Ionizable cationic lipid; mRNA vaccine delivery

To date, three ionizable cationic lipids have been approved for clinical use in RNA-based therapeutics: DLin-MC3-DMA (heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate), ALC-0315 (4-hydroxybutyl) azanediyl)bis (hexane-6,1-diyl)bis(2-hexyldecanoate), and SM-102(heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy) hexyl) amino) octanoate). MC3 was designed for delivery of siRNA to hepatocytes to treat hereditary transthyretin amyloidosis (hATTR). ALC-0315 and SM-102 were designed for mRNA delivery, as they are the ionizable cationic lipids in the mRNA-based SARS-CoV-2 vaccines developed by Pfizer/BioNTech/Acuitas and Moderna, respectively.17,18 All three of these drug formulations use similar “helper” lipids and lipid molar ratios, approximately 50% ionizable cationic lipid, 10% DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine), 38.5% cholesterol, and 1.5% PEG-DMG (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000). MC3 contains major structural differences compared to ALC-0315 and SM-102, therefore differences in potency and toxicity may exist. Here, we report a head-to-head comparison of LNPs containing MC3 and ALC-0315, comparing their hepatotoxicity, and ability to deliver siRNA cargo in vivo to hepatocytes and HSCs. Due to the structural similarity to ALC-0315, SM-102 was not investigated in this study; both ionizable lipids exhibit similar branching, and the same functional groups (one hydroxy, one tertiary amine, two esters and only saturated hydrocarbons)[2].

In this study, researchers evaluated the effect of in vivo codelivery of Cas9 mRNA and guide RNAs (gRNAs) by SM-102-based lipid nanoparticles (LNPs) on HBV cccDNA and integrated DNA in mouse and a higher species. CRISPR nanoparticle treatment decreased the levels of HBcAg, HBsAg and cccDNA in AAV-HBV1.04 transduced mouse liver by 53%, 73% and 64% respectively. In HBV infected tree shrews, the treatment achieved 70% reduction of viral RNA and 35% reduction of cccDNA. In HBV transgenic mouse, 90% inhibition of HBV RNA and 95% inhibition of DNA were observed. CRISPR nanoparticle treatment was well tolerated in both mouse and tree shrew, as no elevation of liver enzymes and minimal off-target was observed. The study demonstrated that SM-102-based CRISPR is safe and effective in targeting HBV episomal and integration DNA in vivo. The system delivered by SM-102-based LNPs may be used as a potential therapeutic strategy against HBV infection[4].

Protocols for mRNA Synthesis and Encapsulation in Ionizable Lipid Nanoparticles: [3]
Basic Protocol 1: Synthesis of mRNA by in vitro transcription and enzymatic capping and tailing
Basic Protocol 2: Encapsulation of mRNA into ionizable lipid nanoparticles
Alternate Protocol: Small-scale encapsulation of mRNA using preformed vesicles
Basic Protocol 3: Characterization and quality control of mRNA ionizable lipid nanoparticles.
For more details, please refer to https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.898

SM-102 (1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate) is an amino cationic lipid that has been tailored for the formation of lipid nanoparticles and it is one of the essential ingredients present in the ModernaTM COVID-19 vaccine. However, to what extent it may modify varying types of plasmalemmal ionic currents remains largely uncertain. In this study, we investigate the effects of SM-102 on ionic currents either in two types of endocrine cells (e.g., rat pituitary tumor (GH3) cells and mouse Leydig tumor (MA-10) cells) or in microglial (BV2) cells. Hyperpolarization-activated K+ currents in these cells bathed in high-K+, Ca2+-free extracellular solution were examined to assess the effects of SM-102 on the amplitude and hysteresis of the erg-mediated K+ current (IK(erg)). The SM-102 addition was effective at blocking IK(erg) in a concentration-dependent fashion with a half-maximal concentration (IC50) of 108 μM, a value which is similar to the KD value (i.e., 134 μM) required for its accentuation of deactivation time constant of the current. The hysteretic strength of IK(erg) in response to the long-lasting isosceles-triangular ramp pulse was effectively decreased in the presence of SM-102. Cell exposure to TurboFectinTM 8.0 (0.1%, v/v), a transfection reagent, was able to inhibit hyperpolarization-activated IK(erg) effectively with an increase in the deactivation time course of the current. Additionally, in GH3 cells dialyzed with spermine (30 μM), the IK(erg) amplitude progressively decreased; moreover, a further bath application of SM-102 (100 μM) or TurboFectin (0.1%) diminished the current magnitude further. In MA-10 Leydig cells, the IK(erg) was also blocked by the presence of SM-102 or TurboFectin. The IC50 value for SM-102-induced inhibition of IK(erg) in MA-10 cells was 98 μM. In BV2 microglial cells, the amplitude of the inwardly rectifying K+ current was inhibited by SM-102. Taken together, the presence of SM-102 concentration-dependently inhibited IK(erg) in endocrine cells (e.g., GH3 or MA-10 cells), and such action may contribute to their functional activities, assuming that similar in vivo findings exist[5].

Bioluminescence imaging[4]
To detect the tissue distribution of SM-102-based LNPs formulation containing mRNA upon intravenous administration in mice, bioluminescence imaging (BLI) analysis was performed by using SM-102-based LNPs containing a firefly luciferase (FLuc) reporter mRNA. Briefly, 6–8 weeks old C57BL/6 mice were inoculated with 20 μg SM-102-based LNPs containing FLuc mRNA via the i.v. route. Six hours after injection, animals were given an intraperitoneal injection of luciferase substrate, and fluorescent signals were collected with an IVIS Spectrum instrument. The heart, liver, spleen, lung, and kidney tissues were collected, and the fluorescence signal of each tissue was detected by IVIS Spectrum instrument.[4]

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Mouse experiments[4]
C57BL/6 mice and HBV transgenic mice were used. For AAV-HBV1.04 transduced mouse model, 6–8 weeks C57BL/6 male mice were injected with 5 × 1010 viral genome equivalents/mouse D genotype AAV-HBV1.04 through tail vein. After 10 days, the model was successfully built. To compare the efficiencies of CKK-E12- and SM-102-based nanoparticles, one dose of these two kinds of LNPs (3 mg/kg body weight) were injected into the mice respectively, and mice received PBS as control group. Then blood samples were collected at 2, 4 and 6 days after treatment. Livers were collected at 6 days after therapy. And in D genotype AAV-HBV1.04 transduced mice, we also evaluated SM-102-based LNPs encapsulating Cas9 mRNA and HBV targeting gRNAs effect with different doses. The mice received 3 or 1.5 mg/kg body weight SM-102-based LNPs encapsulating Cas9 mRNA and HBV targeting gRNAs. And mice received PBS as control group. The blood samples were collected at 2, 4 and 6 days after treatment. The livers were collected at 6 days after LNPs treatment. For HBV transgenic mice, 6 weeks old male mice were used to detect the therapeutic effect of LNPs. Two doses of nanoparticles encapsulating Cas9 mRNA with HBV or GFP targeting gRNAs were injected via i.v. into the mice at 1.5 mg/kg body weight. The blood samples were collected at day 2, 4, 11, 18 and 25 after therapy. The mice were sacrificed on day 25 post LNPs treatment. The blood was centrifuged at 1800 rpm for 15 min at 4 °C. Next, we collected the serum obtained to examine HBeAg, HBsAg, ALT and AST. The levels of HBV RNA, DNA and protein in the liver were determined. [4]

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Tree shrew experiments[4]
The tree shrews used in this study were originated from the Kunming Institute of Zoology, Chinese Academy of Science (Yunnan, China). Tacrolimus was dissolved in 5% DMSO, 40% PEG300, 5% Tween-80 and 50% saline. The resulting tacrolimus solution was injected via i.m. into the tree shrews at 0.08 mg/day/kg body weight in a total volume of 50 μl for the first 14 days. Tree shrews were inoculated i.p. with dexamethasone 10 mg/kg 2 days before and after HBV infection. Liposomal alendronate (CAS 121268-17-5) was injected via i.v. into tree shrews at 0.5 mg/kg the day before HBV infection. After Tacrolimus treated for 7 days, tree shrews were infected with HBV DNA positive human serum via the tail vein and the dose was adjusted to 106 copies/tree shrew. Three days later, tree shrews received 106 copies/tree shrew HBV via i.p. again. After the model successfully built, two doses of SM-102-based LNPs were injected into tree shrews at 1.5 mg/kg body weight for therapy. For control group, the tree shrews received LNPs encapsulating Cas9 mRNA and GFP targeting gRNAs. On day 2 and 4 after the treatment, the blood samples were collected. Tree shrews were sacrificed on day 5 post the nanoparticles treatment to determine the level of HBV RNA, DNA, protein and SMC5 in liver. And hepatitis B e antigen (HBeAg), HBsAg and HBV DNA levels in serum were also detected.

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SM-102-based LNP formulation[4]
LNPs were prepared by microfluidic techniques as reported previously (Mol. Ther., 26 (2018), pp. 1509-1519, 10.1016/j.ymthe.2018.03.010). In brief, SM-102 lipids were dissolved in ethanol at molar ratios of 50: 10: 38.5: 1.5 (ionizable lipid: DSPC: cholesterol: PEG-lipid). Chemically modified (2’ O-Methyl RNA/Phosphorothioated) HBV targeting gRNA and GFP targeting gRNA was synthesized. The RNA cargo (1: 1 wt ratio Cas9 mRNA: gRNA) was dissolved in 6.25 mM sodium acetate buffer (pH 5.0). Then, one volume of lipid mixture and three volume of RNA cargo were injected in to a NanoAssemblr microfluidic mixing device. The LNPs were dialyzed against PBS (pH 7.4) for overnight. The LNPs were passed through a 0.22 μm filter, and stored at 4 °C until use. RNA encapsulation efficiency was characterized by Ribogreen assay. The LNP size and ζ-potential were measured using a dynamic light scattering (DLS) technique.

[1]. A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates. Mol Ther. 2018 Jun 6;26(6):1509-1519.
[2]. Comparison of DLin-MC3-DMA and ALC-0315 for siRNA Delivery to Hepatocytes and Hepatic Stellate Cells. Mol Pharm. 2022;19(7):2175-2182.
[3]. mRNA Synthesis and Encapsulation in Ionizable Lipid Nanoparticles. Curr Protoc. 2023;3(9):e898.
[4]. Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus episomal and integration DNA in mouse and tree shrew models. Antiviral Res . 2023 Jul:215:105618.
[5]. Effective Perturbations on the Amplitude and Hysteresis of Erg-Mediated Potassium Current Caused by 1-Octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6(undecyloxy)hexyl]amino]-octanoate (SM-102), a Cationic Lipid. Biomedicines . 2021 Oct 1;9(10):1367.

Refer to Figure 1 for safe stopping points indicated by red arrows.
Basic Protocol 1: Synthesis of mRNA by in vitro transcription and enzymatic capping and tailing
Allow 2 to 4 days to complete the entire protocol including the production and assessment of capped and tailed mRNA. Four days will be required if precipitations are planned overnight. The IVT, capping and tailing reactions all take approximately half a day. All reactions can be set up in ∼1 hr followed by the required incubation time of two hours for IVT and one hour for capping/tailing. Precipitation of the mRNA requires a 30 min centrifugation step and resuspension of the RNA takes ∼10 min. Quality assessment of the mRNA by Nanodrop, agarose gel and automated gel electrophoresis takes 1 hr.
Note when scaling up, more time is required particularly when multiple or larger mRNA pellets need to be resuspended in nuclease-free water.

Basic Protocol 2: Encapsulation of mRNA into iLNPs
Preparation of lipid solutions may be carried out in advance of the formulation step. Otherwise, the entire encapsulation protocol must be carried out on the same day. Allow an hour for all the reagents to come to room temperature before use and an hour to carry out the formulation and dilution into DPBS step. The centrifugal concentration step is dependent on the particle size, total sample volume and desired end volume. Typically allow 1 to 4 hr. Once concentrated, the iLNP solution can be stored in the fridge until the dilution requirements are determined by the RiboGreen assay.
Alternate Protocol: Small-scale encapsulation of mRNA using preformed vesicles
Same as for Basic Protocol 2.

Basic Protocol 3: Characterization and quality control of mRNA iLNPs
Allow 1 to 2 hr for the RiboGreen assay including allowing the kit to warm to room temperature from the fridge. DLS (size, PDI, zeta potential) should be carried out on the final, diluted sample before it is used for biological evaluation. Preparation of samples takes a few minutes and analysis time varies across different instruments, but it is typically 5 to 10 min per sample.
The buffers used in the TNS assay must be at room temperature before use. Depending on the size of the aliquot this may take several hours. Buffers can be moved to the fridge the night before the assay is to be run to reduce the time required to warm the buffers. The assay requires 40 aliquots in a 96-well plate per iLNP sample therefore allow 10 to 15 min per iLNP sample to prepare the plate and 10 min to read the plate.

Allow 30 min for the mRNA extraction protocol. See above (Basic Protocol 1) for analysis of extracted mRNA by automated gel electrophoresis.[3]

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With 296 million chronically infected individuals worldwide, hepatitis B virus (HBV) causes a major health burden. The major challenge to cure HBV infection lies in the fact that the source of persistence infection, viral episomal covalently closed circular DNA (cccDNA), could not be targeted. In addition, HBV DNA integration, although normally results in replication-incompetent transcripts, considered as oncogenic. Though several studies evaluated the potential of gene-editing approaches to target HBV, previous in vivo studies have been of limited relevance to authentic HBV infection, as the models do not contain HBV cccDNA or feature a complete HBV replication cycle under competent host immune system. In this study, we evaluated the effect of in vivo codelivery of Cas9 mRNA and guide RNAs (gRNAs) by SM-102-based lipid nanoparticles (LNPs) on HBV cccDNA and integrated DNA in mouse and a higher species. CRISPR nanoparticle treatment decreased the levels of HBcAg, HBsAg and cccDNA in AAV-HBV1.04 transduced mouse liver by 53%, 73% and 64% respectively. In HBV infected tree shrews, the treatment achieved 70% reduction of viral RNA and 35% reduction of cccDNA. In HBV transgenic mouse, 90% inhibition of HBV RNA and 95% inhibition of DNA were observed. CRISPR nanoparticle treatment was well tolerated in both mouse and tree shrew, as no elevation of liver enzymes and minimal off-target was observed. Our study demonstrated that SM-102-based CRISPR is safe and effective in targeting HBV episomal and integration DNA in vivo. The system delivered by SM-102-based LNPs may be used as a potential therapeutic strategy against HBV infection.[4]

C44H87NO5

710.1653

709.66

C, 74.42; H, 12.35; N, 1.97; O, 11.26

2089251-47-6

126697616

Colorless to light yellow oily liquid

15.5

1

6

43

50

686

0

BGNVBNJYBVCBJH-UHFFFAOYSA-N

InChI=1S/C44H87NO5/c1-4-7-10-13-16-17-18-24-32-41-49-43(47)35-29-25-31-38-45(39-40-46)37-30-23-19-22-28-36-44(48)50-42(33-26-20-14-11-8-5-2)34-27-21-15-12-9-6-3/h42,46H,4-41H2,1-3H3

heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate

SM102; SM-102; heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; SM-102 (Excipient); T7OBQ65G2I; 1-Octylnonyl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate; Heptadecan-9-yl 8-[(2-Hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate; SM 102

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 :≥ 100 mg/mL (~140.81 mM)
DMSO : ~100 mg/mL (~140.81 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.52 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 2: 2.5 mg/mL (3.52 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 3: ≥ 2.5 mg/mL (3.52 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.)