Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells

Summary

The protocol presents in vitro transcription (IVT) of chemically modified mRNA, cationic liposome preparation, and functional analysis of liposome enabled mRNA transfections in mammalian cells.

Abstract

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic applications, including a novel class of vaccines, protein replacement therapies, and immune therapies. Among delivery vectors, lipid nanoparticles are found to be safer and more effective in delivering RNA molecules (e.g., siRNA, miRNA, mRNA) and a few products are already in clinical use. To demonstrate lipid nanoparticle-mediated mRNA delivery, we present an optimized protocol for the synthesis of functional me1Ψ-UTP modified eGFP mRNA, the preparation of cationic liposomes, the electrostatic complex formation of mRNA with cationic liposomes, and the evaluation of transfection efficiencies in mammalian cells. The results demonstrate that these modifications efficiently improved the stability of mRNA when delivered with cationic liposomes and increased the eGFP mRNA translation efficiency and stability in mammalian cells. This protocol can be used to synthesize the desired mRNA and transfect with cationic liposomes for target gene expression in mammalian cells.

Introduction

As a therapeutic molecule, mRNA offers several advantages due to its non-integrative nature and its ability to transfect non-mitotic cells when compared to plasmid DNA (pDNA)¹. Although mRNA delivery was demonstrated in the early 1990s, therapeutic applications were limited due to its lack of stability, its lack of immune activation, and poor translational efficiency². Recently identified chemical modifications, such as pseudouridine 5'-triphosphate (Ψ-UTP) and methyl pseudouridine 5'-triphosphate (me1Ψ-UTP) on mRNA, helped to overcome these limitations, revolutionized mRNA research, and in turn, made mRNA a promising tool in both basic and applied research. The range of applications covers the generation of iPSCs to vaccination and gene therapy^3,4.

In parallel to advancement in mRNA technology, significant advances in non-viral delivery systems made the delivery of mRNA effective, making this technology feasible for multiple therapeutic applications⁵. Among the non-viral vectors, lipid nanoparticles have been found to be effective in delivering nucleic acids^6,7. Recently, Alnylam has received FDA approval of lipid-based siRNA drugs for treating liver diseases, including Patisiran for hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis) and Givosiran for acute hepatic porphyrias (AHP)⁸. During the COVID19 pandemic, lipid encapsulated mRNA based vaccines from Pfizer-BioNtech and Moderna demonstrated their efficacy and received FDA approvals^9,10. Thus, lipid enabled mRNA delivery has a great therapeutic potential.

Here, we describe a detailed protocol for the production of chemically modified, in vitro transcribed eGFP mRNA, cationic liposome preparation, mRNA-lipid complex optimization and transfections into mammalian cells (Figure 1).

Protocol

1. Production of me1 Ψ-UTP modified mRNA

1. In vitro transcription (IVT) DNA template preparation
   NOTE: For IVT DNA template (T7 promoter- open reading frame (ORF) of the gene) preparation, design a gene-specific primer set for the gene of interest. Add the T7 promoter (5'-NNNNNNTAATACGACTCACTATAGGGNNNNNN-3') sequence before gene-specific forward primer.
   1. Prepare PCR reaction mixture as described in Table 1.
      NOTE: Run at least four PCR reactions to increase the IVT DNA template concentration and quality for IVT.
   2. Completely mix the reaction mixture with a micropipette and spin down using a microfuge.
   3. Run the PCR cycling protocol given in Table 2 on a thermocycler.
2. Purification of IVT DNA template by organic extraction/ethanol precipitation
   1. Adjust the amplified PCR reaction mixture to 200 µL total using DEPC-treated water in a 1.5 mL microfuge tube (nuclease-free).
   2. Add 200 µL of TE-saturated phenol/chloroform, pH 8.0. Vortex vigorously for 10seconds.
   3. Centrifuge at 12,000 x g for 5 minutes to separate thephases and transfer the aqueous upper phase (approximately 200 µL) to a new 1.5 mL microfuge tube.
   4. Add 1/10^th (20 µL) volume of 3 M sodium acetate, pH 5.5 and two-volumes (400 µL) of 99-100% ethanol. Mix well and then incubate for at least 30 minutes at -20 °C.
   5. Pellet the DNA template by centrifugation at 12,000 x g for 15 minutes at 4 °C.
   6. Remove the supernatant completely without disturbing the pellet using a micropipette.
   7. Add 0.5 mL of 75% ethanol to the pellet and invert 5-10 times.
   8. Centrifuge for 2 minutes at 12,000 x g at 4 °C. Then remove the ethanol completely with a pipette without disturbing the DNApellet.
   9. Allow the pellet to dry at room temperature until the pellet becomes a little translucent.
   10. Add 20 µL of nuclease-free water and re-suspend thoroughly for few seconds.
3. Quality control for the purified IVT DNA template
   1. Quantification
      1. Measure purified IVT DNA template concentration and quality using a micro-spectrophotometer.
         NOTE: The expected DNA concentration will be around 300-600 ng/µL. Store the IVT DNA template at -20 °C for the long term.
   2. DNA agarose gel electrophoresis
      NOTE: This experiment is to verify whether the purified IVT DNA template is the correct size and devoid of non-specific product contamination.
      1. To prepare a 1% agarose gel, add 0.5 g of agarose and 50 mL of 1x TAE in a conical flask. Microwave it until the agarose dissolves completely. Cool the agarose at room temperature for 5 minutes.
      2. Add 1 µL of nucleic acid stain (SafeView dye) for a 50 mL agarose solution.
      3. Pour the agarose solution into a gel casting tray with a comb and leave it until the gel becomes solidified.
      4. Take out the comb from the gel and keep the gel in the 1x TAE buffered tank.
      5. Mix 10 µL of 100-10000 bp DNA ladder and 100-200 ng of PCR purified template product with 2 µL of 6x DNA loading buffer to a total volume of 12 µL.
      6. Load each sample on respective wells and run at 100 V for at least 45-60 minutes.
      7. Visualize the DNA bands on a gel documentation instrument (Figure 2).
4. Synthesis of me1Ψ-UTP modified RNA
   ​NOTE: Before starting this experiment, the working area (laminar airflow) should be cleaned with 70% ethanol in DEPC-treated water. Use sterile nuclease and endotoxin free, low retention tubes and filter barrier tips. Frequently apply 70% ethanol to gloved hands.
   1. Prepare the IVT reaction mixture as given in Table 3 at room temperature in a 0.2 mL tube and mix it thoroughly using a micropipette.
   2. Spin the tube for 10 seconds in a microfuge.
   3. Incubate at 37 °C for 3 hours in a thermocycler.
5. Degradation of IVT DNA template by DNase 1 treatment
   1. Add 1 µL of 1 U/µL DNase 1 (RNase free) into the IVT reaction mix and incubate at 37 °C for 30 minutes.
6. Purification of RNA by organic extraction/ammonium acetate precipitation
   1. Adjust the volume of the IVT reaction mix to 200 µL with 179 µL of DEPC-treated water.
   2. Add 200 µL of TE-saturated phenol/chloroform pH 8.0. Vortex it for 10seconds.
   3. Centrifuge at 12,000 x g for 5 minutes at 25 °C to separate the two phases.
   4. Transfer the aqueous upper phase (200 µL) to a 1.5 mL tube and add 200 µL of 5 M ammonium acetate. Mix well and then incubate for 15 minutes on ice to precipitate the RNA.
   5. Pellet the precipitated RNA by centrifugation at 12,000 x g for 15 minutes at 4 °C and remove the supernatant completely with a micropipette.
   6. Wash the RNA pellet by using 70% ethanol and invert 5-10 times. Centrifuge at 12,000 x g for 5 minutes at 4 °C.
   7. Remove the supernatant completely with a micropipette without disturbing the RNA pellet.
   8. Allow the pellet to dry at room temperature till the pellet become semi-translucent. Then re-suspend the pellet in 60-75 µL of RNase-free water.
7. Quality control for purified RNA
   1. Quantification
      1. Measure the purified RNA concentration and quality using a micro-spectrophotometer.
         NOTE: The expected RNA yield will be 140-180 µg for unmodified RNA and 100-150 µg for me1Ψ-UTP modified RNA per reaction, depending on the size of the gene of interest and quality of IVT DNA template. The optimal quality should be a OD₂₆₀/OD₂₈₀ ratio around 1.9-2.0 and OD₂₆₀/ OD₂₃₀ ratio >2.0. Store the RNA at -20 °C for a short time.
   2. Denaturing RNA agarose gel electrophoresis
      ​NOTE: This experiment is performed to verify whether the synthesized RNA is of the correct length and devoid of IVT by-product contamination.
      1. To prepare a 1% agarose gel, add 0.5 g of agarose and 50 mL of 1x TAE in a conical flask. Microwave the solution until the agarose gets dissolved completely. Keep the agarose solution in a room temperature for 5 minutes to cool down.
      2. Add 1 µL of nucleic acid stain for 50 mL of 1% agarose solution.
      3. Pour the agarose solution into the gel casting tray with a comb and leave it until the gel becomes solidified.
      4. Take out the comb from the gel and keep the gel in the 1x TAE buffered tank.
      5. Prepare the RNA loading dye sample as described in Table 4.
      6. Heat the samples at 65 °C for 10 minutes and then keep samples on ice.
      7. Load each sample on the respective wells and run at 100 V for at least 45-60 minutes.
      8. Visualize the RNA bands on a gel documentation instrument (Figure 3).
8. Synthesis of me1Ψ-UTP modified mRNA by enzymatic based capping & poly-A tailing
   NOTE: The capping efficiency of IVT RNA using the enzymatic method is 100%. Hence, we used enzymatic capping of Cap-1 in mRNA synthesis in this protocol. We added poly-A tails at a length of >150 A bases per molecule to improve the translational efficiency of mRNA.
   1. Add 55-60 µg of purified IVT RNA and make it up to 72 µL with the RNase free water in a 1.5 mL tube.
   2. Denature the RNA at 65 °C for 10 minutes in a thermomixer and then immediately place the tube on ice for 5 minutes.
   3. Meanwhile, prepare the capping reaction mixture as shown in Table 5.
   4. Add the capping reaction mixture and 4 µL of capping enzyme to the denatured RNA and mix well by micropipette. Spin the tube for 10 seconds in a microfuge.
   5. Incubate the reaction mixture at 37 °C for 2 hours.
   6. After 2 hours, keep the tube on ice and prepare the Poly A tailing master mix as given in Table 6.
   7. Add the Poly A tailing master mix to the capped RNA solution and mix well by micropipette. Spin the tube for 10 seconds in a microfuge.
   8. Incubate the reaction mixture at 37 °C for 2 hours.
      NOTE: mRNA can be further subjected to purification immediately, or crude mRNA can be stored at -20 °C overnight.
9. IVT mRNA purification
   1. Purify the mRNA by organic extraction/ammonium acetate precipitation as given in protocol section 1.6.
   2. Re-suspend the mRNA pellet with 60 µL of RNase-free water.
10. Quality control for the purified mRNA
    1. Follow quality control protocol as described in protocol section 1.7.
       NOTE: The mRNA band should appear above the RNA band due to the addition of Poly-A tailing in the denaturing RNA agarose gel electrophoresis (Figure 3). Also, Poly-A tailing increases the mRNA yield (should be > RNA concentration). After quantification, put multiple aliquots of mRNA at 1 µg/µL concentration and store immediately at -80 °C. Avoid multiple freeze and thaw cycles of RNA to prevent the degradation of synthesized mRNA.

2. Preparation of cationic liposomes and evaluation of in vitro mRNA transfection properties

1. Liposome preparation
   1. For the preparation of 1 mM cationic liposome, use cationic lipid: DOPE: cholesterol in the molar ratio of 1:1:0.5.
   2. Dissolve appropriate molar ratios of the cationic lipid, cholesterol, and DOPE (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine) in chloroform (200 µL) in a glass vial.
   3. Use a thin flow of moisture-free nitrogen gas for solvent removal.
   4. Keep dried lipids under a high vacuum for further drying for 2 hours.
   5. Add 1 mL of sterile deionized water to dried lipids after vacuum-drying and allow the mixture to swell overnight.
   6. Vortex the vial at room temperature to make multi-unilamellar vesicles (MUVs).
   7. Use bath sonication followed by probe sonication at 25 W power to make small unilamellar vesicles (SUVs) from multi unilamellar vesicles (MUVs).
      ​NOTE: The SUVs should look like a translucent liposome solution. If not, increase the number of 30-second pulses on and off with an interval of 1 min. Hydrodynamic diameters and surface potentials are measured (Figure 4) in a particle size analyzer.
2. mRNA/liposomes complex formation and gel retardation assay
   1. For the preparation of different lipid-RNA charge ratios from 1:1 to 8:1, dilute the mRNA and cationic liposome in deionized water separately as given in Table 7.
   2. Mix the diluted mRNA to liposome solution as indicated in Table 7 and incubate it for 10 minutes at room temperature for lipoplex formation.
   3. Add 20 µL of 2x RNA loading dye into the complex and load onto the well. mRNA alone serves as a control.
   4. Load the samples on a 1% agarose gel in 1x TAE buffer and run at 100 V for 45 minutes.
   5. Visualize the RNA bands on a gel documentation instrument (Figure 5).
3. In vitro mRNA transfection
   1. Seed 45,000 mammalian cells per well of 48 well plates in complete media and then incubate at 37 °C for 16 to 20 hours of transfection.
   2. After 16 to 20 hours, check the cell density. At the time of transfection, the cell confluence should be around 80%.
   3. To 0.5 mL tubes, add 150 ng of GFP protein-encoding mRNA complex with a 1:1 charge ratio of cationic liposomes and mRNA in DMEM medium without serum. The total volume makes up to 20 µL.
   4. Incubate at room temperature for 10 minutes.
   5. Add lipoplex into the cells and incubate it for 4 hours in a 37 °C and 5% CO₂ incubator.
   6. Remove the media without disturbing the cells. Add 250 µL of complete media with 10% FBS (Table 8) into each well.
   7. After 72 hours of transfection, view GFP expression under a fluorescent microscope (Figure 6, 7).
   8. To quantify the GFP expression, process the cells for flow cytometer analysis.
   9. Remove the media and wash with 1x PBS twice. Trypsinize the cells and process the cells to quantifying the percentage of GFP positive cells in a flow cytometer.
   10. Acquire the cells in a flow cytometer using Laser 488. Gate the live population from that and analyze the percentage of GFP positive cells. Quantify the mean fluorescent intensity (MFI) (Figure 6, 7).

Results

We optimized the protocol for me1Ψ-UTP modified mRNA production, liposome preparation, and mRNA transfection experiments with cationic liposomes into multiple mammalian cells (Figure 1). To synthesize mRNA, the mammalian codon-optimized eGFP IVT template was amplified from the mEGFP-N1 mammalian expression vector and purified by organic extraction/ethanol precipitation method (Figure 2). Later, me1Ψ-UTP modified RNA and mRNA were produced by the IVT pr...

Discussion

Therapeutic applications of unmodified mRNAs have been limited due to their shorter half-life and their ability to activate intracellular innate immune responses, which in turn lead to poor protein expression in transfected cells¹¹. Katalin et al. demonstrated that RNA containing modified nucleosides such as m5C, m6A, ΨU, and me1Ψ-UTP could avoid TLR activation¹². More importantly, incorporation of ΨU or me1Ψ-UTP in IVT mRNA showed superior translational...

Materials

Name	Company	Catalog Number	Comments
Agarose	Lonza	50004
Bath sonicator	DNMANM Industries	USC-100
Cationic lipid	Synthesized in the lab
Chlorofrom	MP biomedicals	67-66-3	"Caution"
Cholesterol	Himedia	GRM335
DEPC water	SRL BioLit	66886
DMEM	Lonza	12-604F
DNA Ladder	GeneDireX	DM010-R50C
DOPE	TCI	D4251
EDTA sodium salt	MP biomedicals	194822
Ethanol	Hayman	F204325	"Caution"
Fetal bovine serum	Gibco	10270
Flow cytometry	BD	FACS Celesta
Fluroscence Microscope	Leica	MI6000B
Gel documentation system	Cell Biosciences	Flurochem E
Glacial acetic acid	Fisher Scientific	85801	"Caution"
mEGFP-N1, Mammalian expression vector	Addgene	54767
N1-Methylpseudo-UTP	Jena Bioscience	NU-890
Phenol:chloroform:isoamyl alchol (25:24:1), pH 8.0	SRL BioLit	136112-00-0	"Caution"
Phosphate Buffer Saline (PBS), pH 7.4	CellClone	CC3041
Probe sonicator	Sonics Vibra Cells	VCX130
RNA ladder	NEB	N0362S
RNase inhibitor	Thermo Scientific	N8080119
SafeView dye	abm	G108
Sodium acetate	Sigma	S7545
Thermocycler	Applied biosystems	4375786
Thermomixer	Eppendrof	22331
Tris buffer	SRL BioLit	71033
Trypsin	Gibco	25200056