Experiment Summary

Recently, interest has been garnered for the use of nucleoside-modified mRNA-LNP (mRNA-LNP) due to their safe and non-integrative nature. Unlike viral gene delivery systems that come with a risk of mutagenesis and systemic adverse effects, intravenously administered mRNA-LNP are directed to the liver, with robust yet transient protein expression induced for only about 5 days. mRNA-LNPs also provide an advantage over recombinant protein replacement therapy, which has limitations due to short protein half-life, requiring repetitive dosing to achieve benefit. Other methods for delivering protein expression to the liver, such as ASGPR-mediated hepatocyte targeting, also come with limitations such as off-target effects and reduced effectiveness in diseased conditions.

Fig. 1 Experimental Design of mRNA-LNP IV Injection and Analysis of Liver Cell Specificity and Efficiency of Transfection.

Materials and Reagents

A. mRNA-LNP production:

1. T7 RNA polymerase
2. One-methylpseudouridine (m1Ψ)-5'-triphosphate
3. m7G capping kit with 2'-O-methyltransferase
4. Fast Protein Liquid Chromatography (FPLC)
5. Agarose
6. TAE buffer
7. mRNA-LNP to be stored at -80°C

B. In vivo administration of mRNA-LNP:

1. Dulbecco's Phosphate Buffered Saline (DPBS 1×), Sterile, (-) Calcium Chloride, (-) Magnesium Chloride
2. Isothesia Isoflurane, USP
3. 1/2cc Lo-Dose Insulin Syringe, 0.5 ml, 29 G, sterile

C. Tissue fixation, cryopreservation, sectioning, and immunofluorescence on sections:

4. FluorSave reagent
5. Triton-X 100
6. 16% Paraformaldehyde Aqueous Solution, EM Grade
7. Tissue Plus 4585 O.C.T. Compound, Clear
8. Sucrose
9. Normal Donkey Serum
10. DAPI, 4',6-Diamidino-2-Phenylindole, Dilactate

D. Liver perfusion and flow cytometry:

1. Intravenous (I.V.) administration set
2. Catheter, IV, 24 G × 3/4", Sterile, SURFLO®
3. Pump Tubings, 3P Set of 12 PVC Flow Tubes of 1.52 mm
4. 40 µm Sterile Cell Strainer
5. 100 µm Sterile Cell Strainer
6. 60 mm Petri Dish
7. 1cc Insulin Syringe, 1.0 ml, 28 G, sterile
8. Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap
9. Liver Perfusion Medium 1×, called "Solution 1" in procedure, ~30 ml per mouse
10. Earl's Balanced Salt Solution (EBSS), Ca²⁺, Mg²⁺, phenol red, pH 7.4, called "Solution 2" in procedure, ~20 ml per mouse
11. Liver Digest Medium, called "Solution 3" in procedure, ~50 ml per mouse
12. Hepatocyte Wash Medium
13. Collagenase Type IV
14. DNase I
15. Trypsin-EDTA (1×), 0.05%, phenol red
16. Phosphate Buffered Saline (PBS), 10× solution
17. Small Cotton-Tipped Applicators, 3 inches, Wooden Shaft, Sterilized
18. Ketamine Hydrochloride
19. Dulbecco's Phosphate Buffered Saline
20. Fc Block, CD16/CD32 Purified Rat anti-Mouse
21. Ethylenediaminetetraacetic acid (EDTA), 0.5M, pH 8.0
22. Fetal Bovine Serum (FBS) Characterized
23. Zombie NIR Fixable Viability Dye
24. Red Blood Cell Lysing Buffer Hybri-Max
25. Antibodies



26. DNase I stock solution (10 mg/ml)
27. NPC Digest Solution
28. Wash Medium
29. Ketamine Anesthetic Solution
30. FACs Buffer
31. 4% Paraformaldehyde
32. 3% Normal Donkey Serum
33. DAPI stock solution

Equipment

1. Magnetic Stirring Bars
2. HotHands Hand Warmers, used as heating pads for anesthetized mice
3. Flow cytometer
4. Peristaltic perfusion pump
5. High-speed refrigerated centrifuge
6. Fluorescence microscope
7. Cryostat
8. Magnetic stirrer
9. Zetasizer Nano ZS dynamic light scattering instrument
10. Electrophoresis machine

Procedure

A. mRNA production

1. Produce mRNAs using T7 RNA polymerase on a linearized plasmid encoding eGFP.
2. Transcribe mRNAs to contain 101 nucleotide-long poly(A) tails.
3. Cap RNAs using the m7G capping kit with 2'-O-methyltransferase to obtain cap1.
4. Purify mRNA by Fast Protein Liquid Chromatography (FPLC).
5. Analyze all mRNAs by agarose gel electrophoresis and store frozen at -20°C.

B. LNP encapsulation of the mRNA

1. Encapsulate FPLC-purified m1Ψ-containing mRNAs in LNP using a self-assembly process in which an aqueous solution of mRNA at pH = 4.0 is rapidly mixed with a solution of lipids dissolved in ethanol.

C. In vivo administration of mRNA-LNP

1. Thaw mRNA-LNP on ice immediately before use.
2. Dilute mRNA-LNP fresh on ice in sterile Dulbecco's Phosphate Buffered Saline (PBS) prior to each experiment.
3. Anesthetize mice using an isoflurane chamber.
4. Remove mice from the anesthesia chamber and quickly inject mRNA-LNP into the retro-orbital sinus using 1/2cc insulin syringes.

D. Tissue fixation, cryopreservation, sectioning, and immunofluorescence on sections

1. Collect intact liver and other organs including spleen, intestine, and lung directly in 4% paraformaldehyde (PFA) diluted in 1× PBS.
2. Fix for at least 2 h at room temperature.
3. For cryopreservation, wash tissues three times with 1× PBS and dip in 15% sucrose solution for 15 min.
4. Transfer tissues to 30% sucrose solution and keep until they sink to the bottom.
5. Embed the tissues in O.C.T. compound. The blocks can be stored at -80°C until further required.
6. Cut 5 μm liver sections with cryostat and store the slides at -20°C until required for immunostaining.
7. Defrost the frozen slides at room temperature for 30 min and dry.
8. Dip the slides in 1× PBS for 10 min.
9. Permeabilize the tissues using 0.3% Triton-X in 1× PBS for 10 min.
10. Rinse the slides three times in 1× PBS, 10 min each.
11. Block the tissue with 3% normal donkey serum diluted in 1× PBS for 30 min.
12. Incubate the sections overnight at 4°C with chicken anti-mouse GFP antibody diluted in 1× PBS at 1:300 concentration. Other markers can be used to identify specific cell types transfected.
13. Wash the slides three times with 1× PBS, 10 min each.
14. Incubate with fluorescently labeled anti-chicken secondary antibody for 1 h at room temperature protected from light at a concentration of 1:500 diluted in 1× PBS.
15. Wash the slides three times with 1× PBS for 10 min each.
16. Incubate with DAPI (1:3,000 in 1× PBS) for 3-5 min protected from light.
17. Rinse the slides two times with 1× PBS for 5 min each.
18. Mount using FluorSave reagent.
19. Image the slides using a fluorescent microscope.

E. Liver perfusion

1. Warm all solutions to 40°C in water bath.
2. Sterilize pump tubing with 70% ethanol, then wash with Solution 1.
3. Attach I.V. administration set to catheter at one end and to pump tubing at the other end and fill with Solution 1.
4. Anesthetize the mouse using Ketamine Anesthetic Solution at 100 μl/20 g body weight of mice, and place mice on heating pads to maintain their body temperature.
5. Immobilize all four limbs of the mouse with tape once under deep anesthesia on a moisture-absorbing surface. Open the abdomen and the chest area and push intestines to the side with a sterile cotton-tipped applicator, thereby exposing the vessels. Locate the inferior vena cava (IVC) in the chest area and portal vein under the liver.
6. Carefully cannulate the IVC using the catheter and start the flow of the pump at 4 ml/min (pump setting 6.5-6.8). The liver will fill up with the solution turning brown within a few seconds. Quickly locate and cut the portal vein. Tape down the catheter and tubing securely.
7. After about 30 ml of Solution 1, run approximately 10 ml of Solution 2, and finally run Solution 3 for 10-15 min.
8. When the liver is digested, remove the catheter, excise the gallbladder, and then carefully remove the liver without disrupting the liver capsule if possible.
9. Transfer the liver to a 60 mm Petri dish containing 10 ml of Solution 3. Disrupt the liver capsule with forceps and wash out the hepatocytes by swirling the dish. Collect hepatocytes and filter through a 100 µm filter unit into a sterile 50 ml conical tube that contains 10 ml of cold Wash Medium.
10. For studies of non-parenchymal cells (NPC), collect the tissue retained on the filter and digested further as directed in Step E12.
11. Pellet hepatocytes by centrifuging at 50 × g for 2 min at 4°C. Collect the supernatant and centrifuge it at 300 × g for 5 min and resuspend in cold Wash Medium. This is NPC Fraction 1. Resuspend the hepatocyte pellet in 50 ml cold Wash Medium.
12. Transfer tissue fragments collected in Step E10 to a 60 mm Petri dish. Add 5 ml of NPC Digest Solution. Add a magnetic stirring bar and stir at 37°C for 10 min. After 10 min, pipette the solution up and down to assist the breakup of remaining tissue fragments.
13. Collect dissociated cells by passage through a 40 µm strainer. Spin at 300 × g and resuspend the pellet in cold Wash Medium. This is NPC Fraction 2.
14. Transfer remaining tissue from Step E13 to a new dish with 5 ml of 0.05% trypsin, and incubate at 37°C with stirring for 10-15 min. After stirring, pipette the solution up and down to assist the breakup of remaining tissue fragments.
15. Collect newly dissociated cells by passage through a 40 µm strainer. Spin at 300 × g and resuspend in cold Wash Medium. This is NPC Fraction 3.
16. Pool all NPC Fractions collected in Steps E11, E13, and E15, pellet the cells by centrifugation, and resuspend in 5 ml cold Wash Medium. This gives the total NPC fraction.

F. Flow Cytometry

1. Centrifuge hepatocytes at 50 × g and NPCs at 300 × g and resuspend in 1-2 ml RBC lysis buffer.
2. Keep in the lysis buffer for 2 min at room temperature, then add 10 ml to 20 ml Wash Medium.
3. Wash cells by centrifuging hepatocytes at 50 × g and NPCs at 300 × g. Resuspend hepatocytes and NPCs in 1-2 ml PBS depending upon the number of wells/samples required for analysis.
4. Incubate fractions in Zombie NIR dye for 30 min at room temperature. Wash with Wash Media, centrifuge appropriately, then resuspend in FACS Buffer.
5. Dispense 100 μl cell suspension in each well of a 96 well plate, as necessary.
6. Add 2 μl Fc block to each well and wait for 10 min.
7. Add respective flow cytometry antibodies and incubate for 20 min at room temperature. Figure 3B outlines markers for endothelial cells, leukocytes, and pan-immune cells, for example.
8. Centrifuge plate at 700 × g for 4 min.
9. Remove supernatant, and add 100 μl FACS Buffer to each well.
10. Centrifuge plate at 700 × g for 4 min.
11. Remove supernatant, and add 100 μl FACS Buffer to each well.
12. Resuspend thoroughly, then transfer cells into the filter top of FACS tubes prepared with 300 μl FACS buffer.
13. Run cells on a flow cytometer.

Fig. 2 Identification of transfected liver cell types 5 h after IV eGFP mRNA-LNP injection.