narayanannair J.K. et al.Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.Angew. Chem. Int. Ed. Engl. 2012; 51: 8529-8533Crossref PubMed Scopus (666) Google Scholar, 19Love K.T. Mahon K.P. Levins C.G. Whitehead K.A. Querbes W. Dorkin J.R. Qin J. Cantley W. Qin L.L. Racie T. et al.Lipid-like materials for low-dose, in vivo gene silencing.Proc. Natl. Acad. Sci. USA. 2010; 107: 1864-1869Crossref PubMed Scopus (656) Google Scholar, 20Yin H. Kanasty R.L. Eltoukhy A.A. Vegas A.J. Dorkin J.R. Anderson D.G. Non-viral vectors for gene-based therapy.Nat. Rev. Genet. 2014; 15: 541-555Crossref PubMed Scopus (2211) Google Scholar, 21Wang M. Glass Z.A. Xu Q. Non-viral delivery of genome-editing nucleases for gene therapy.Gene Ther. 2017; 24: 144-150Crossref PubMed Scopus (72) Google Scholar Among them, cationic lipid-like nanoparticles are most advanced in terms of clinical translation, as demonstrated by the FDA approval of patisiran, a lipid nanoparticle (LNP)-formulated RNAi-based therapy for the treatment of the hereditary transthyretin amyloidosis.22Adams D. Gonzalez-Duarte A. O’Riordan W.D. Yang C.C. Ueda M. Kristen A.V. Tournev I. Schmidt H.H. Coelho T. Berk J.L. et al.Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis.N. Engl. J. Med. 2018; 379: 11-21Crossref PubMed Scopus (1424) Google Scholar Recently, we have used the combinatorial library strategy to synthesize lipid-like materials for delivering various biologics, including proteins and nucleic acids, both in vitro and in vivo.23Chang J. Chen X. Glass Z. Gao F. Mao L. Wang M. Xu Q. Integrating Combinatorial Lipid Nanoparticle and Chemically Modified Protein for Intracellular Delivery and Genome Editing.Acc. Chem. Res. 2019; 52: 665-675Crossref PubMed Scopus (71) Google Scholar,24Altınoglu S. Wang M. Xu Q. Combinatorial library strategies for synthesis of cationic lipid-like nanoparticles and their potential medical applications.Nanomedicine (Lond.). 2015; 10: 643-657Crossref PubMed Scopus (46) Google Scholar The subset of synthetic lipids, which contain disulfide bonds, has been demonstrated to be biodegradable and has shown excellent biocompatibility.25Takeda Y.S. Wang M. Deng P. Xu Q. Synthetic bioreducible lipid-based nanoparticles for miRNA delivery to mesenchymal stem cells to induce neuronal differentiation.Bioeng. Transl. Med. 2016; 1: 160-167Crossref PubMed Google Scholar, 26Wang M. Alberti K. Varone A. Pouli D. Georgakoudi I. Xu Q. Enhanced intracellular siRNA delivery using bioreducible lipid-like nanoparticles.Adv. Healthc. Mater. 2014; 3: 1398-1403Crossref PubMed Scopus (66) Google Scholar, 27Sun S. Wang M. Alberti K.A. Choy A. Xu Q. DOPE facilitates quaternized lipidoids (QLDs) for in vitro DNA delivery.Nanomedicine (Lond.). 2013; 9: 849-854Crossref PubMed Scopus (30) Google Scholar, 28Wang M. Zuris J.A. Meng F. Rees H. Sun S. Deng P. Han Y. Gao X. Pouli D. Wu Q. et al.Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles.Proc. Natl. Acad. Sci. USA. 2016; 113: 2868-2873Crossref PubMed Scopus (397) Google Scholar The high concentration of glutathione (GSH) in cytoplasm tends to intracellularly cleave and degrade LNPs through disulfide bond exchange once the nanoparticles reach the reductive intracellular environment.29Shirazi R.S. Ewert K.K. Leal C. Majzoub R.N. Bouxsein N.F. Safinya C.R. Synthesis and characterization of degradable multivalent cationic lipids with disulfide-bond spacers for gene delivery.Biochim. Biophys. Acta. 2011; 1808: 2156-2166Crossref PubMed Scopus (67) Google Scholar,30Wang M. Alberti K. Sun S. Arellano C.L. Xu Q. Combinatorially designed lipid-like nanoparticles for intracellular delivery of cytotoxic protein for cancer therapy.Angew. Chem. Int. Ed. Engl. 2014; 53: 2893-2898Crossref PubMed Scopus (181) Google Scholar In this study, we investigated the potential application of this novel class of synthetic lipids for ASO delivery both in vitro and in vivo. For in vivo study, we chose proprotein convertase subtilisin/kexin type 9 (PCSK9) as a therapeutic target, because it plays an essential role in cholesterol metabolism by regulating low-density lipoprotein (LDL) receptor degradation. The antibody against PCSK9 has been developed and approved by the FDA for the treatment of hyperlipidemia.31Schmitz J. Gouni-Berthold I. Anti-PCSK9 Antibodies: A New Era in the Treatment of Dyslipidemia.Curr. Pharm. Des. 2017; 23: 1484-1494Crossref PubMed Scopus (6) Google Scholar ASO or RNAi may be a better strategy to manage the hyperlipidemia than antibody therapeutics, due to the potential longer-lasting effect and thus less-frequent dosing of ASOs and RNAi compared with antibody. However, this strategy relies on a robust, targeted delivery method that specifically delivers the ASO to the liver. Herein, we explored the capability of the bioreducible lipids to deliver ASO for efficient mRNA silencing both in vitro and in vivo (Figure 1A). We conducted a screening of a small library of the bioreducible lipids by delivering GFP silencing ASO in GFP-expressing cells and identified three lipids, 113-O14B-3, 113-O16B-3, and 306-O12B-3, showing high efficiency in gene knockdown. They all showed superior delivery efficacy than Lipofectamine 2000 (LPF 2000), a commercially available transfection reagent. These lipid nanoparticles also exhibit lower toxicity than that of LPF 2000 in vitro. Further, we evaluated the capability of these synthetic lipids in delivering PCSK9 silencing ASO in mouse liver for efficient PCSK9 mRNA knockdown. The top-performing lipid was found to be 306-O12B-3, with a median effective dose (ED50) around 0.034 mg·kg-1. The knockdown of the PCSK9 mRNA level by ASO/LNP complexes was also demonstrated to effectively reduce the total PCSK9 protein and serum cholesterol level in mice, demonstrating a functional knockdown. Meanwhile, no hepatotoxicity or nephrotoxicity was observed. This study showed that the bioreducible lipids can be good candidates for ASO delivery and may pave the way for the advancement of ASO-based therapeutics. The bioreducible lipids were synthesized by the Michael addition between the primary or secondary amines and acrylate containing a disulfide bond (Figure 1BI), according to our published procedure.26Wang M. Alberti K. Varone A. Pouli D. Georgakoudi I. Xu Q. Enhanced intracellular siRNA delivery using bioreducible lipid-like nanoparticles.Adv. Healthc. Mater. 2014; 3: 1398-1403Crossref PubMed Scopus (66) Google Scholar Bioreducible lipids were named “R-O[x]B-n” using the following method: the amine head is indicated by a two- or three-digit serial number (R). The letter “O” indicates an acrylate linker between the amine head and hydrophobic tail. The number [x] indicates the number of carbon atoms in the hydrophobic tail of the acrylate. The “B” refers to a bioreducible lipid, i.e., containing a disulfide bond in the tail. The last number “n” indicates the total number of hydrophobic tails of the synthetic lipids and is usually either 3 or 4. For example, 306-O12B-3 indicates the lipid prepared by reacting amine 306 with acrylate O12B, resulting in a lipid with 3 tails. Figure 1BII shows the chemical structure of the lipid, termed “306-O12B-3.” For in vitro screening of the lipids, the HEK cell line stably expressing GFP (GFP-HEK cells) was used as a model to evaluate the efficiency of the ASO delivery using these bioreducible lipids. Because the activity of ASO depends on many factors, such as chemical linkages, guanine and cytosine (GC) content, and secondary structure of mRNA targeting, it is necessary to optimize the sequences and chemistry pattern of ASO to achieve optimal gene silence of a target gene. We first designed five ASO sequences targeting the GFP gene, each 20 nucleotides in length (identified as G-ASO-1 to G-ASO-5) with phosphodiester linkages between each nucleotide (Figure S1A). We screened the delivery of these ASOs to the GFP-HEK cells using the commercial transfection reagent LPF 2000. As shown in Figure S1B, the #5 ASO showed higher GFP silencing efficiency than other ASOs, so we chose the #5 ASO for further study. This sequence was optimized as described in literature using chemically modified phosphorothioate bonds and 2′-O-methyl (2-OME)-modified ribose (Figure S1C). The modified 5th ASO showed a 55.6% improvement in GFP mRNA repression, demonstrating that the chemical modification of ASO played a crucial role in improving the capacity and stability in ASO (Figure S1D). The chemically modified scrambled ASO, delivered by LPF 2000, showed no effect in GFP silencing, indicating ASO sequence-specific gene silence. Thus, all ASOs are phosphorothioate bonds and 2-OME modified in the following experiments. For in vitro ASO delivery, we used the pure lipids, referred as “nonformulated LNPs,” in which no other helper lipids were added to the formulation. The ASO and the lipid sample were complexed at a 1/15 (w/w) ratio. The particle size and zeta potential were measured using dynamic light scattering (DLS). As shown in Figure S2A, the blank, nonformulated LNPs ranged from 90 to 300 nm effective diameter, whereas the sizes increased to 150 nm to 500 nm after complexing with ASO. The size increase is due to the complexation between the lipid and ASO. The zeta potential of the nanocomplex (Figure S2B) showed an obvious surface-charge decrease from positive (0.2 to 28 mV) before complexation with ASO to negative (−31.7 to −12.2 mV) after ASO complexation. This demonstrates that the nanocomplexation is driven by the electrostatic interaction between the negative-charged ASO and positive-charged LNPs. For in vivo ASO delivery, in addition to the active bioreducible lipid, we also added helper lipids, including cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and polyethylene glycol (PEG)ylated lipid in the formulation. The LNPs produced with colipids were referred as “formulated LNPs.” The ASO and the formulated LNPs were mixed at a 1/15 (w/w) ratio of ASO to bioreducible lipid. The nanocomplex size and surface charge were determined using DLS, and the morphology was visualized using transmission electronic microscopy (TEM). The sizes of the ASO/LNP nanocomplexes are in the range of 90 to 150 nm. Figures S2C–S2F showed the typical morphology of the LNP formulation with bioreducible lipid (306-O12B-3), before and after complexing with ASO. Both showed spherical shape, and the particle size is consistent with the DLS analysis. The ASO encapsulation efficiency using formulated LNPs was measured by the Quant-iT OliGreen ssDNA assay kit, and all of the lipid formulations exhibited high ASO encapsulation efficiency above 97% (Figure S3). For in vitro study, lipid nanoparticles were fabricated via directly dissolving the pure lipids in sodium acetate buffer (25 mM, pH 5.2). For the in vitro screening, we fixed the weight ratio of synthetic lipids to GFP-ASO at 15/1 and delivered the GFP-ASO to the GFP-HEK cells at a concentration of 0.1 μg/mL. The GFP expression level of the HEK cells was analyzed by flow cytometry after 16 h of post-transfection. The ASO delivery efficacy was determined by the ratio of the mean fluorescent intensity of treated cells to that of untreated ones. As shown in Figure 2A, GFP-ASO-alone treatment exhibits negligible GFP suppression, similar to PBS vehicle control treatment, indicating the inability to penetrate the cell membranes by ASO itself. The cells treated with the ASO/LNP complexes showed different GFP silencing levels. We found that 113-O16B-3 and 306-O16B-3 showed higher GFP silencing than LPF 2000, a commercial in vitro gene transfection reagent, with 53.5% of GFP knockdown efficacy. The amine heads (113 and 306 shown in Figure 1C), used to synthesize these lipids, share some structural similarities. Both amine heads contained three amino groups and two of them evenly distributed at the end of the structures with the potential to induce different numbers of carbon tails. Further, the synthetic lipids showed the most efficient in vitro ASO delivery when the lipids were synthesized with three branches of carbon tails instead of two or four tails. To investigate whether the carbon tail length also affected the ASO delivery, we synthesized lipids derived from amines 113 and 306 with three tails using different tail lengths (O12B, O14B, O16B, and O18B, shown in Figure 1C). As shown in Figure 2B, three of eight synthetic lipids (113-O14B-3, 113-O16B-3, and 306-O12B-3) in the expanded library showed higher delivery efficiency than LPF 2000 in vitro. In particular, 306-O12B-3 nanoparticles exhibited the highest GFP silencing efficiency, with 81.5% of GFP-negative cells observed. Also, those lipids showed high cell viability compared to that of LPF 2000, demonstrating low cell toxicity of those different tail lengths (Figure S4). We further investigated the influence of the ratio of the lipid to ASO (N/P ratio). The N/P ratio is the molar ratio of the nitrogen in synthetic lipid to the phosphate groups in the ASO. The top three performing lipids from the screening (113-O14B-3, 113-O16B-3, and 306-O12B-3) were chosen for this study. The N/P ratio of lipid to ASO was varied from 2.5 to 20. As shown in Figure 2C, when N/P ratios increased from 2.5 to 15, the GFP silencing efficiency was improved from 10.9% to 79.5%, whereas the increase of N/P ratios above 15 did not augment ASO delivery efficiency. Hence, we chose the N/P ratio of 15/1 for following experiments to achieve sufficient delivery efficacy with good biocompatibility. Interestingly, while N/P ratio is defined as a molar ratio, given the exact molecular weights of our particular lipids and ASOs, a 15/1 N/P ratio was found to correlate to the 15/1 weight ratio as well. Thus, for the remainder of this paper, we report delivery parameters of our synthetic lipids as a weight/weight ratio for practical purposes. We next checked the in vitro cytotoxicity and gene-knockdown efficiency of the LNP/ASO nanocomplexes in a dose-dependent matter. Commercially available transfection reagent LPF 2000 is used as control for comparison. The N/P ratio of LNP/ASO is fixed at 15/1, and the dose of GFP-ASO varying from 0.3 to 30 nM was used to treat the GFP-HEK cells. The GFP gene-knockdown efficiency was evaluated using a flow cytometer, and the cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As Figure 2D shows, all three lipids showed higher GFP silencing efficiency than that of LPF 2000 at each dose of GFP-ASO, except for the 0.3-nM concentration, which is too low to show significant gene silencing in all lipids. To eliminate the potential of unspecific targeting, we also formulated the scrambled GFP-ASO sequence with our LNPs as a negative. No GFP silencing effect was observed, indicating the specificity of silencing. Figure 2E showed the dose-dependent cell-viability results from the three synthetic lipids and control LPF 2000. The three top candidates showed the similar cell viability trend as the GFP-ASO doses increased. Even at the highest LNP/ASO complex concentration (30 nM), the cell viability from all three bioreducible lipid-treated cells maintained above 76.4%, whereas the cells treated with LPF 2000 at the same dose reduced to 47.9%. These results suggested that our lipids were not only highly effective but also more biocompatible and less cytotoxic than LPF 2000 for ASO delivery. For in vivo delivery, in addition to the active synthetic lipid, excipients, including cholesterol, DOPE, and PEGylated lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 [DSPE-PEG2000]), were used to formulate the LNPs. ASO labeled with near-infrared fluorescent dye (ASO-Alexa 750) was used as a cargo to form nanocomplexes with LNPs. The top three lipids (113-O14B-3, 113-O16B-3, and 306-O12B-3) identified from the in vitro study were chosen for in vivo delivery of ASO-Alexa 750. The active lipid to the Alexa 750-labeled ASO ratio was fixed at 15/1 (w/w). At 1 h of postinjection, major organs were collected and then visualized using IVIS (in vivo imaging system). As shown in Figure 3, all of the ASO-LNP-treated mice exhibited most fluorescence signals in the liver, indicating the LNP/ASO complexes mainly targeted and accumulated to this organ. It is noticeable that the ASO-Alexa 750s complexed with 306-O12B-3 showed the highest fluorescence intensity in the liver, which is about 2-fold higher than that from mice injected with ASO/113-O14B-3 complexes (Table S1). Besides liver, we also observed the fluorescent signals in the organs, such as spleen, kidney, and lung, although much less than liver. As expected, no fluorescence was detected from the PBS-injected control mice. The PCSK9 expression is involved in hypercholesterolemia and premature atherosclerotic cardiovascular disease (ASCVD), and antibody against PCSK9 has gained FDA approval for the management of hypercholesterolemia.31Schmitz J. Gouni-Berthold I. Anti-PCSK9 Antibodies: A New Era in the Treatment of Dyslipidemia.Curr. Pharm. Des. 2017; 23: 1484-1494Crossref PubMed Scopus (6) Google Scholar,32Fitzgerald K. Frank-Kamenetsky M. Shulga-Morskaya S. Liebow A. Bettencourt B.R. Sutherland J.E. Hutabarat R.M. Clausen V.A. Karsten V. Cehelsky J. et al.Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled, phase 1 trial.Lancet. 2014; 383: 60-68Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar Here, we evaluated whether our lipid formulation can deliver PCSK9-targeted ASO (PCSK9-ASO) for efficient PCSK9 knockdown in mice. The top three synthetic lipids (113-O14B-3, 113-O16B-3, and 306-O12B-3) identified from in vitro study were used to form LNPs for PCSK9-ASO delivery in vivo. To increase the serum stability and circulation time of the LNP/ASO complexes, we formulated LNPs with other helper lipids. The helper lipids cholesterol, DOPE, and DSPE-PEG2000 were used in the LNP formulation. In a typical formulation, the synthetic lipid, cholesterol, DOPE, and DSPE-PEG2000 were set at the weight ratio of 16/4/1/1. The synthetic lipid to PCSK9-ASO ratio was set as 15/1 (w/w). The PCSK9-ASO/LNP complexes were injected in mice through the tail vein at the PCSK9-ASO doses of 0.05, 0.1, 0.5, 1, and 1.5 mg·kg-1. A control group was injected with saline via the tail vein and another control group with scrambled ASO encapsulated in the same lipid formulations. Livers were harvested after 72 h after administration for PCSK9 mRNA quantization using RT-PCR. As shown in Figure 4, the PCSK9 mRNA level in mice liver was found to be ASO dose dependent. In general, the PCSK9 mRNA level decreased as the injected PCSK9-ASO dose increased for all three lipid formulations. Among them, 306-O12B-3 LNP formulation showed the best silencing effect with an ED50 of 0.088 mg·kg-1 in the mRNA level. Thus, we chose 306-O12B-3 as the leading lipid for further study, including formulation optimization and in vivo biocompatibility. It has been reported that the formulation ratios of the helper lipids may influence the delivery efficiency of the nanoparticle system. Thus, we investigated whether increasing the amount of the DOPE in the LNP formulation can improve the ASO delivery in vivo. In the study shown in Figure 4, the weight ratio of cationic lipid/cholesterol/DOPE/DSPE-PEG2000 was set as 16/4/1/1. To evaluate the influence of the DOPE on the in vivo delivery, we kept the ratio of cationic lipid (306-O12B-3), cholesterol, and DSPE-PEG2000 not changed, whereas we increased the DOPE. The final ratio of cationic lipid/cholesterol/DOPE/DSPE-PEG2000 was set as 16/4/4/1 in the formulation. We kept the active cationic lipid-to-ASO ratio the same at 15/1 (w/w) in both cases. The formulations were injected into mice via tail vein at PCSK9-ASO doses of 0.025, 0.05, 0.1, or 0.5 mg·kg-1. After 72 h of postinjection, livers were harvested for PCSK9 mRNA determination, and the blood was collected for serum PCSK9 protein and total cholesterol measurement. As shown in Figure 5A, we found that increasing the DOPE fraction in the LNP improved the in vivo ASO delivery efficiency. For 16/4/4/1 formulation (cationic lipid/cholesterol/DOPE/DSPE-PEG2000), an ED50 of 0.034 mg·kg-1 PCSK9-ASO was obtained, compared with an ED50 of 0.088 mg·kg-1 in the 16/4/1/1 formulation for the PCSK9 mRNA knockdown in the mouse liver. Both PCSK9 protein in serum and serum cholesterol level were significantly decreased after the LNP/ASO treatment in a dose-dependent manner (Figures 5B and 5C). Meanwhile, no PCSK9 silencing effect was observed in the scrambled PCSK9-ASO (S-PCSK9-ASO) delivered by the 306-O12B-3 in either 16/4/4/1 or 16/4/1/1 formulation (Figure S5). This result demonstrated the specific binding of functional PCSK9-ASO to the corresponding mRNA sequence. At the dose of 0.1 mg·kg-1 of ASO, the PCSK9 protein and serum cholesterol level also decrease to 50% of the normal level. We also tested some other lipid composition and ratios (Figure S6), but none of them exhibited higher PCSK9 silencing efficiency than the 16/4/4/1 formulation. We also investigated the tolerability of the optimized 306-O12B-3 formulation in mice. Animals were intravenously (i.v.) injected with a PCSK9-ASO dose up to 5 mg·kg-1. The LNPs comprising 306-O12B-3 were well tolerated at all dose levels and observed no clinically significant change in mouse behavior, spleen size, and key hepatotoxic or nephrotoxic parameters (Table 1).Table 1Clinical Chemistry and Hematology Parameters for ASO/LNP Complex-Treated MiceGroupsALT (U/L)AST (U/L)Cr (mg/dL)BUN (mg/dL)Spleen (mg)0 mg·kg-120.24 ± 2.78129.14 ± 17.890.35 ± 0.0920.28 ± 2.3976.8 ± 3.61 mg·kg-120.53 ± 2.03131.76 ± 15.880.35 ± 0.0920.11 ± 3.6977.3 ± 3.13 mg·kg-117.88 ± 3.38129.95 ± 20.210.36 ± 0.0720.9 ± 2.5179.7 ± 1.65 mg·kg-119.84 ± 3.5133.34 ± 15.080.37 ± 0.121.99 ± 2.3480.9 ± 2.2ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; Cr, creatinine. Data points are expressed as a percentage of saline control animals and represent group mean ± SD (n = 3). Open table in a new tab ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; Cr, creatinine. Data points are expressed as a percentage of saline control animals and represent group mean ± SD (n = 3). The therapeutic application of oligonucleotides, including ASOs and small interfering RNA (siRNA), has been recognized as potent tools against myriad diseases. Due to the stability and the low off-target effect of ASO, some ASO-mediated gene silencing has achieved a successfully clinical translation.33Porensky P.N. Burghes A.H. Antisense oligonucleotides for the treatment of spinal mu" @default.
• W3002543944 created "2020-01-30" @default.
• W3002543944 creator A5019138735 @default.
• W3002543944 creator A5049711127 @default.
• W3002543944 creator A5058274340 @default.
• W3002543944 creator A5074618791 @default.
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• W3002543944 date "2020-03-01" @default.
• W3002543944 modified "2023-10-11" @default.
• W3002543944 title "Efficient Delivery of Antisense Oligonucleotides Using Bioreducible Lipid Nanoparticles In Vitro and In Vivo" @default.
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• W3002543944 doi "https://doi.org/10.1016/j.omtn.2020.01.018" @default.
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