ionizable cationic lipid; RNA delivery

Protocols for mRNA Synthesis and Encapsulation in Ionizable Lipid Nanoparticles: [2]
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

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By means of a steric mechanism, the polyethylene glycol (PEG) moiety of ALC-0159 aids in the stability of nanoparticles [1].
The LNPs in the Pfizer-BioNTech COVID-19 vaccine contain low levels (<2 mol %) of ALC-0159, which contributes to nanoparticle stabilization by a steric mechanism through its poly(ethylene glycol) (PEG) moiety. In the Moderna COVID-19 vaccine, ALC-0159 is replaced with another PEGylated lipid (1,2-dimyristoyl-rac-glycero-3-methoxyPEG2000). There are speculations on a possible role for ALC-0159 (the PEGylated lipid) in triggering anaphylaxis, based on earlier reported anaphylactic reactions in some recipients of intravenously infused PEGylated nanomedicines.[1]

mRNA vaccines have recently generated significant interest due to their success during the COVID-19 pandemic. Their success is due to advances in mRNA design and encapsulation into ionizable lipid nanoparticles (iLNPs). This has highlighted the potential for the use of mRNA-iLNPs in other settings such as cancer, gene therapy, or vaccines for different infectious diseases. Here, we describe the production of mRNA-iLNPs using commercially available reagents that are suitable for use as vaccines and therapeutics. This article contains detailed protocols for the synthesis of mRNA by in vitro transcription with enzymatic capping and tailing and the encapsulation of the mRNA into iLNPs using the ionizable lipid DLin-MC3-DMA. DLin-MC3-DMA is often used as a benchmark for new formulations and provides an efficient delivery vehicle for screening mRNA design. The protocol also describes how the formulation can be adapted to other lipids. Finally, a stepwise methodology is presented for the characterization and quality control of mRNA-iLNPs, including measuring mRNA concentration and encapsulation efficiency, particle size, and zeta potential.[2]

[1]. Allergic Reactions and Anaphylaxis to LNP-Based COVID-19 Vaccines. Mol Ther. 2021 Mar 3;29(3):898-900.

ALC-0315 is an ionisable aminolipid that is responsible for mRNA compaction and aids mRNA cellular delivery and its cytoplasmic release through suspected endosomal destabilization. The LNPs in the Pfizer-BioNTech COVID-19 vaccine contain low levels (<2 mol %) of ALC-0159, which contributes to nanoparticle stabilization by a steric mechanism through its poly(ethylene glycol) (PEG) moiety. Considering their low ALC-0159 content, LNPs in the Pfizer-BioNTech COVID-19 vaccine most likely display a weak steric barrier of PEG. [1]

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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]

C33H70N2O3

542.9205

542.538

1849616-42-7

155977658

White to off-white solid

1

4

31

38

415

0

BPWFJNQUTKVHIR-UHFFFAOYSA-N

InChI=1S/C33H67NO3/c1-4-6-8-10-12-14-16-18-20-22-24-26-28-34(33(35)32-37-31-30-36-3)29-27-25-23-21-19-17-15-13-11-9-7-5-2/h4-32H2,1-3H3

2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide

ALC0159; ALC 0159; ALC-0159; Azane;2-(2-methoxyethoxy)-N,N-di(tetradecyl)acetamide; mPEG-DTA; PEG-N,N-ditetradecylacetamide; ALC-0159

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)

DMSO : ~100 mg/mL
Ethanol :≥ 50 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (Infinity 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 (Infinity mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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 (Infinity 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.)