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

Gokulnath Mahalingam; Aruna Mohan; Porkizhi Arjunan; Ajay Kumar Dhyani; Kanimozhi Subramaniyam; Yogapriya Periyasamy; Srujan Marepally

doi:10.3791/62407

JoVE Journal > Biochemistry

Bioquímica

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

Published: June 10, 2022

doi:

10.3791/62407

Gokulnath Mahalingam*¹, Aruna Mohan*¹, Porkizhi Arjunan*¹, Ajay Kumar Dhyani¹, Kanimozhi Subramaniyam¹, Yogapriya Periyasamy¹, Srujan Marepally¹

¹Centre for Stem Cell Research,Christian Medical College Campus

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 therapy³^,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 acids⁶^,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 approvals⁹^,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

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.
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.
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).
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.
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.
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.
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).
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.
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.
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

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.
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).
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).

Representative 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 process. The denaturing RNA agarose gel electrophoresis data showed that these synthesized RNAs had good integrity and the correct length (750 base RNA, and ~1000 base mRNA with respect to the RNA ladder) (Figure 3).

To prepare cationic liposomes, a thin-film hydration method and sonication were used to form small unilamellar vesicles (SUVs). Physico-chemical characterization of the liposomes revealed that the hydrodynamic diameters were observed around 65 nm and the surface potentials were around +20 meV (Figure 4). A gel retardation assay was performed with liposome-mRNA complexes at varying charge ratios of lipid/base from 1:1 to 8:1. The cationic liposomes showed a high binding efficiency to mRNAs even at a 1:1 charge ratio (Figure 5). Hence, we used a 1:1 charge ratio for mRNA transfection experiments. Liposome mediated eGFP mRNA transfection experiments were performed in HEK-293T and NIH/3T3 cell lines. eGFP expression was analyzed with flow cytometry. me1Ψ-UTP modified mRNA showed superior and stable eGFP protein expression when compared to unmodified mRNA in HEK-293T and NIH/3T3 cells on the 3rd-day post-transfection (Figure 6, 7).

Figure 1: Schematic presentation of me1Ψ-UTP modified mRNA production, liposome preparation and transfection protocol. The IVT DNA Template (T7 promoter-Gene ORF) is amplified by PCR and purified. me1Ψ-UTP modified mRNAs are generated by the IVT process using the IVT DNA template and purified. The cationic liposome is prepared and complexed with me1Ψ-UTP modified mRNA (Lipoplex) and can be transfected into mammalian cells. Please click here to view a larger version of this figure.

Figure 2: Determination of IVT DNA template quality in agarose gel electrophoresis. The purified eGFP IVT DNA template was run on a 1% agarose gel and visualized by gel. Please click here to view a larger version of this figure.

Figure 3: Analysis of size and quality of me1Ψ-UTP modified IVT RNA by denaturing RNA electrophoresis in agarose gel. The purified me1Ψ-UTP modified RNAs were denatured and loaded on a 1% TAE-agarose gel, and the size and quality of RNAs were determined by gel. Lane 1: ssRNA ladder, Lane 2: me1Ψ-UTP modified IVT RNA, Lane 3: me1Ψ-UTP modified IVT RNA with Cap1 and Poly A tail.Please click here to view a larger version of this figure.

Figure 4: Physico-chemical characterization of the liposomes: Surface potentials (A) and hydrodynamic diameters (B). Please click here to view a larger version of this figure.

Figure 5: mRNA binding ability of liposome was determined by denaturing agarose gel retardation assay. Liposome-mRNA complexes (Lipoplexes) were prepared at different lipid/base charge ratios and loaded on a 1% agarose gel and gel documented. Please click here to view a larger version of this figure.

Figure 6: Protein expression efficiency of me1Ψ-UTP modified eGFP mRNA in human HEK-293T cells. (A) Fluorescence images of unmodified eGFP mRNA and me1Ψ-UTP-modified eGFP mRNA transfected with cationic liposomes into HEK-293T cells obtained on 3^rd-day post-transfection (100x Magnification). % of eGFP protein expression (B) and mean fluorescent intensity (MFI). (C) of the transfected cellswere analyzed using flow cytometry. (N=3) Please click here to view a larger version of this figure.

Figure 7: Translation efficiency of me1Ψ-UTP modified eGFP mRNA in mouse NIH/3T3 cells. (A) Fluorescence images of unmodified eGFP mRNA and me1Ψ-UTP-modified eGFP mRNA transfected into NIH/3T3 cells obtained on the 3^rd-day post-transfection (100x Magnification). % of eGFP protein expression (B) and mean fluorescent intensity (MFI). (C) of the transfected cellswere analyzed using flow cytometry. (N=3) Please click here to view a larger version of this figure.

Components	25 µL reaction	Final concentration
5x Q5 buffer	5 µL	1x
10 mM dNTP	0.5 µL	200 µM
10 µM Forward primer	1.25 µL	0.5 µM
10 µM Reverse primer	1.25 µL	0.5 µM
Q5 polymerase	0.25 µL	0.02 U/µL
Gene of interest in plasmid (template)	1-5 ng	variable
Nuclease free water	To 25 µL

Table 1: PCR reaction mixture preparation

Steps	Duration	Temperature	Cycle number
Initial denaturation	30 seconds	98 °C	1
Denaturation	10 seconds	98 °C
Annealing	20 seconds	variable	18-25
Extension	variable	72 °C
Final extension	2 minutes	72 °C	1
Hold	∞	1 5°C

Table 2: PCR cycling conditions

Components	Unmodified RNA	me1Ψ-UTPmodified RNA
RNase-Free Water	variable	variable
Linearized template DNA with T7 RNAP promoter	variable (1 µg)	variable (1 µg)
10x T7 TranscriptionBuffer	2 µL	2 µL
100 mM N1-methyl Pseudouridine	–	1.5 µL
100 mM ATP	1.8 µL	1.8 µL
100 mM UTP	1.8 µL	–
100 mM CTP	1.8 µL	1.8 µL
100 mM GTP	1.8 µL	1.8 µL
100 mM DTT	2 µL	2 µL
40 U/µL RNase Inhibitor	0.5 µL	0.5 µL
T7 Enzyme Solution	2 µL	2 µL
Total Reaction Volume	20 µL	20 µL

Table 3: IVT reaction mixture preparation

Components	RNA ladder	RNA sample
2x RNA loading dye (NEB)	6 µL	6 µL
RNA	2 µL	1 µL (0.5-1 µg )
DEPC treated water	4 µL	5 µL
Total volume	12 µL	12 µL

Table 4: RNA loading dye preparation

Components	Quantity
10x CappingBuffer	10 µL
20 mM GTP	5 µL
20 mM SAM	2.5 µL
RNase Inhibitor	2.5 µL
2'-O-Methyltransferase	4 µL
Total Volume	24 µL

Table 5: Enzymatic Cap-1 synthesis reaction mixture

Components	Quantity
5’-Capped IVT RNA	100 µL
RNase Inhibitor	0.5 µL
10x A-PlusTailingBuffer	12 µL
20 mM ATP	6 µL
4 U/µL A-Plus Poly(A) Polymerase	5 µL
Total Volume	123.5 µL

Table 6: Poly A tailing reaction mixture

Charge ratio	Liposome	DI water	mRNA(500ng)	DI water
1:1	1.5 μL	8.5 μL	1 μL	9 μL
2:1	3 μL	7 μL	1 μL	9 μL
4:1	6 μL	4 μL	1 μL	9 μL
8:1	12 μL	–	1 μL	9 μL
Total volume (20 μL)	10 μL		10 μL

Table 7: Preparation of lipoplex based on charge ratios

Name	Components
50x TAE buffer	Dissolve 50 mM EDTA sodium salt, 2 M Tris, 1 M glacial acetic acid in 1 L of water
HEK-293T and NIH/3T3 cell culture medium	DMEM with 4.5 g/L glucose, L-glutamine, 1% penicillin/streptomycin and 10% FBS

Table 8: Preparation of buffer and media

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 efficiency of target proteins, improved stability at room temperature, and prevented degradation from nucleases¹³^, ¹⁴.

In this video, we demonstrated the protocol for lipid enabled me1Ψ-UTP modified mRNA delivery into multiple cultured cells. The protocol includes production of me1Ψ-UTP modified mRNA, cationic liposome preparation, transfection into cells, and evaluation of protein expression. We used the mammalian codon-optimized eGFP reporter gene for transfection experiments to analyze protein expression levels by measuring fluorescent intensity. Cationic liposomes were prepared to complex mRNA, and their electrostatic complexation was analyzed at varying lipid/base charge ratios from 1:1 to 8:1. Since at 1:1, the cationic liposomes could completely complex mRNA, we used a 1:1 charge ratio for transfection. We demonstrated that transfection of mRNA with cationic liposomes could efficiently deliver both modified and unmodified eGFP mRNAs with 90% transfection efficiency in HEK-293T cells, whereas there were 80% efficiency with modified and 60% efficiency with unmodified mRNAs in NIH/373 cells. More importantly, me1Ψ-UTP modified mRNA showed superior eGFP protein expression for 3 days in mammalian cells compared to unmodified mRNA (>6 fold in HEK-293T and >2 fold in NIH/3T3 cells). These studies demonstrated that modification of me1Ψ-UTP on mRNA could improve translation and stability of mRNA in mammalian cells.

The transfection efficiency of cationic liposome and translation efficiency of synthesized mRNA vary with different cell types. Hence, it is important to optimize mRNA concentration for each different cell type. Using the protocol, we synthesized functional me1Ψ-UTP modified mRNA, size up to 6 kb but the IVT DNA template concentration and time could be optimized to get good mRNA yield and correct length.

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

MS thanks the Department of Biotechnology, India, for the financial support (BT/PR25841/GET/119/162/2017), Dr Alok Srivastava, Head, CSCR, Vellore, for his support and Dr Sandhya, CSCR core facilities for imaging and FACS experiments. We thank R. Harikrishna Reddy and Rajkumar Banerjee, Applied Biology Division, CSIR-Indian Institute of Chemical Technology Uppal Road, Tarnaka, Hyderabad, 500 007, TS, India, for their help in analyzing physico-chemical data of the liposomes. Vigneshwaran V, and Joshua A, CSCR for their help in video making.

Materials

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

Referencias

Sahin, U., Kariko, K., Tureci, O. mRNA-based therapeutics–developing a new class of drugs. Nature Reviews Drug Discovery. 13 (10), 759-780 (2014).
Schlake, T., Thess, A., Fotin-Mleczek, M., Kallen, K. J. Developing mRNA-vaccine technologies. RNA Biology. 9 (11), 1319-1330 (2012).
Carlile, T. M., et al. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature. 515 (7525), 143-146 (2014).
Kariko, K., Buckstein, M., Ni, H., Weissman, D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity. 23 (2), 165-175 (2005).
Guan, S., Rosenecker, J. Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems. Gene Therapy. 24 (3), 133-143 (2017).
Srujan, M., et al. The influence of the structural orientation of amide linkers on the serum compatibility and lung transfection properties of cationic amphiphiles. Biomaterials. 32 (22), 5231-5240 (2011).
Dharmalingam, P., et al. Transfection: Cationic Lipid Nanocarrier System Derivatized from Vegetable Fat, Palmstearin Enhances Nucleic Acid Transfections. ACS Omega. 2 (11), 7892-7903 (2017).
Hoy, S. M. Patisiran: First Global Approval. Drugs. 78 (15), 1625-1631 (2018).
Anderson, E. J., et al. Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults. New England Journal of Medicine. , (2020).
Polack, F. P., et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. New England Journal of Medicine. , (2020).
Schlee, M., Hartmann, G. Discriminating self from non-self in nucleic acid-sensing. Nature Reviews Immunology. 16 (9), 566-580 (2016).
Kariko, K., Buckstein, M., Hi, H., Weissman, D. Suppression of RNA recognition by Toll-like receptors: The impact of nucleoside modification and evolutionary origin of RNA. Immunity. 23 (2), 165-175 (2005).
Mauger, D. M., et al. mRNA structure regulates protein expression through changes in functional half-life. Proceedings of the National Academy of Sciences of the United States of America. 116 (48), 24075-24083 (2019).
Vaidyanathan, S., et al. Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification. Molecular Therapy – Nucleic Acids. 12, 530-542 (2018).