ionizable cationic lipid; siRNA delivery

Preparation of plasmid-containing LNP[1]
LNP-pDNA was prepared as previously described in J Phys Chem B, 119 (28) (2015), pp. 8698-8706. Amino-lipids, helper lipids, cholesterol and PEG-DMG were dissolved in ethanol at a molar ratio of 50/10/39/1. Purified pCI-FLuc (expressing Firefly Luciferase) or pCAX-eGFP (expressing eGFP) was dissolved in 25 mM sodium acetate pH 4 to 0.116 mg/ml. The solutions were mixed using a micromixer. For large-scale formulations, mixing was performed through a T-junction mixer as previously described in Pharm Res, 22 (3) (2005), pp. 362-372. All formulations were produced at 0.029 mg DNA per μmol lipid (corresponding to N/P charge ratio of 6.0), unless otherwise stated.

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Analysis of lipid particles containing DNA[1]
Particle size, lipid concentration, pDNA entrapment, and total pDNA concentration were measured as previously described in J Phys Chem B, 119 (28) (2015), pp. 8698-8706 and J Control Release, 196 (2014), pp. 106-112. Zeta potential was measured as previously described in Mol Ther Nucl Acids, 3 (2014), p. e210.

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Lipid nanoparticles (LNPs) containing distearoylphosphatidlycholine (DSPC), and ionizable amino-lipids such as dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) are potent siRNA delivery vehicles in vivo. Here we explore the utility of similar LNP systems as transfection reagents for plasmid DNA (pDNA). It is shown that replacement of DSPC by unsaturated PCs and DLin-MC3-DMA by the related lipid DLin-KC2-DMA resulted in highly potent transfection reagents for HeLa cells in vitro. Further, these formulations exhibited excellent transfection properties in a variety of mammalian cell lines and transfection efficiencies approaching 90% in primary cell cultures. These transfection levels were equal or greater than achieved by Lipofectamine, with much reduced toxicity[1].

Effective internal siRNA delivery vehicles are phosphate nanoparticles (LNP) that contain ionizable phosphate esters, such as dilinoleylmethyl-4-dimethylphosphate (DLin-MC3-DMA), and distearyl acetylphosphatidylcholine (DSPC). With a molar ratio of 50/10/38.5/1.5 (PEG)-Pallet, the LNP-siRNA system procedure is tuned to achieve maximum gene silencing efficacy in hepatocytes following intravenous injection. It contains DLin-MC3-DMA (MC3), DSPC, solution, and polymerization solution. The improved pKa value of DLin-MC3-DMA greatly increases efficacy [1].

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At a dose of 1 mg/kg siRNA in mice, LNPs with ALC-0315 achieved a 2- and 10-fold greater knockdown of FVII and ADAMTS13, respectively, compared to LNPs with MC3. At a high dose (5 mg/kg), ALC-0315 LNPs increased markers of liver toxicity (ALT and bile acids), while the same dose of MC3 LNPs did not. These results demonstrate that ALC-0315 LNPs achieves potent siRNA-mediated knockdown of target proteins in hepatocytes and HSCs, in mice, though markers of liver toxicity can be observed after a high dose. This study provides an initial comparison that may inform the development of ionizable cationic LNP therapeutics with maximal efficacy and limited toxicity.[2]

In vitro transfection studies of cultured cells[1]
HeLa, HepG2 and Hep3B cells were cultured in DMEM with 10% FBS. PC12 and MCF7 cells were cultured in RPMI 1640 with 10% FBS. LNPs were diluted into medium at 0.75-6.0 μg/ml pDNA. The luciferase assay was performed 24 h post-treatment. In the case of serum-free treatments, LNP-pDNA systems were diluted into medium alone. To study the role of ApoE, wild-type (C57BL/6) and ApoE−/− (B6.129P2-Apoetm1Unc/J) mouse sera were purchased from The Jackson Laboratory. LNP-pDNA systems were diluted into medium containing 10% of each serum. Measured luminescence was normalized to protein content as measured by the Pierce BCA Protein Assay.

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In vitro LNP uptake measurement[1]
Cellular uptake of LNP formulations was performed as previously described.29 LNP-pDNAs labeled with DiI-C18 (0.2 mol% total lipid) were used. Plates were imaged using a Cellomics Arrayscan VTI HCS reader.

In vivo transfection by injection of LNP-pDNA[1]
Stage ~19-20 white leghorn chicken embryos were stained with neutral red dye, and a small tear was made in the extraembryonic membranes over the forelimb. LNP formulations mixed with ~0.1% Fast Green dye was injected at the distal forelimb at 10 μg/ml pDNA, using a Picospritzer II microinjector (Parker Hannifin Corp). Eggs were incubated overnight. Embryos were rinsed and fixed in 4% paraformaldehyde in PBS, and GFP fluorescence was recorded with a Leica MZFLIII stereofluorescence microscope.

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Transfection of primary embryonic mesenchyme and analysis of reagent toxicity[1]
White leghorn chicken embryos at stage 24 were removed and dissected in Hanks buffered saline solution (HBSS). Forelimbs were pooled and treated for 1 h in dispase to remove epithelial ectoderm. Limb mesenchyme was dissociated, and resuspended in DMEM with 5% FBS and 1% penicillin/streptomycin. Cells were plated onto 96-well plates at a density of 2 × 105 or 2 × 107 cells/ml and treated with 0-40 μg/ml LNP-pDNA. Lipofectamine was used as per manufacturer's protocol. Cultures were incubated overnight at 37 °C and resuspended in PBS buffer for expression analysis using a BD LSRII flow cytometer. Cell survival was assessed in low-density cultures after 24 h of treatment, by counting the adherent cells remaining after PBS washes. Viability was determined based on average counts of all adherent cells within a single field of view (100×) normalized to cell counts of untreated cultures.

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LNP-siRNA injections[2]
siFVII, siADAMTS13, and siLuc were encapsulated in LNPs containing either ALC-0315 or MC3 as the ionizable cationic lipid. We injected mice with 1 mg siRNA per kg body weight (mg/kg) for knockdown studies, and 5 mg/kg dose for toxicity studies. A dose of 1 mg/kg siRNA in mice is standard for inducing knockdown of mRNA for proteins made in hepatocytes using siRNA-LNPs, whereas 5 mg/kg is a higher dose than the one that would normally be used in mice.3 The recommended dose of ONPATTRO (the clinically approved siRNA for hATTR) is 0.3 mg/kg, which corresponds to a human equivalent dose (HED) of 3.69 mg/kg in mice when using body surface area conversion. One week after administration, liver tissue and blood were collected to measure target mRNA and protein levels, respectively, and compared to siLuc-treated mice; half-lives of plasma FVII and ADAMTS13 are 3–6 hours, and 2–3 days, respectively.23,24 mRNA and protein quantification, and toxicity studies are described further below.

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Toxicological analysis[2]
Mice were injected IV with either PBS, or with siLuc encapsulated in LNPs with ALC-0315 (siLuc-ALC-0315) or MC3 (siLuc-MC3) at 5 mg/kg (N = 4). While a dose of any LNP at 10 mg/kg usually causes severe toxicity, such as inflammation and liver necrosis, the toxicity after a 5 mg/kg dose depends on the lipid formulation.26,27 Five hours after the injection, mice were sacrificed, and serum samples were collected as described above. Serum samples were submitted to Idexx BioAnalytics for a toxicology panel. Aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), bile acids, total bilirubin (TBIL), blood urea nitrogen (BUN), creatine (CREA), gamma-glutamyl transferase (GGT) levels were analyzed. To note, data regarding bile acid levels in mice treated with PBS and with siLuc-ALC-0315 had N = 3 due to the presence of an outlier in each group (data not shown). The presence of the outliers would have not altered the conclusion, siLuc-ALC-0315 treated mice would have had an even higher bile acid mean and would have been more statistically significant from the PBS-treated mice. Outliers were determined via the ROUT method using GraphPad Prism although limitations such as our small sample size were considered. Bile acid levels commonly range from 0 to 6 μmol/L; however, our results were likely not biologically possible (>130 μmol/L).

[1]. Design of lipid nanoparticles for in vitro and in vivo delivery of plasmid DNA. Nanomedicine. 2017 May;13(4):1377-1387.

[2]. Ferraresso F, Strilchuk AW, Juang LJ, Poole LG, Luyendyk JP, Kastrup CJ. Comparison of DLin-MC3-DMA and ALC-0315 for siRNA Delivery to Hepatocytes and Hepatic Stellate Cells. Mol Pharm. 2022;19(7):2175-2182.

Lipid nanoparticles (LNPs) containing distearoylphosphatidlycholine (DSPC), and ionizable amino-lipids such as dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) are potent siRNA delivery vehicles in vivo. Here we explore the utility of similar LNP systems as transfection reagents for plasmid DNA (pDNA). It is shown that replacement of DSPC by unsaturated PCs and DLin-MC3-DMA by the related lipid DLin-KC2-DMA resulted in highly potent transfection reagents for HeLa cells in vitro. Further, these formulations exhibited excellent transfection properties in a variety of mammalian cell lines and transfection efficiencies approaching 90% in primary cell cultures. These transfection levels were equal or greater than achieved by Lipofectamine, with much reduced toxicity. Finally, microinjection of LNP-eGFP into the limb bud of a chick embryo resulted in robust reporter-gene expression. It is concluded that LNP systems containing ionizable amino lipids can be highly effective, non-toxic pDNA delivery systems for gene expression both in vitro and in vivo.In summary, the results presented in this study show that LNP formulations of pDNA containing ionizable cationic lipids can be highly effective transfection reagents in vitro that are significantly less toxic than commercial reagents such as Lipofectamine. LNP systems containing DLin-KC2-DMA and unsaturated helper lipids are also potent systems for transfecting primary cells in vitro and in vivo. It is anticipated that these systems will be of considerable utility for transfection of developing tissues in vitro with attendant potential for gene editing applications.[1]

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Ionizable cationic lipids are essential for efficient in vivo delivery of RNA by lipid nanoparticles (LNPs). DLin-MC3-DMA (MC3), ALC-0315, and SM-102 are the only ionizable cationic lipids currently clinically approved for RNA therapies. ALC-0315 and SM-102 are structurally similar lipids used in SARS-CoV-2 mRNA vaccines, while MC3 is used in siRNA therapy to knock down transthyretin in hepatocytes. Hepatocytes and hepatic stellate cells (HSCs) are particularly attractive targets for RNA therapy because they synthesize many plasma proteins, including those that influence blood coagulation. While LNPs preferentially accumulate in the liver, evaluating the ability of different ionizable cationic lipids to deliver RNA cargo into distinct cell populations is important for designing RNA-LNP therapies with minimal hepatotoxicity. Here, we directly compared LNPs containing either ALC-0315 or MC3 to knock-down coagulation factor VII (FVII) in hepatocytes and ADAMTS13 in HSCs. At a dose of 1 mg/kg siRNA in mice, LNPs with ALC-0315 achieved a 2- and 10-fold greater knockdown of FVII and ADAMTS13, respectively, compared to LNPs with MC3. At a high dose (5 mg/kg), ALC-0315 LNPs increased markers of liver toxicity (ALT and bile acids), while the same dose of MC3 LNPs did not. These results demonstrate that ALC-0315 LNPs achieves potent siRNA-mediated knockdown of target proteins in hepatocytes and HSCs, in mice, though markers of liver toxicity can be observed after a high dose. This study provides an initial comparison that may inform the development of ionizable cationic LNP therapeutics with maximal efficacy and limited toxicity. This work represents proof-of-concept that ionizable cationic LNPs can be used to access and knockdown HSCs-specific targets like ADAMTS13. It shows there are differences in efficacy of MC3 and ALC-0315, which are two of the three clinically approved ionizable cationic lipids used in the LNP delivery platform. Insights from this head-to-head comparison may enable the optimization of RNA-based agents to modulate expression of proteins that were previously inaccessible in vivo for research and therapeutic purposes.[2]

C43H79NO2

642.0929

641.611

C, 80.43; H, 12.40; N, 2.18; O, 4.98

1224606-06-7

1224606-06-7 ; 1258299-72-7 (deleted)

49785164

Colorless to light yellow liquid

0.886±0.06 g/cm3(Predicted)

670.2±43.0 °C(Predicted)

13.647

0

3

36

46

687

0

O(C(CCCN(C)C)=O)C(CCCCCCCC/C=C\C/C=C\CCCCC)CCCCCCCC/C=C\C/C=C\CCCCC

NRLNQCOGCKAESA-KWXKLSQISA-N

InChI=1S/C43H79NO2/c1-5-7-9-11-13-15-17-19-21-23-25-27-29-31-33-35-38-42(46-43(45)40-37-41-44(3)4)39-36-34-32-30-28-26-24-22-20-18-16-14-12-10-8-6-2/h13-16,19-22,42H,5-12,17-18,23-41H2,1-4H3/b15-13-,16-14-,21-19-,22-20-

(6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate

D-Lin-MC3-DMA; MC 3; RV-28; MC3; DLin-MC3-DMA; (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate; Q0J6FQ6FKP; D-Lin-MC3-DMA (Excipient); RV 28; MC-3; RV28; DLin-MC3-DMA; Dlin-mc3-dma

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Ethanol : ~125 mg/mL (~194.68 mM)
DMSO : ~100 mg/mL (~155.74 mM)

Solubility in Formulation 1: 6.25 mg/mL (9.73 mM) in 10% DMSO + 90% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 5 mg/mL (7.79 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: 5 mg/mL (7.79 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (3.89 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: 2.5 mg/mL (3.89 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 6: ≥ 2.5 mg/mL (3.89 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 7: ≥ 2.5 mg/mL (3.89 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)