Protocol for the development of mRNA lipid nanoparticle vaccines and analysis of immunization efficiency in mice

Summary

Here, we present a protocol for the development of mRNA-loaded lipid nanoparticle (LNP) vaccines for target antigen sequences of interest. We describe key steps required to design and synthesize mRNA constructs, their LNP encapsulation, and mouse immunization. We then detail quality control assays to determine RNA purity, guidelines to measure RNA immunogenicity using in vitro reporter systems, and a technique to evaluate antigen-specific T cell responses following immunization.

Graphical abstract

Highlights

• •Protocol for the generation of mRNA-encapsulated lipid nanoparticle (LNP) vaccine
• •In vitro assays to measure mRNA immunogenicity, LNP integrity, and antigen presentation
• •Immune assays to measure antigen-specific T cell responses ex vivo after vaccination

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for the development of mRNA-loaded lipid nanoparticle (LNP) vaccines for target antigen sequences of interest. We describe key steps required to design and synthesize mRNA constructs, their LNP encapsulation, and mouse immunization. We then detail quality control assays to determine RNA purity, guidelines to measure RNA immunogenicity using in vitro reporter systems, and a technique to evaluate antigen-specific T cell responses following immunization.

Before you begin

Considerations for mRNA sequence design

The mRNA sequence used in this study includes a 5′ untranslated region (UTR), a sequence encoding 5 antigens, a GFP reporter gene, a 3′UTR and a poly adenylation (PolyA) tail. An adenine (A) nucleotide was engineered upstream of the 5′UTR to enable effective capping of the in vitro transcribed mRNAs using the CleanCap AG analog cap from TriLink BioTechnologies. The antigens incorporated into this mRNA vaccine construct include murine shared tumor-associated antigens, a tumor neoantigen, and a model epitope derived from ovalbumin (OVA), each separated with 10-mer glycine-serine (GS) linker sequences. 5′UTR and 3′UTR sequences are important for optimal mRNA stability and translation. In mRNA vaccines, these sequences are often derived from the UTRs of highly expressed genes to encourage effective translation, for example α-globin. The UTR regions in the mRNA vaccines BNT162b2 and mRNA1273 from BioNTech or Moderna¹ respectively, provide two examples, with UTRs based on the α-globin, AES/TLE5 and mtRNR1 genes. A polyA tail also promotes effective translation and mRNA stability and can be encoded in the DNA template or added after in vitro transcription (IVT). In this protocol, the polyA tail consists of a 30-nucleotides A stretch, followed by a stabilizing 5′-GCATATGACT-3′ sequence, itself followed by another 70-nucleotides A stretch. When designing the mRNA-encoding plasmid sequence, it is also important to consider the inclusion of a unique restriction enzyme site in the plasmid, which facilitates the linearization of plasmid DNA 3′ downstream of the mRNA construct sequence, prior to IVT. As such, we integrated a Type IIS restriction enzyme recognition site (BsPQI) that cuts at the 3′ extremity of the polyA tail.² Finally, the coding region of the RNA construct can be codon optimized to ensure optimal expression in the host organism. Here, the antigen’s sequence was codon optimized using GENEWIZ bioinformatics software tools prior to cloning into the mRNA encoding plasmid.

Institutional permissions

All animal experiments in this protocol were performed per procedures approved by the Icahn School of Medicine at Mount Sinai’s Institutional Animal Care and Use Committee (IACUC). Before you begin, all animal experiments must be approved by the IACUC of your relevant institutions.

Preparatory steps

The following protocol outlines three key preparatory steps, namely.

• 1.Bacterial transformation to amplify plasmid DNA encoding the mRNA template.
• 2.Generation of bone-marrow-derived dendritic cells (BMDC) for in vitro testing of mRNA nanoparticles.
• 3.Culture and collection of B16F10 murine melanoma cells for engraftment in vivo.

Preparation of plasmid DNA

Timing: 3days

• 4.For bacterial transformation, thaw competent STBL3 E. coli cells on ice, this usually takes approximately 20 min.
• 5.Mix a small amount of plasmid DNA (<10 ng, 1–2 μL) with 50 μL of the bacterial cells in an Eppendorf.
• 6.Gently flick the tube to mix the solution and incubate the mixture on ice for 30 min.
• 7.Heat shock the cells by incubating the tube in a water bath at 42°C for 45 s.
• 8.Place the tube back on ice and incubate for 2 min.
• 9.Add 500 μL of Luria-Bertani (LB) broth (without any antibiotic) and incubate in a shaking incubator (37°C, 250 rpm) for 1 h.
• 10.Plate 100 μL of the incubated mixture on a 10 cm LB agar plate containing 0.1% ampicillin and incubate the plate for 12–14 h at 37°C.
• 11.After incubation, check for bacterial colony growth (the amount and quality of the bacterial plasmid will depend upon the plasmid design).
• 12.Screen for well-isolated bacterial colonies.
• 13.Pick the colony using a sterile spatula or pipette tip. Add the colony to a 5 mL LB broth with 0.1% ampicillin and incubate in a shaking incubator (37°C, 250 rpm) for 6 h.
• 14.Check for bacterial growth (the LB broth should turn turbid indicating bacterial growth).
• 15.Add 1 mL of the culture into 250 mL of LB broth (with 0.1% ampicillin antibiotic).
• 16.Incubate 8–10 h on a shaking incubator (37°C, 250 rpm).
• 17.Following incubation, proceed with isolating plasmid DNA using a plasmid DNA isolation kit.

Note: This step can be performed using any commercially available endotoxin-free plasmid DNA isolation kit. We use a kit from Promega - PureYield Plasmid Maxiprep System for this experiment.

Generating bone-marrow-derived dendritic cells (BMDCs)

Timing: 9days

This step involves isolating bone marrow cells, culturing them with GM-CSF to drive their differentiation into dendritic cells. The cultures are subjected to washing and differentiation steps via depletion of macrophages over the course of 7–9 days to obtain a population of BMDCs suitable for experimental use.

Note: For all centrifugation steps involving cells throughout this protocol, unless otherwise specified, the centrifuge should be set to an acceleration of 9 and a deceleration of 5.

• 18.Euthanize a 6 to 8-week-old mouse.
• 19.Without damaging the bone, isolate the leg, remove soft tissue, and separate the tibia from the femur.
• 20.Clean the bones and disinfect them by placing them in an aqueous solution of 70% ethanol for 1 min, before putting the bones in 2 mL 1X PBS on ice.
• 21.Cut the bone edges and flush the bone marrow cells into an empty 50 mL falcon tube using a 27G needle and syringe using 25 mL 1X PBS.
• 22.Centrifuge the bone marrow cells at 500 × g for 5 min at 4°C.
• 23.Aspirate the supernatant and re-suspend the cells in 2 mL pre-warmed Iscove’s Modified Dulbecco’s Medium (IMDM) complete culture medium before counting using trypan blue exclusion staining.
• 24.Plate 15 × 10⁶ cells in non-tissue culture-treated 100 mm petri dishes with 10 mL IMDM culture media supplemented with 20 ng/mL of GM-CSF (BMDC culture media).
• 25.Incubate in a cell culture incubator at 37°C under a 5% CO₂ atmosphere until the cells start differentiating.

Note: It will take approximately 3 days for the cells to start differentiating.

• 26.On day 4, collect the media in a 15 mL falcon tube.
• 27.Add 3–4 mL dissociation media, incubate for 7 min at 37°C and gently collect the dissociated cells using a pipette. Add them to the supernatant collected in 8.

Note: The BMDC culture should be dissociated gently to limit cell activation due to rupture of E-cadherin adhesion. When looking at the plate under a microscope, you should see a mix of adherent cells with dendrites and others that remain round. Most tightly adhered cells are macrophages, these cells will not become dissociated and will be depleted during this step.

• 28.Centrifuge at 500 × g for 5 min at room temperature.
• 29.Aspirate the supernatant and resuspend the cells in 1 mL culture media.
• 30.Count the cells and plate 5 × 10⁶ cells on non-tissue culture-treated 100 mm petri dishes with 10 mL BMDC culture media.
• 31.Place the dish back in the cell culture incubator until day 7.
• 32.On day 7, aspirate and discard the culture media.
• 33.Add 3–4 mL dissociation media, incubate for 7 min at 37°C and gently collect the dissociated cells using a pipette.
• 34.Centrifuge the dissociated cells at 500 × g for 5 min at room temperature and proceed to repeat step 11.

Note: After 7–9 days of culturing the cells with repeated washing and differentiation steps, the bone-marrow-derived dendritic cells can be utilized for downstream experiments.

• 35.Aspirate and discard the culture media.
• 36.Add 3–4 mL dissociation media, incubate for 7 min at 37°C and gently collect the dissociated cells using a pipette.
• 37.Centrifuge the dissociated cells at 500 × g for 5 min at room temperature, resuspend, count the cells, and proceed with downstream experiments.

Collection of B16F10 melanoma cells for tumor engraftment

Timing: 30 min

B16F10 murine melanoma cells are cultured in Dulbecco’s Modified Eagle Medium (DMEM) culture media. Cells should be cultured for at least 3–7 days after thawing before being collected for engraftment to allow recovery from thawing process. To maintain 30%–80% confluency during culture of B16F10 cells, split the cells 1 in 10 (i.e., keep 10% of the cells in culture) every 2–3 days. When mice have been prepared as detailed in section 10 of the step-by-step methods detailed below and B16F10 cells are 70%–80% confluent in culture, collect the B16F10 cells for engraftment as follows.

• 38.
  Aspirate culture media and wash cells once with 1x PBS before adding TrypLE Express to cover the bottom of the flask (2 mL of TrypLE is sufficient to cover a T75 flask).

  • a.
    Incubate at room temperature (25°C) for 3–6 min until the cells detach from the flask when agitated.
  • b.
    Quench with 4x the volume of TrypLE in cell culture media.
  • c.
    Spin at 500 × g for 5 min at 4°C.
• 39.
  Aspirate the supernatant and resuspend cells in 1 mL of 1X PBS.

  • a.
    Count the cells.
  • b.
    Transfer enough cells for the number of mice to be engrafted to a new tube, including 20% extra (e.g., in preparing cells to engraft 1.5 × 10⁵ cells per mouse in 10 mice, prepare 1.8 × 10⁶ cells total).
• 40.Spin down collected cells as above, resuspend in 5 mL 1X PBS to wash cells, and spin again.
• 41.Resuspend cells in 100 μL 1X PBS per 1.5 × 10⁵ cells (e.g., resuspend 1.8 × 10⁶ cells in 1200 μL).
• 42.Engraft cells immediately following collection and keep cells on ice until injected.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-dsRNA monoclonal antibody J2 (1:5,000)	Jena Bioscience	# RNT-SCI-10010200
LIVE/DEAD fixable aqua dead cell stain kit (1:1,000)	Invitrogen	#L34957
PerCP/Cyanine5.5 anti-mouse CD45 antibody (1:200 or 1:400)	BioLegend	#103131
Brilliant Violet 421 anti-mouse CD3 antibody (1:400)	BioLegend	#100227
Brilliant Violet 711 anti-mouse CD4 atibody (1:400)	BioLegend	#100549
Anti-CD8 (mouse) mAb-Alexa Fluor 647 (1:800)	MBL	#K0227-A64
OVA257-264 (SIINFEKL) peptide bound to H2-Kb monoclonal antibody (eBio25-D1.16 (25-D1.16)), PE (1:200)	Invitrogen	#12-5743-82
iTAg MHC tetramer H2-Kb OVA SIINFEKL PE (1:200)	MBL	# TB-5001-1
CD11c monoclonal antibody (N418), APC-eFluor 780, eBioscience (1:200)	Invitrogen	# 47-0114-82
Bacterial and virus strains
STBL3 E.coli	New England Biolabs	#C2987H
Chemicals, peptides, and recombinant proteins
BsPQI	New England Biolabs	#R0712S
DNase I	Zymo Research	#E1010
Whatman Nytran SuPerCharge membrane	Sigma-Aldrich	#WHA10416296
Whatman GB005 blotting paper	Millipore Sigma	#WHA10426994
1 mL slip tip (Becton Dickinson) syringe	Fisher Scientific	# 14-826-88
TBS 10x	Bio-Rad	#1706435
Tween-20 Fisher	Cole-Parmer	#CAS 9005-64-5
UltraPure DNase/RNase-free distilled water	Life technologies	#10977015
Triton X-100	Sigma-Aldrich	#CAS 9036-19-5
Absolute ethanol (100%)	Sigma-Aldrich	#E7023
Horseradish peroxidase (HRP)-conjugated donkey anti-mouse immunoglobulin G (IgG) (1:10,000)	Thermo Fisher Scientific	#SA1-100
DNA digestion buffer	Zymo research	#E1010
dsRNA ladder	New England Biolabs	#N0363S
ssRNA ladder	New England Biolabs	#N0362S
UltraPure ethidium bromide, 10 mg/mL	Invitrogen	# 15585011
Trypan blue solution, 0.4%	Gibco	# 15250061
Amersham ECL prime western blotting detection reagent	Millipore Sigma	#GERPN2232
Polyinosinic:polycytidylic acid (Poly(I:C)) HMW	InvivoGen	#tlrl-pic
Opti-MEM I reduced serum medium	Gibco	#31985088
SIINFEKL peptide	InvivoGen	#vac-sin
TE buffer, Tris-EDTA, 1X solution	Fisher Scientific	# BP2473100
Fisherbrand syringe filters - sterile	Fisher Scientific	# 09-719C
NxGen cartridges	Precision NanoSystems	#NIN0061
GenVoy lipid mix	Precision NanoSystems	# NWW0041
Sodium pyruvate (100 mM)	Life Technologies	11360-070
PureYield plasmid maxiprep system	Promega	#A2392
Monarch RNA cleanup kit	New England Biolabs	#T2050L
DNA clean and concentrate	Zymo research	#D4033
MEM non-essential amino acids solution (NEAA)	Gibco	#11-140-050
Penicillin-streptomycin (10,000 U/mL)	Life Technologies	#15140-122
Fetal bovine serum, heat inactivated	Gibco	#10082147
NEB HiScribe T7 high yield RNA synthesis kit	New England Biolabs	#E2040S
B-mercaptoethanol	Fisher Scientific	#ICN19470583
Opti-MEM I reduced serum medium	Gibco	#31985088
Lipofectamine 2000 transfection reagent	Thermo Fisher Scientific	#11668027
10x Tris-buffered saline (TBS)	Bio-Rad	#1706435
TAE buffer (10x)	Thermo Fisher Scientific	#AM9869
TrypLE express enzyme (1x), phenol red	Thermo Fisher Scientific	#12605010
Trypsin-EDTA (0.05%), phenol red	Life Technologies	#25300-054
Ficoll-Paque PLUS (500 mL)	Fisher Scientific	#45-001-750
Sodium pyruvate (100 mM)	Life Technologies	#11360-070
HEPES	Life Technologies	#15630-130
RPMI 1640 (L-glutamine)	Life Technologies	#11875-119
Dulbecco’s modified Eagle’s medium (DMEM)	Life Technologies	#10569-044
MEM non-essential amino acids solution (100X)	Life Technologies	#11140-050
IMDM (Iscove’s modified Dulbecco’s medium)	Life Technologies	#12440-061
ACK lysing buffer	Life Technologies	#A1049201
LB Broth, Miller	Fisher Scientific	#BP1426-2
LB agar powder	Fisher Scientific	#BP1425-500
Ampicillin ready-made solution	Sigma-Aldrich	#A5354
DNA gel loading dye (6X)	Thermo Fisher Scientific	#R0611
Recombinant murine GM-CSF	PeproTech	#315-03
Ketamine hydrochloride injection (100 mg/mL)	Dechra Veterinary Products	#1000001250
AnaSed xylazine injection (20 mg/mL)	Akorn Animal Health	#NDC 59399-110-20
Critical commercial assays
QUANTI-Luc	InvivoGen	#rep-qlc1
Quant-iT RiboGreen assay kit	Invitrogen	#R11490
Experimental models: Cell lines
B16F10	ATCC	#CRL- 6475
THP-1 dual cells	InvivoGen	#thpd-nfis
Experimental models: Organisms/strains
C57Bl/6 mice (male and female)	JAX	Jax strain #000664
OT-I mice (male and female)	JAX	Jax strain #003831
Oligonucleotides
NEB HiScribe T7 high yield RNA synthesis kit	New England Biolabs	#E2040S
N1-Methylpseudouridine-5′-Triphosphate	TriLink BioTechnologies	#N-1081
CleanCap reagent AG	TriLink BioTechnologies	#N-7113
Software and algorithms
GraphPad Prism 9
ImageJ
FlowJo v10
Other
Agilent 2100 bioanalyzer	Agilent Technologies	N/A
Agilent RNA 6000 nano kit	Agilent Technologies	#5067-1511
BRAND-UV cuvettes	Millipore Sigma	#Z628026
BD Microtainer MAP K2E 1.0 mg	Becton Dickinson	#363706
Slide-A-Lyzer dialysis casette	Thermo Fisher Scientific	#87732
The NanoAssemblr Ignite+	Precision NanoSytems	#1001413
Attune NxT flow cytometer	Thermo Fisher Scientific	N/A
Litesizer 500	Anton Paar GmbH, Austria	#173050
Gel Doc XR + imaging system	Bio-Rad	#170-8170

Materials and equipment

Slide-A-Lyzer Dialysis G2 Casette by Thermo Fisher Scientific (cat. # 87732 has been discontinued by the company since June 1^st, 2023). Alternatives are available on company website. Tetramers to quantify T cells against the HSF2 antigen were custom ordered from MBL technologies. Another relevant source for tetramer production is the NIH Tetramer Core Facility (TCF) at Emory University, which can provide reagents to investigators at no cost other than shipping and handling fees.

Reagent and media preparations

(Dulbecco’s Modified Eagle Medium) DMEM culture media

Reagent	Amount
Dulbecco’s Modified Eagle Medium (DMEM)	Up to 500 mL
Fetal bovine serum (FBS)	50 mL
Penicillin/Streptomycin (1,000 U/mL)	5 mL
HEPES	10 mL
Sodium Pyruvate	5 mL
MEM NEAA (1x)	5 mL
Total	500 mL

[Note on storage conditions: store at 4°C for up to 2 weeks]

(Iscove’s Modified Dulbecco’s Medium) IMDM culture media

Reagent	Amount
Iscove’s Modified Dulbecco’s Medium (IMDM)	Up to 500 mL
Fetal bovine serum	50 mL
Penicillin/Streptomycin	5 mL
Glutamine	5 mL
2-mercaptoethanol	500 μL
Total	500 mL

[Note on storage conditions: store at 4°C for up to 2 weeks]

THP-1 cell media

Reagent	Amount
RPMI	445 mL
Fetal bovine serum (FBS)	50 mL
Penicillin/Streptomycin (1000U/mL)	5 mL
Total	500 mL

[Note on storage conditions: store at 4°C for up to 2 weeks]

TBS-T buffer

Reagent	Amount
TBS 10x	100 mL
Tween	1 mL
DNase/RNase-free water	899 mL
Total	1000 mL

[Note on storage conditions: store at room temperature for up to 2 weeks]

• •Prepare 1% (w/v) non-fat dried milk in (TBS)-T buffer.

[Note: Store at 4°C for a week].

• •Prepare 5% (w/v) non-fat dried milk in (TBS)-T buffer.

[Note: Store at 4°C for a week].

• •Staining buffer: 2% FBS in 1x PBS.

[Note: Store at 4°C for 2 weeks].

• •Dissociation media: 500 mL 1X PBS + 5 mL EDTA.

[Note: Store at 4°C for 2 weeks].

Step-by-step method details

Synthesis of mRNA lipid nanoparticles and in vitro and in vivo characterization

Linearization of plasmid DNA and quality control

Timing: 3 h

This step will generate a linearized DNA template for use in the IVT reaction.

Note: The choice of restriction enzyme will depend on the restriction sites designed in a plasmid and desired linearization product. In our case, we used the Type IIS enzyme BspQI, which we positioned to generate a 5′ cut at the 3′ extremity of the PolyA sequence (Figure 1A).

• 1.
  Add the following into an Eppendorf tube:

  • a.
    5 μL 10X buffer r3.1.
  • b.
    1 μg DNA.
  • c.
    1 μL BspQI enzyme.
  • d.
    Nuclease free water – complete up to 50 μL.
• 2.Mix gently via pipetting and incubate at 50°C for 15 min.
• 3.Purify the linearized DNA using Zymo Research’s DNA Clean and Concentrate kit.
• 4.Elute the linear DNA with 10 μL RNase/DNase purified water in a RNase/DNase free Eppendorf.
• 5.
  Check the linearization of the plasmid using Gel Electrophoresis on a 1% agarose gel as follows (Figure 1B):

  • a.
    Prepare a solution of 1% w/v agarose gel in TAE buffer and microwave for 90 s.
  • b.
    Prepare the electrophoresis assembly with the stage and comb for loading samples.
  • c.
    After the agarose solution is cooled, add 5 μL Ethidium bromide or SYBR safe DNA gel stain per 50 mL of solution.
  • d.
    Carefully add the solution into the assembly and let the agarose solution sit for approximately 30 min to solidify into a gel.
  • e.
    Remove the comb and place the gel with the casket in the electrophoresis machine basket. Add TAE buffer until the gel is submerged.
  • f.
    In an Eppendorf tube, mix 200 ng DNA with the DNA loading dye in 1:6 ratio. Samples should include both digested and undigested plasmid DNA to confirm linearization, and a DNA ladder control.
  • g.
    Load the mix carefully into the comb slot for each sample and the DNA ladder.
  • h.
    Set the migration voltage depending on the length of your gel (10 V per centimeter).
  • i.
    Run the gel for approximately 30–40 min (until a successful band separation is achieved on imaging as shown in Figure 1B) and acquire an image using a Gel imaging system (Gel Doc XR + Imaging system).

CRITICAL: The linearized DNA samples can be stored at −20°C and used later for IVT mRNA synthesis.

Figure 1.

Characterization of mRNA purity and immunogenicity

(A) Schematic depiction of the plasmid DNA sequence.

(B) Gel electrophoresis of linearized plasmid compared to non-digested plasmid (ND).

(C) Bioanalyzer electrophoresis mRNA (D). J2 Dot-blot assay quantifying dsRNA quantity in IVT samples using a dsRNA standard (E). Flow cytometry gating strategy quantifying GFP⁺ transduced THP-1 cells.

(F) Comparison of percent GFP translation in THP-1 cells transfected with capped or uncapped mRNAs synthesized using unmodified uridine or modified N1-Methyl-pseudouridine nucleotides.

(G) Type-I interferon response measure after transfection of THP-1 reporter cells with same RNAs as in (F).

One-step capping and in vitro transcription of mRNA followed by mRNA quality control

Timing: 4 h and 30 min (including 2:30 h incubation)

This step uses linearized DNA as a template to synthesize mRNA.

Note: The 5′ cap can be added during in vitro synthesis or post-transcriptionally. We here describe a protocol for a ‘one step’ co-transcriptional addition of the 5′-cap, utilizing CleanCap AG. The protocol includes the incorporation of a modified nucleotide N1-Methylpseudouridine-5′-Triphosphate (m1ψ) to decrease the immunogenicity of the mRNA and improve its translation. Finally, it includes a DNase I treatment to remove residual plasmid DNA after IVT.

• 6.Thaw the components from NEB High Yield RNA Synthesis Kit, N1-Methylpseudouridine-5′-Triphosphate and CleanCap Reagent AG and keep on ice.
• 7.Set up the reaction by combining the components in the order mentioned in Table 1 below.
• 8.Mix gently by pipetting up and down and incubate at 37°C for 2 h.
• 9.To remove the template DNA from the reaction, prepare a DNase I buffer mix with 5 μL DNase I and 5 μL reaction buffer. Add 10 μL of this mix to the IVT RNA reaction and incubate at 37°C for 15 min.
• 10.Purify the in vitro transcribed mRNA using the Monarch RNA cleanup kit and elute the mRNA using 15 μL nuclease-free water.
• 11.Perform a quality control test of the mRNA using the Agilent RNA 6000 Nano kit assay (Figure 1C).

Table 1.

One-step in vitro transcription mRNA reaction mix

Component	Volume
Nuclease free water	Make up to 40 μL
10x Reaction buffer	2 μL
100 mM ATP	3 μL
100 mM GTP	0.54 μL
100 mM N1-Methylpseudouridine-5′-Triphosphate	3 μL
100 mM CTP	3 μL
100 mM CleanCap AG reagent	2 μL
Linear DNA template	1 μg
T7 RNA polymerase mix	4 μL

Expected yield: Approximately 50 μg of IVT-mRNA is typically produced from 1 μg of linearized DNA. (See Troubleshooting Problem 1).

CRITICAL: Maintaining an RNAse-free work environment is critical, as protocols like dot blotting can expose RNA to degradation. Use RNAse-free consumables and reagents and minimize RNA exposure to potential contaminants. Always work swiftly when handling RNA solutions and wear clean gloves. Keep RNA samples on ice when not actively being used to slow enzymatic degradation.

Enzymes like RNA polymerases should always be kept on ice when not in use to maintain optimal activity. IVT mRNA can be stored at −80°C. After thawing, it is critical to keep the mRNA on ice to avoid its degradation by RNAse contaminants.

Detection of double-stranded RNA using dot-blot analysis

Timing: 2 days

Double stranded RNA (dsRNA) that may be generated during the in vitro transcription can be immunogenic.³ In this step, we analyze the amount of dsRNA in the IVT RNA prep (Figure 1D).

Note: To remove dsRNA, a cellulose-based purification protocol can be followed.⁴

• 12.
  Prepare the following reagents:

  • a.
    Prepare TBS-T buffer by mixing 20 mM Tris [pH 7.4], 150 mM NaCl and 0.1% (v/v) Tween-20.
  • b.
    Prepare 1% and 5% (w/v) non-fat dried milk in Tris-buffered saline (TBS)-T buffer.
  • c.
    Dilute the IVT mRNA samples in nuclease-free water to a final concentration of 10, 30, 100 and 300 ng/5 μL.
  • d.
    Dilute dsRNA ladder to a final concentration of 0.2, 0.4, 1, 2 and 4 ng/μL (corresponding to 1, 2, 5, 10 and 20 ng/5 μL).
• 13.
  Incubation of mRNA samples with primary antibody:

  • a.
    Place the Nytran membrane on a sheet of Whatman GB005 blotting paper and fix with tape.
  • b.
    Mark the location for each sample on the membrane approximately 1 cm apart.
  • c.
    Load 5 μL (/dot) of IVT-mRNA samples, ssRNA control and dsRNA ladder dropwise carefully on the marked area.
  • d.
    Allow the liquid to drain through the Nytran membrane onto the Whatman paper. The negatively charged RNA will bind to the Nytran membrane.
  • e.
    Air dry the membrane. Once the membrane is completely dried, the samples will not be visible on the membrane.
  • f.
    Carefully remove the membrane from the Whatman paper.
  • g.
    Lay down the membrane in a clean petri dish.
  • h.
    Block the membrane by incubating it in 5% (w/v) non-fat dried milk in Tris-buffered saline (TBS)-T buffer. Place this assembly on a horizontal shaker at room temperature for 30 min.
  • i.
    After 30 min, discard the 5% (w/v) non-fat dried milk in Tris-buffered saline (TBS)-T buffer.
  • j.
    Wash using 10–15 mL TBS-T buffer and place this assembly back on a horizontal shaker at 25°C for 10 min. Discard the (TBS)-T buffer.
  • k.
    Dilute the J2 anti-dsRNA murine antibody (1:5000) in 5% non-fat dried milk in TBS-T buffer.
  • l.
    Incubate the membrane with the antibody solution on shaker overnight (8–10 h) at 4°C.
• 14.
  Secondary antibody treatment and chemiluminescent imaging:

  • a.
    Wash the membrane thrice using 10–15 mL (TBS)-T wash buffer for 15 min each.
  • b.
    After the last wash, discard the wash buffer and incubate the membrane with horseradish peroxidase (HRP)-conjugated donkey anti-mouse immunoglobulin G (IgG) (1:10,000) in 1% (w/v) non-fat dried milk in (TBS)-T at 25°C for 1 h.
  • c.
    Wash the membrane thrice as before.
  • d.
    After the last wash, remove the membrane from the petri dish and hold it in the air for a few seconds to remove any excess buffer.
  • e.
    Proceed to detect the chemiluminescence using Amersham ECL Prime Western Blotting Detection Reagent.
  • f.
    Image using chemiluminescent imaging system.

CRITICAL: All the steps should be performed in an RNase free environment. Do not let the membrane dry out after blocking the mRNA on the membrane. If the chemiluminescence intensity read outs are not as desired, it is possible to wash the membrane thrice and repeat the detection step using Blotting Detection Reagent with higher or lower sensitivity. The mRNA embedded membrane can be washed with buffer, air dried and stored at −20°C if needed (for approximately 7 days). (See Troubleshooting Problem 2).

Detecting type-1 interferon (IFN-I) response using the dual reporter THP-1 cell line

Timing: 2 days

This step outlines how to assess the immunogenicity of the IVT mRNA encapsulated in lipoplexes through the quantification of induced type-I interferon responses.⁵ This method takes advantage of the ability of lipoplexes to be formed with minimal amounts of mRNA, allowing testing of multiple construct designs during the initial stages of development. This step was also repeated after encapsulating mRNA into lipid nanoparticles (Figures 1E–1G).

Note: This assay functions as a quality control step and should be performed at the following stages of the workflow: 1) before mRNA encapsulation and 2) before in vivo experimentation.

• 15.Plate THP-1 cells in a 96-well U-bottom plate at a cell density of 50k/well in 50 μL THP-1 cell media.
• 16.
  Preparation of lipoplexes:

  • a.
    Sample preparation: To test different concentrations of mRNA, prepare 3 mRNA solutions in Opti-MEM with the following concentrations: 1.2 μg/mL, 4 μg/mL, and 12 μg/mL. Prepare a solution of high molecular weight (HMW) Polyinosinic:polycytidylic acid (Poly I:C) in Opti-MEM at a concentration of 4 μg/mL.
  • b.
    Lipofectamine-OptiMEM solution: Prepare a 10 μL/mL solution of lipofectamine-2000 in Opti-MEM solution.
  • c.
    Prepare the lipoplexes by mixing 100 μL of the sample solution with 100 μL of Lipofectamine-OptiMEM solution (ratio of 1:1).
  • d.
    Mix using a pipette and incubate the lipoplexes for 20 min at 25°C .
• 17.After 20 min, add 30 μL of mRNA-lipoplexes and controls to each well of the THP-1 cells (use 1x PBS or Lipofectamine alone as negative control and HMW Poly I:C as positive control).
• 18.Incubate for 10 h at 37°C, 5% CO₂.
• 19.Centrifuge the plate at 500 g for 5 min.
• 20.
  For type-I interferon analysis:

  • a.
    Carefully collect 20 μL cell supernatant on a white bottom plate and add equal volume of QUANTI-Luc reagent (InvivoGen).
  • b.
    Mix by shaking the plate gently.
  • c.
    Immediately read the plate on a luminometer following the provider’s instructions.
• 21.
  If your mRNA sequence encoded a fluorescent reporter (such as GFP), you can use the same THP-1 cells to measure the efficacy of mRNA translation by FACS analysis:

  • a.
    Wash the cells twice by adding 200 μL staining buffer to the wells and centrifuge at 500 g for 5 min.
  • b.
    Stain using blue fluorescent dye (1:1000) in staining buffer.
  • c.
    Wash the cells again twice and proceed to FACS acquisition. A cell viability > 80% is desired (Figures 1E and 1F).

CRITICAL: Do not use a vortex to resuspend lipoplexes and/or LNPs. To mix the nanoparticles, use a pipette or gently turn the nanoparticle containing tube upside down a few times. QUANTI-Luc analysis on fresh supernatant is more sensitive. However, the cell supernatant can be stored at −20°C and measured after one freeze-thaw cycle if needed.

Preparation of IVT-mRNA lipid nanoparticles (LNPs)

Timing: 2 days

Once the mRNA is synthesized and has passed the quality check, the mRNA is encapsulated in LNPs (in this protocol, utilizing the GenVoy-ILM lipid mix) using The Nanoassemblr Ignite+. The LNPs protect the mRNA from degradation and facilitate its entry into cells via the endocytosis/phagocytosis pathways. This step describes the encapsulation of mRNA into LNPs and the subsequent buffer exchange to minimize solvent content, which is required for the transition of mRNA-LNPs to a stable state prior to in vitro or in vivo use. The mRNA-LNPs can be stored for at least a month at 4°C. The mRNA-LNPs can be stored by freeze-drying using 20% w/v sucrose as a cryoprotectant.

• 22.
  Sample preparation and dialysis set up:

  • a.
    Dilute mRNA into 1.5 mL formulation buffer at a concentration of 200 μg/mL. Load this solution in a 3 mL Luer-Lok (Beckton Dickinson) syringe.
  • b.
    Add 300 μL of 100% ethanol to 300 μL of GenVoy lipid mix. Load this solution in a 1 mL Slip Tip (Beckton Dickinson) syringe.
  • c.
    Immerse the 10k MWCO dialysis cassette in a beaker containing 500 mL 1X PBS (10 mM, pH 7.4) to hydrate the membranes.
• 23.
  Preparation of Lipid nanoparticles (LNPs):

  • a.
    Turn on the NanoAssemblr Ignite and select “Quick Run” from the main menu.
  • b.
    Enter the parameters in the screen menu: Flow rate Ratio, C:R (RNA:Lipid) = 3:1; Total volume = 2 mL; Total flow rate = 12 mL/min; Start waste volume = 0.35 mL; and End waste volume = 0.05 mL).
  • c.
    Open the lid of the NanoAssemblr Ignite and load the NxGen mixing cartridge in the cartridge slot until soft click is felt. Label two 15 mL conical tubes: one for waste, and one for mRNA-LNP collection. Load them into the clip labeled “waste” and “sample”, respectively.
  • d.
    Load the syringes into respective slots in the cartridge: 3 mL syringe loaded with mRNA solution into the “C” inlet, and 1 mL syringe loaded with lipid mix into the “R” inlet. Twist clockwise to engage the Luer Lok.

    Note: Make sure to clear air bubbles from the syringes and use the plunger to advance the liquid to the tip avoiding sample dipping.

  • e.
    Close the Ignite lid and tap “Next”, confirm the parameters and press “Start”.
  • f.
    After the instrument is finished running, open the lid and remove the 15 mL conical collection tube containing the mRNA-LNPs.
  • g.
    Add 10 mL 10 mM 1x PBS to the mRNA-LNPs solution and mix well.

    Note: Volumes are provided to make a 2 mL mRNA-LNP solution. The mRNA-LNP must be diluted 5x with 1x PBS.

  • h.
    Add the diluted mRNA-LNP solution to the dialysis cassette (10k MWCO) for buffer exchange. Keep the cassette immersed in the 1x PBS solution while adding the mRNA-LNP solution.
  • i.
    Incubate the sample 8–10 h at 4°C, under slow stirring.
  • j.
    Remove the LNP solution from the dialysis cassette using a pipette and transfer it to the top compartment of a 15 mL ultra-centrifuge filter 30k MWCO Vivaspin.
  • k.
    Concentrate the mRNA-LNP solution by centrifuging the solution at 4000 × g for 10 min. Reduce the volume to approximately 1 mL.
  • l.
    Pipette the mRNA-LNP solution against the walls of the ultra-centrifuge; filter a few times to minimize LNP loss.
  • m.
    Discard the flow-through from the bottom compartment and filter the mRNA-LNPs with 200 nm syringe filter.
  • n.
    Transfer the volume into a 1.5 mL RNase- and DNase-free Eppendorf tube.
• 24.
  Storage of mRNA-LNPs.

  • a.
    The mRNA-LNPs should be stored at 4°C for 1 month.
  • b.
    For longer periods of time, mRNA-LNP can also be freeze-dried in presence of a cryoprotectant and stored at −80°C. Add 20% (w/v) sucrose to mRNA-LNP solution prior freezing at −80°C.⁶

Characterizing the mRNA-LNP’s physicochemical properties

Timing: Total 3 h

This section describes the quality control steps used to determine the size, charge, encapsulation efficiency and fraction of free RNA of the generated mRNA-LNPs. Dynamic light scattering (DLS) via a Zetasizer instrument (Malvern) is used to determine the LNP hydrodynamic size, dispersity (Đ) and ζ potential (surface charge) of the mRNA-LNPs. The Quant-it Ribogreen assay is performed to assess mRNA-LNP integrity, encapsulation efficiency and RNA release (Figure 2).

• 25.
  mRNA-LNP characterization: Particle size, polydispersity and ζ potential measurements (See Troubleshooting Problem 3).

  • a.
    Dilute an aliquot of the mRNA-LNP sample 1:100 in 1x PBS to obtain a final volume of 1 mL. Add the solution into a semi-micro polystyrene disposable cuvette (10 mm path length) and insert it into the DLS instrument.
  • b.
    Set up an operating procedure in the instrument software including measurement type:

    • i.
      size (conducted at an angle of 175°).
    • ii.
      cell type: disposable (Millipore-sigma Brand-UV cuvettes).
    • iii.
      sample details: temperature (25°C).
    • iv.
      dispersant media (1x PBS), material (phospholipids).
    • v.
      Select number of runs (3) and.
    • vi.
      click “Start” to begin with the measurement acquisition.
  • c.
    Hydrodynamic diameter as well as dispersity values are provided (Figures 2A and 2B).
  • d.
    For ζ potential measurements, mRNA-LNPs were diluted 1:100 in nuclease free water to obtain a final volume of 1 mL.
  • e.
    Transfer the volume to a folded capillary zeta cell, up to the fill line.
  • f.
    Insert the cell into the instrument ensuring that the electrodes are in contact with the instrument electrodes.
  • g.
    Set up an operating procedure in the instrument software including measurement type: ζ potential, cell type (Omega cuvette), sample details: temperature (25°C), voltage (20 V), dispersant media (water). Then click “Start”.
  • h.
    Smoluchowski approximation was used for the estimation of the ζ potential value. The results are reported as an average of three independent measurements along with standard deviation.
• 26.
  Measure encapsulation efficiency using Quant-it Ribogreen assay (Invitrogen):

  • a.
    Prepare a standard curve by making 2-fold serial dilutions in 1x PBS of the mRNA stock solution, starting with 5 μg/mL as the highest concentration.
  • b.
    Prepare the mRNA-LNP sample dilutions.
    Dilute mRNA-LNP samples with 1x PBS, to achieve an approximate theoretical concentration that lies around mid-point of the standard curve (e.g., Estimated initial concentration = 100 μg/mL, dilution 1:50 to get ∼ 2 μg/mL of mRNA).
  • c.
    In a 96-well black fluorescence capable plate, place 50 μL of the standard curve and 50 μL of mRNA-LNP diluted samples. For the samples, prepare at least 6 replicates.
  • d.
    Add 50 μL of 2% Triton X-100 to each well of standards.
  • e.
    To half of the sample replicates (3) add 50 μL of 2% Triton X-100.
  • f.
    To the remaining wells of sample replicates (3) place 50 μL of 1X TE.
  • g.
    Repeat the same for at least 4 reference wells with 50 μL 1x PBS (2 wells with 50 μL of 2% Triton X-100, 2 wells 50 μL 1X TE).

    Note: The wells containing Triton X-100 are used to quantify the total amount of mRNA while the ones containing 1X TE are used quantify the mRNA not encapsulated inside LNPs.

  • h.
    Slightly shake the plate to ensure through mixing of the reagents. Allow the plate to incubate at 37°C for 10 min. During this time, thaw the RiboGreen 100x reagent (stored at −20C) and dilute it 1:100 with 1X TE buffer.
  • i.
    After the 10 min incubation, add 100 μL of diluted RiboGreen to each well. Avoid creating bubbles when mixing and take precautions to avoid light exposure.
  • j.
    Measure the fluorescence using a microplate reader with an excitation wavelength of 500 nm, emission wavelength of 525 nm and gain of 50.
  • k.
    After subtracting the fluorescence values of the blanks from each sample well, calculate the concentration of free mRNA (from wells without Triton X-100) using the standard curve and multiplying by the dilution factor used in 25b.
  • l.
    Repeat the same to calculate the concentration of mRNA both inside and outside the LNP (from wells with Triton X-100).
  • m.
    The concentration of mRNA in the final formulation is determined from subtracting the concentration of free mRNA from the total concentration of mRNA both inside and outside LNP (calculated from 25k-l).
  • n.
    The fraction of free mRNA is determined by dividing the fluorescence of the intact particle sample (no Triton X-100) by the fluorescence value of the disrupted particle sample (with Triton X-100).
  • o.
    Quantify the encapsulation efficiency = 1 – free mRNA fraction (calculated in 25k) (Figure 2C).
• 27.
  mRNA-LNP stability

  • a.
    Stability over time is evaluated by measuring periodically, once per week, mRNA-LNP particle size distribution, hydrodynamic diameter, dispersity and ζ potential, as detailed in section 6.1 (Figures 2D and 2E).
  • b.
    mRNA release from LNP over time is determined by calculating mRNA-LNP encapsulation after a month, as detailed in Measure encapsulation efficiency using Quant-it Ribogreen assay (Invitrogen) section (Figure 2F).

Figure 2.

Determination of the physicochemical and stability properties of fresh and frozen mRNA-LNPs

(A) Size distribution of three independent mRNA-LNPs formulations as measured by DLS.

(B) Mean hydrodynamic diameter and polydispersity index (PDI) measured via DLS. Results are displayed as mean ± SD.

(C) Encapsulation efficiency of mRNA within LNPs of three independent formulations. Results are displayed as mean ± SD.

(D) Mean diameter and PDI of mRNA-LNPs after storage at 4^∘C for 1, 2, 3, or 4 weeks.

(E) Zeta-potential of the mRNA-LNP after storage at 4^∘C for 1, 2, 3, or 4 weeks.

(F) Comparison of mRNA encapsulation efficiency after 1 month of mRNA-LNP storage at 4°C.

(G) Size distribution of fresh vs. frozen mRNA-LNPs.

(H) Mean diameter and PDI of fresh and frozen LNPs following thawing.

(I) Encapsulation efficiency of mRNA-LNP after thawing.

Stability of mRNA-LNP formulation after freezing cycles is determined by following the size distribution (Figure 2G), hydrodynamic diameter and dispersity (Figure 2H) and mRNA encapsulation efficiency (Figure 2I).

Characterization of antigen presentation after transfection of mRNA vaccine into the B16F10 cancer cell line

Timing: 3 days

After cellular uptake, the mRNA is translated by the ribosomal cellular machinery and the antigens are presented at the surface of the cell on major histocompatibility complex class I (MHC-I). The C57BL/6 mouse strain (and cancer cells line derived from this strain such as B16F10), express 2 MHC-I alleles: H2-K^b and H2-D^b. This assay harnesses the specificity of an anti-H2-K^b/SIINFEKL-PE-conjugated antibody that detects the SIINFEKL peptide when presented on H2-K^b. This allows the induction of antigen presentation by B16F10 or C57BL/6-derived BMDCs transfected with an mRNA vaccine encoding the SIINFEKL peptide to be quantified (Figure 3A).

Note: Characterization of antigen presentation is a quality control step and should be performed at the following stages of the workflow: 1) before mRNA encapsulation and 2) before in vivo experimentation with mRNA-LNP.

Figure 3.

B16F10 and BMDC antigen presentation assay following stimulation with RNA-lipoplexes and RNA-LNP formulations

(A) The principle underlying the antigen (SIINFEKL) presentation assay.

(B) Flow cytometry gating strategy to quantify antigen presenting (H2-K^b/SIINFEKL⁺) B16F10 cells.

(C) % H2-K^b /SIINFEKL⁺ cells and (D). Mean fluorescence intensity of H2-K^b /SIINFEKL in B16F10 transduced with fresh or previously frozen mRNA-LNPs.

(E) Flow cytometry gating strategy to quantify antigen presenting (H2-K^b/SIINFEKL⁺) BMDCs.

(F) % H2-K^b/SIINFEKL⁺ cells and (G). Mean fluorescence intensity of H2-K^b /SIINFEKL in BMDCs transduced with fresh or previously frozen mRNA-LNPs.

For all centrifugation steps involving cells throughout this protocol, unless otherwise specified, the centrifuge should be set to an acceleration of 9 and a deceleration of 5.

• 28.
  Day 0: Seeding the cells.

  • a.
    Seed B16F10 WT and OVA expressing cells in a 48 well plate at a density of 15k cells per well in 200 μL warm DMEM complete media (supplemented with FBS, HEPES, sodium pyruvate and NEAA).

    Note: The cells should be approximately 65%–70% confluent before transfection. It will take approximately 24 h for the cells to reach 65% confluency.

  • b.
    Place the plate in a cell culture incubator at 37°C, under a 5% CO₂ atmosphere.
• 29.
  Day 1: Transfection of the cells.

  • a.
    Prepare mRNA lipoplexes as described under step 2 in detection of type-I interferon response using THP-1 cell line.
  • b.
    LNPs should be diluted with Opti-MEM to a desired concentration.
  • c.
    Treat the plated cells with mRNA-lipoplexes, mRNA-LNPs and controls (PBS or lipofectamine as negative control and 10 μM SIINFEKL peptide as positive control).
  • d.
    Incubate the cells at 37°C for 2 h.
  • e.
    Treat the cells with recombinant IFN-γ to reach a final IFN-γ concentration of 25 IU/mL and incubate the mixture for 10 h at 37°C.
• 30.
  Day 2: Flow cytometry analysis for antigen presentation.

  • a.
    Carefully remove the media from the wells with a pipette and wash with 1x PBS.
  • b.
    Resuspend the cells using 50 μL trypsin pre-warmed to 37°C.
  • c.
    After the cells are dissociated from the plate, add 50 μL warm DMEM culture media to neutralize the trypsin.
  • d.
    Transfer the cells to a 96-well v-bottom plate, spin at 500 g for 5 min, and discard the supernatant.
  • e.
    Wash the cells by adding 200 μL staining buffer to each well, centrifuging at 500 xg for 5 min and discarding the supernatant. Repeat this washing step one more time.
  • f.
    Stain the cells with blue fluorescent dye (1:1000) and anti-H2-K^b/SIINFEKL-PE (1:200) in staining buffer. Incubate the plate at 25°C for 20 min and then transfer to 4°C for another 10 min.
  • g.
    Wash the cells with staining buffer twice as mentioned in step e before proceeding with flow cytometry analysis of mRNA-encoded ova antigen in B16F10 melanoma cells (Figures 3B, 3C, and 3D).

Characterizing antigen presentation on BMDC

Timing: 2days

Dendritic cells (DCs) are antigen presenting cells (APC) that are essential for initiating an immune response to mRNA vaccines.⁷ This in vitro assay is designed to quantify the induction of mRNA-encoded antigen expression and presentation in DCs. This assay also serves as a quality control step, as DCs express high levels of innate immune sensors which will modulate their response to mRNA vaccines, e.g., if dsRNA or uncapped RNAs contaminants are present.

Note: This is a quality control step and should be performed at the following stages of the workflow- 1) before mRNA encapsulation and 2) before in vivo use of the mRNA-LNPs.

• 31.
  Day 1: Seeding and transfection.

  • a.
    Seed 25 × 10³ BMDCs in 150 μL of pre-warmed to 37°C IMDM medium supplemented with HEPES, FCS, P/S and BMT in a 96-well u-bottom low adherence plate using IMDM media.
  • b.
    Prepare mRNA lipoplexes as described under Step-by-Step method’s section 4, point #2 (a-d).
  • c.
    LNPs should be diluted with Opti-MEM to a desired concentration.

    Note: In this study, the working concentrations of mRNA-lipid nanoparticles (mRNA-LNPs) employed are 10 ng, 30 ng, 100 ng, and 300 ng in a volume of 50 μL of Opti-MEM.

  • d.
    Treat the plated cells with mRNA-lipoplexes, mRNA-LNPs, 1x PBS or lipofectamine as negative control and SIINFEKL peptide as positive control.
  • e.
    Incubate the cells at 37°C.
• 32.
  Day 2: Flow cytometry analysis for antigen presentation.

  • a.
    Tilt the plate and hold it still. Remove the media by inserting a 200 μL pipette at a 45° angle and without disturbing the cells. Wash the cells with 1x PBS.
  • b.
    Re-suspend the cells using 100 μL dissociation media warmed up to 37°C.
  • c.
    After the cells are dissociated from the plate, add 100 μL of IMDM media.
  • d.
    Spin the plate at 500 × g for 5 min.
  • e.
    Discard the supernatant and wash the cells twice by adding 200 μL staining buffer to the wells and centrifuging at 500 × g for 5 min.
  • f.
    Discard the supernatant.
  • g.
    Stain all the cells with blue fluorescent dye (1:1000), CD11c- APC-eFluor 780 (1:200) and CD45 – BV510 (1:200) for BMDCs and anti-H2-K^b/SIINFEKL-PE (1:200) in 200 μL staining buffer.
  • h.
    Let the plate sit at 25°C for 15 min before placing it at 4°C for another 15 min.
  • i.
    Wash the cells with staining buffer twice as mentioned above (point e.) and proceed with flow cytometry analysis.

CRITICAL: After day 1 incubation, BMDC cells become activated and adherent, and it can take 10–30 min for the cells to dissociate completely. Frozen LNPs were thawed on ice.

Induction of antigen-specific T cells following mRNA-LNP vaccination

Timing: 12 days

This step describes the immunization of mice with the mRNA-LNP vaccine and subsequent analysis of antigen-specific CD8⁺ T cell responses (Figures 4A–4E). Tetrameric peptide–MHC-class-I complexes (tetramers) are used to detect antigen-specific T cells. In this example, we use tetramer specific for SIINFEKL and for a B1610 tumor neoantigen encoded by a mutation in the gene Hsf2.⁸ These 2 antigens are encoded in our mRNA vaccine construct.

Note: The tetramers used here are specific to MHC class I.

• 33.
  Day 1–5: Immunization of mice.

  • a.
    Anesthetize mice by intraperitoneally injecting 0.1 mL of a 20% ketamine, 10% xylazine cocktail in distilled water per mouse (this assumes a mouse of 25 g, the volume of anesthesia cocktail injected should be adjusted based on weight and observed response to anesthesia).
  • b.
    Immunize each mouse with 10 μg of mRNA-LNPs diluted in 125 μL 1x PBS via retro-orbital injection. For negative control, immunize mice with vehicle control (here 1X PBS) (Figure 4A).

    Note: Other routes of administration can be used to deliver mRNA-LNP vaccines, such as intramuscular, intradermal, subcutaneous, or intranasal. Different routes of administration will differentially impact the kinetics and properties of the immune response.

  • c.
    A booster dose was given on day 5 with 10 μg mRNA-LNPs or PBS as negative control. On Day 8, the mice were sacrificed (details in next section- Day 8: Isolation of PBMC and splenocytes).
• 34.
  Day 8: Isolation of PBMC and splenocytes.

  • a.
    Add 5 mL cold RPMI in 6-well plates and prepare label microtainer tubes for spleen and blood collection.
  • b.
    Sacrifice a mouse via lethal CO₂ inhalation followed by cervical dislocation. Immediately begin the dissection.

    • i.
      Add 100% CO₂ to a closed chamber in a well-ventilated area at a displacement rate of 30%–70%. Adjust the flow rate of 100% CO₂ from the compressed CO₂ cylinder based on the volume of the container the mice are in.

      Note: Generally, the mice should fall unconscious 2–3 minutes after CO₂ is added and should be kept in the chamber for another 2–3 minutes while observing the breathing rate for reliable euthanasia.

    • ii.
      Remove the mice from the chamber once the breathing has stopped and perform cervical dislocation by holding down on the neck of the mouse just behind the ears and pulling up quickly on the tail at a 45-degree angle.
  • c.
    For blood collection:

    • i.
      Perform a cardiac puncture immediately following death by inserting a needle into the left ventricle of the heart and slowly pulling back on the syringe to collect blood (expect to collect up to 1 mL of blood per mouse using this method).
    • ii.
      Immediately transfer collected blood to 0.5 mL BD K2EDTA microtainer tubes and mix slowly with a pipette to prevent blood clots. Place blood on a slow-moving rotator until all tissues are harvested and processing can begin.
  • d.
    For spleen collection:

    • i.
      Following cardiac puncture, remove the spleen using forceps, cutting connective tissue connecting the spleen to the abdominal cavity.
    • ii.
      Immediately transfer the collected spleen to a prepared 6-well plate with 5 mL RPMI in each well. Keep the plate on ice until all tissues are harvested and processing can begin.
  • e.
    For PBMC isolation:

    • i.
      Transfer blood to 15 mL falcon tubes and dilute up to 6 mL with 1x PBS.
    • ii.
      Fill a 15 mL falcon tube with 3 mL ficoll-paque (density 1.077 gm/mL).
    • iii.
      Slightly tilt the falcon tube and slowly layer the diluted blood on top of the Ficoll-paque layer. Two distinct layers should be visible in the tube.
    • iv.
      Centrifuge the tubes at 450 × g, 25°C, 35 min (acceleration: 1, and deceleration: 0).
    • v.
      Following centrifugation, the PBMC layer should be visible as a cloudy band in between the serum and ficoll-paque. Carefully pipette it out using a p1000 micropipette into a clean 15 mL falcon tube.
    • vi.
      Add 1x PBS and make the volume up to 15 mL. Wash the cells twice using PBS (wash spin at 500 × g, 5 min, 4°C).
    • vii.
      Aspirate the supernatant.
    • viii.
      Freeze down the cells at a cell density of 10 M/mL freezing media in a cryovial.
    • ix.
      Place the cryovials in a Nalgene Mr. Frosty holder and store them at −80°C for 24 h before transferring the cryovials to liquid nitrogen. [Expected cell count: approximately 5–8 million cells per mouse from 1–1.5 mL of blood].
  • f.
    For Splenocyte isolation:

    • i.
      Dissociate and homogenize the spleen using a flat syringe plunger.
    • ii.
      Filter the splenocytes through a 70 μm sterile filter into a 50 mL conical flask.
    • iii.
      Add 10 mL cold 1x PBS and centrifuge at 500 × g for 5 min at 4°C.
    • iv.
      Aspirate the supernatant and wash the cells again with 5 mL 1x PBS at 500 g, 5 min, 4°C.
    • v.
      Aspirate the supernatant. Add ACK lysing buffer 3 mL and incubate for 5 min on ice.
    • vi.
      Add 12 mL 1x PBS and spin the cells at 500 g, 5 min, 4°C.
    • vii.
      Discard the supernatant.
    • viii.
      Freeze the cells in a cryovial at a cell density of 10 M cells/mL freezing media.
    • ix.
      Place the cryovials in a Nalgene Mr. Frosty and store them at −80°C for 24 h before transferring the cryovials to liquid nitrogen. [Expected cell count: 120–150 M cells per spleen].
  • g.
    Day 8+: Multimers staining analysis using frozen PBMCs and splenocytes:

    Note: In this step, prepared PBMCs and splenocytes are stained with MHC-peptide multimers corresponding to antigens encoded by the mRNA-LNP vaccine. In this protocol, we use tetramers and each sample tested requires one well in a 96-well plate per tetramer of interest (e.g. for one mouse to test two tetramers for each PBMCs and splenocytes would require four wells total). Alternatively, multimers can be multiplexed within flow panels. Finally, unstained and fluorescence minus one (FMO) control should be prepared per tissue type to be analyzed (Figures 4B and 4D).

    • i.
      Add 5 mL fresh RPMI media (warmed to 37°C) in a 15 mL Eppendorf tube for each frozen PBMC/splenocyte sample.
    • ii.
      Submerge the frozen cryovial in a 37°C water bath until just thawed, then immediately transfer the contents of the cryovial into the prepared 15 mL tube with RPMI.
    • iii.
      Spin the cells at 500 xg for 5 min at 4°C, resuspend in 10 mL 1x PBS and spin again to wash.
    • iv.
      Resuspend in 1 mL 1x PBS and count. Adjust the concentration of cells to 3 × 10⁶ cells per mL and plate 200 μL sample in each well of a 96-well v-bottom plate.
  • h.
    Tetramer staining analysis using fresh PBMCs and splenocytes:

    • i.
      Add 200 μL of sample in 1x PBS prepared in the prior step to each well of a 96-well v-bottom plate.
    • ii.
      Spin the plate at 500 × g for 5 min at 4°C, flick off the supernatant and resuspend each well in 200 μL of PBS. Spin again as above.

      Note: Dasatinib treatment inhibits TCR internalization and results in enhanced staining intensities. If starting from frozen samples, add 5 μL of 1mg/mL DNAse I per well, 15 minutes into the Dasatinib incubation.

  • i.
    Flick off the supernatant and resuspend each well in 50 μL of tetramer staining buffer (PBS supplemented with 2% FBS) with 50 nM Dasatinib. Incubate at 25°C for 30 min.
  • j.
    Dilute tetramer stock in tetramer staining buffer 1:10, preparing 5 μL of diluted tetramer solution per well to be stained.
  • k.
    Add 5 μL of tetramer solution (containing 0.5 μL of tetramer) per well and resuspend all wells to mix.

    Note: Do not add tetramer to unstained and FMO wells. Staining with 0.5 μL per well is optimal for the tetramers used as examples in this protocol. The optimal tetramer staining concentration should be determined through titration experiments for each individual tetramer prior to their use to validate immunization efficacy.

  • l.
    Incubate for 30 min on ice in the dark.
  • m.
    Add 150 μL of tetramer staining buffer to each well and spin (500 xg, 5 min, 4°C).
  • n.
    Flick off the supernatant and resuspend in 200 μL tetramer staining buffer, then spin again (500 × g, 5 min, 4°C).
  • o.
    Flick off supernatant and resuspend each well in 100 μL surface staining mix.
  • p.
    Incubate for 25 min on ice in the dark. Prepare 100 μL surface staining mix per well with 1 in 1000 live/dead aqua, 1 in 800 anti-CD8 AF647, and 1 in 400 each of anti-CD45 PerCP-Cy5.5, anti-CD3 BV421, and anti-CD4 BV711 in tetramer staining buffer.

    Note: Surface staining mix should be added to all samples except for the unstained wells.

  • q.
    Add 100 μL of tetramer staining buffer to each well, spin (500 xg, 5 min, 4°C).
  • r.
    Flick off supernatant.
  • s.
    Resuspend each well in 200 μL tetramer staining buffer and spin again (500 xg, 5 min, 4°C).
  • t.
    Flick off supernatant and resuspend in 200 μL of tetramer staining buffer.
  • u.
    Keep samples in the dark at 4°C until ready for analysis via flow cytometry (Figures 4A–4E).

    Note: The choice of optimal fluorophores will depend on the flow cytometer used. (See Troubleshooting Problem 4).

    CRITICAL: Tetramer staining on fresh cells gives better results as compared to frozen cells. For each wash cycle of the plate, flick off the supernatant swiftly only once. For tetramer staining assay, spin cycles for less than 10 min may result in lower tetramer positive cells during acquisition. Perform individual tetramer titrations to determine the optimal concentration of tetramer for staining if necessary.

Figure 4.

Induction of antigen-specific T cells and anti-tumor immune responses following mRNA-LNP vaccination

(A) Schematic of the prime-boost vaccination assay.

(B) Flow cytometry gating strategy to identify antigen-specific CD8⁺ T cells in peripheral blood mononuclear cells (PBMCs).

(C) Percentage of antigen-specific CD8⁺ T cells among total CD8⁺ T cells in PBMCs following vaccination with mRNA-LNP. The antigen-specific response to 2 antigens encoded by the vaccines (SIINFEKL/OVA and HSF2) are shown. FMO is a negative control of staining.

(D) Flow cytometry gating strategy to identify antigen-specific CD8⁺ T cells in spleen.

(E) Percentage of antigen-specific CD8⁺ T cells among total CD8⁺ T cells in spleen following vaccination.

(F) Mean tumor growth post engraftment (G). Tumor weight at study endpoint. The significance of the mean tumor weight difference between the control and vaccinated groups is determined by student’s unpaired T-test (p = 0.01).

Functional assay: mRNA-LNP vaccine induced anti-tumor immune responses

Timing: about 27 days (depending on tumor growth)

This step describes the use of a tumor challenge model to assess the functional capacity of antigen-specific responses elicited following immunization with the mRNA-LNP vaccine. This involves the immunization of mice with mRNA-LNP and a subsequent challenge by inoculation with B16F10 melanoma cells. Tumor growth is assessed as an indicator of anti-cancer vaccine efficacy (Figures 4F–4H).

Note: The challenge assay best suited for determining the effectiveness of mRNA-LNP induced immune responses will depend on the antigens encoded in the vaccine. In this example, responses to the B16F10 mutation-encoded neoantigen HSF2 are assessed through engraftment of B16F10 murine melanoma cells.

• 35.
  Day 1–5: Immunization.

  • a.
    Immunize mice as described in the previous step, injecting a prime dose on day 1 and a booster dose on day 5.
  • b.
    Engraft tumors on Day 9.
• 36.
  Day 9–10: Engraft tumors.

  • a.
    On day 9, anesthetize mice as described for immunization.
  • b.
    Shave the right flank of mice using an electric razor in the control and vaccinated groups to allow visualization of the skin for tumor engraftment.
  • c.
    Wait 24 h after shaving before engrafting tumor cells to allow healing of any micro-abrasions that occur during shaving that could interfere with engraftment.
  • d.
    Prepare B16F10 murine melanoma cells as described in ‘preparatory steps’, ending with a cell solution of 1.5 × 10⁵ B16F10 cells in 100 μL of PBS per mouse to be engrafted (including 20% extra cell solution to account for lost volume in loading syringes) on ice.
  • e.
    Anesthetize mice as described for immunization.
  • f.
    Load 100 μL of cell solution in a 28G needle, ensuring no air bubbles are present in the needle.
  • g.
    Pull the skin of the right flank taut and subcutaneously inject 100 μL of cell solution slowly. A bubble should form just under the skin when a subcutaneous injection is performed correctly.

    • i.
      Load the syringe with cell solution slowly, so cells are not stripped by passing through the needle.
    • ii.
      An ear punch can be used to identify individual mice within a cage, allowing tracking of tumor growth throughout the experiment.
    • iii.
      If no bubble is formed following injection, this indicates a deep injection, and it is recommended to exclude this mouse from study.
• 37.
  Day 17–27: Monitor tumor growth.

  Note: Tumor monitoring should commence on day 17, as the tumors are expected to become palpable at that time.

  • a.
    Using calipers, measure tumor width and height every other day.

    • i.
      From width and height, tumor volume can be approximated with the following formula: $Volume={\left(width\right)}^2\times \frac{length}{2}$
  • b.
    Once the largest tumors reach a size of 10 mm in one dimension, sacrifice all the mice via lethal CO2 inhalation followed by cervical dislocation.

    • i.
      Add 100% CO₂ to a closed chamber in a well-ventilated area at a displacement rate of 30%–70%. Adjust the flow rate of 100% CO₂ from the compressed CO₂ cylinder based on the volume of the container the mice are in.

      Note: Generally, the mice should fall unconscious 2–3 minutes after CO₂ is added and should be kept in the chamber for another 2–3 minutes while observing the breathing rate for reliable euthanasia.

    • ii.
      Remove the mice from the chamber once the breathing has stopped and perform cervical dislocation by holding down on the neck of the mouse just behind the ears and pulling up quickly on the tail at a 45-degree angle.
  • c.
    Post-euthanasia, extract the tumors from each mouse as follows:

    • i.
      Using scissors, make a midline incision on the ventral side of the mouse. Using forceps, pull up on the skin and insert the scissors in between the skin and the peritoneal lining, opening the scissors to separate the two layers.
    • ii.
      Make a secondary incision in the skin only (not the peritoneal lining) down the right leg of the mouse and use the forceps to pull a flap of skin along the right flank away from the body of the mouse.
    • iii.
      Isolate the tumor by making small incisions with scissors around the edge of the tumor, taking care to not cut through the skin.

      Note: The tumor should be readily visible in this flap of skin, appearing as a black ball in the skin.

    • iv.
      Press the tumor up from the underside of the skin so it is bulging up and, using large scissors, cut parallel to the skin, scooping the tumor off the skin but leaving the skin intact.
    • v.
      To collect all tumor cells, use a scalpel to gently scrape against the skin where the tumor was growing until little to no black remains.
    • vi.
      Tumor tissue should be transferred to prepared wax paper for weighing.
  • d.
    Weigh each tumor by zeroing a small weighing boat with empty wax paper on a scale (use a scale with sensitivity to measure 0.01 g with a range of 0.0–1.0 g). To measure each tumor, replace the empty wax paper with the paper containing the tumor and record the weight.

    Optional: Following weighing, the tumors can be dissociated to a single cell suspension and tetramer staining of the tumor-infiltrating T cells can be performed as described in step 9.

Expected outcomes

Characterization of mRNA purity and immunogenicity

Prior to IVT, successful linearization of the DNA template should be confirmed by gel electrophoresis (Figure 1B). The mRNA vaccine construct generated following IVT should show a band at the anticipated size and mRNA integrity should also be determined through RNA electrophoresis, where it should show as a single band with minimal presence of smaller bands that may indicate RNA degradation (Figure 1C). The RNA generated should have minimal dsRNA content (<5%) as determined through J2 dot blot analysis (Figure 1D). To confirm RNA functionality, successful transfection of cells of interest and translation of encoded reporter genes such as GFP should be observed following lipoplex-mediated delivery (Figure 1E), the magnitude of translation will likely be lower when using uncapped RNAs or non-modified nucleoside bases (Figure 1F). The immunogenicity of the mRNA should be strongly reduced with the use of capped RNAs and modified nucleoside bases as determined via IFN response (Figure 1G).

Analysis of the physicochemical and stability properties of fresh and frozen mRNA-LNPs

mRNA-LNP size and charge will depend on the type, ratio, and concentration of lipids used and the flow rates with which they are mixed. Regardless, mRNA-LNPs should have a narrow size dispersity (Đ) (PDI < 0.2), and this should be relatively consistent between batches of the same formulation (Figures 2A and 2B; Table 2). The mRNA cargo should be encapsulated in the LNP with efficiencies higher than 95% (Figure 2C; Table 2). RNA-LNPs should be stable at least for 1 month at 4°C (Figure 2D). The mean diameter, dispersity (Đ), zeta-potential and encapsulation efficiency should remain stable for at least a month (Figures 2E and 2F). The process of cryopreservation should not have a significant impact on RNA-LNP size and PDI after thawing (Figures 2G and 2H). Some RNA release can be expected following each freezing cycle (Figure 2I).

Table 2.

Physiochemical characteristics of mRNA -LNP

Characteristic	Values
Size	73.1 ± 6.6
PDI	0.15 ± 0.002
Z-potential2 (mV)	−6.0 ± 2.3
Encapsulation efficiency (%)	96.3% ± 0.9

Induction of antigen-specific T cells and anti-tumor immune responses

The mRNA-LNPs generated should effectively transfect cells of interest, here B16F10 and BMDCs, and induce translation and subsequent antigen presentation (Figures 3C, 3D, 3F, and 3G). This functionality should be confirmed in freeze-thawed batches of mRNA-LNPs. The in vivo functionality of these mRNA-LNP vaccines can be confirmed in murine vaccination models. Following vaccination (Figure 4A) antigen-specific CD8⁺ T cells should be detectable in the peripheral blood and splenic compartments (Figures 4B–4E). To confirm the functionality of these vaccine-induced responses, in vivo challenge models can be utilized. Here, we used a B16F10 murine melanoma model which naturally presents the antigens encoded in the mRNA vaccine. Mice vaccinated prophylactically should display some protection against tumor challenge, indicating the induction of functional anti-tumor immune responses (Figures 4G and 4H).

Limitations

There are several limitations associated with this protocol. First, only IFN-I responses are measured to evaluate the mRNA immunogenicity. Similarly, our assays are not designed to evaluate how the LNP contributes to LNP-mRNA immunogenicity. A third limitation is the reliance on anti-H2-K^b/SIINFEKL-PE antibodies to measure mRNA-encoded antigen presentation. Such antibodies might not exist for other antigens of interest and our approach requires including the SIINFEKL antigen in the mRNA sequence. Similarly, tetramers against the SIINFEKL antigens are available commercially but other antigens might require developing custom tetramers. Alternative approaches such as ELIPOST could also be used to measure antigen specificities.

Troubleshooting

Problem 1

In the instance of low RNA yield from IVT.

Potential solution

• •Use a brief spin with a microfuge to minimize poor yield due to reagents retained on the side of reaction vessel. It is critical to ensure the reagents are properly mixed through pipetting prior to incubation – this mixing should be visible to the naked eye as the T7 polymerase mix is viscous when initially added.
• •IVT reagents can become oxidized following multiple uses. 5 mM DTT (Dithiothreitol) can included in the reaction mix to offset the impact of oxidation.
• •Ensure that the RNA column used for purification has sufficient capacity for anticipated yield.
• •When using a new DNA template – run a control template concurrently in separate samples to confirm other IVT reagents are working adequately.
• •If the yield from a new DNA template remains low following confirmation of IVT reagents, further QC of template to confirm integrity, size, and the presence of Poly A tail should be carried out through restriction digest and gel electrophoresis.
• •If the yield from a new DNA template remains low following confirmation of IVT reagents and DNA template quality – consider increasing the concentration of DNA in reaction mix or increasing the incubation time for IVT.

Problem 2

In the instance of presence of high percentage of contaminant dsRNA.

Potential solution

• •Reduce the temperature used in IVT reaction or the Mg²⁺ concentration in IVT reaction.
• •Proceed to cellulose purification to remove dsRNA content.

Problem 3

In case of LNP degradation/rupture.

Potential solution

• •mRNA-LNP complexes should be handled gently to avoid disruption of the lipid membrane; vortexing can damage particle integrity.

Problem 4

In the instance of low induction of antigen-specific T cell responses measured via tetramer staining.

Potential solution

• •Consider the inclusion of a positive control to confirm tetramer staining efficacy. For example – splenocytes from OT-I mice can be used as positive control for SIINFEKL-specific T cells tetramers.
• •Consider orthogonal assays to measure T cell responses (e.g.,: ELISPOT).

Resource availability

Lead contact

Nicolas Vabret (nicolas.vabret@mssm.edu).

Technical contact

Nicolas Vabret (nicolas.vabret@mssm.edu).

Materials availability

This study did not generate new unique reagent.

Data and code availability

The protocol includes all datasets generated or analyzed during this study.