MRNA Transfection

Generation Of Carbonate Apatite Particles

Addition of only 3 mM Ca2+ to the HCO3  buffered cell culture medium (DMEM or Willium E, pH 7.5) and incubation at 370C for 30 min, resulted in microscopically visible particles. Generation of these particles only in HCO3, but not in Hepes-buffered media or solution (pH 7.5) containing the same amount of exogenous Ca2+ and phosphate (0.9 mM), indicates the possible involvement of carbonate along with phosphate and Ca2+ in particle formation.

1. Mix 3 to 6 μl of 1 M CaCl2 with 2 μg of plasmid DNA in 1 ml of fresh serum-free HCO3- –buffered (pH 7.5) medium (DMEM)

2. Incubation for 30 min at 37 °C for complete generation of DNA/carbonate apatite particles.

3. Add  particles to the rinsed cultured cells.

4. Change medium after 4-h incubation

5. Assay after 1 day.

Liposome Formation

1. dry solid DOTAP from chloroform solution.

2.  disperse in δH 2O.

3. shake at a temperature above the gel-to-liquid-crystalline transition temperature of the lipid (ca. 0°C) for 15 min

4. Sonication of the milky solution for 10 min.

Complex Formation

1. Dilute three micrograms of heat-denatured mRNA (10 min at 65°C) and 6—10 μg of DOTAP  separately in 100 μL DMEM media (pH 7.5). 2. Incubate for 15 min.

3. Add DOTAP to the RNA solution.

4. Incubate for 15 min at room temperature.

5. Add 4 m M CaCl2 and 0 to 50 μg/mL fibronectin solution and then  adjust final volume to 1 mL

6. Incubate for 30 min at 37°C.

7. Add transfection complexes to cells (medium with serum).

8. Incubate at 37°C in a 5% CO2 humidified environment for 2—4 h.

9. Remove the transfection mix and replace with complete medium (with serum )

Lipid-coated nanoparticles with a poly-1 core were synthesized via two different processes:
a double emulsion/solvent evaporation approach or a solvent diffusion/nanoprecipitation
strategy. For double emulsion synthesis, we adapted a previous approach we used for
preparing lipid-enveloped PLGA particles37: 30 mg of poly-1 (or PLGA for pH-insensitive
control particles) and 2 mg of the phospholipids DOPC, DOTAP, and DSPE-PEG in a 7:2:1
molar ratio were co-dissolved in 1 ml of dicholoromethane (DCM). PBS (200 μl) was then
added to the mixture on ice during a 1 min sonication step at 7 W using a probe tip sonicator
(Misonix XL2000, Farmingdale, NY) to form a first emulsion. The primary emulsion was
then dispersed into 6 ml of distilled, deionized nuclease-free water with sonication at 12 W
for 5 min, before leaving on a shaker for 18 h at 25 °C to evaporate the organic solvent. For
nanoprecipitation synthesis, the same quantities of DOPC, DOTAP, and polymer were co-
dissolved in 4 ml of ethanol and added drop-wise to 40 ml of distilled, deionized nuclease-
free water, followed by gentle stirring for 5 h to evaporate ethanol. For particles prepared by
nanoprecipitation, we found that adding DSPE-PEG in the organic phase together with
DOPC, DOTAP and polymer reduced particle yield significantly, so DSPE-PEG was
introduced into the lipid coating via a post-insertion process: DSPE-PEG lipid was added at
1 mM to 0.5 mg/ml particles in distilled, deionized nuclease-free water and the suspension
was stirred for 16 h at 25 °C. The particles were collected and washed once via
centrifugation, resuspended in fresh water and stored at 4 °C until use. Lipid-free PVA-
stabilized particles were prepared by similar processes except the organic emulsion or
solution containing polymer only was dispersed into a 2% w/vol PVA aqueous solution.
A fraction of each particle batch was dried in a vacuum oven to determine the particle
concentration (mg/ml) by measuring the dry mass. Dynamic light scattering (DLS) and zeta
potential measurements were used to determine the particle size and surface charge using a
ZetaPALS dynamic light scattering detector (Brookhaven Instruments). To estimate the
percentage of PEG-lipid incorporated and the resultant mol% of PEG-lipid present in the
lipid surface coating, lipid-enveloped particles were prepared by nanoprecipitation as
described above with 1 mol% DOPE-rhodamine added as a tracer for measuring the total
amount of lipid incorporated into the particles, before the post-insertion of a DSPE-PEG
lipid labeled with carboxyfluorescein (DSPE-PEG-CF). The lipids were then stripped from
the particle surface by treatment with 2% triton X-100 for 15 min and the supernatant was
measured for both rhodamine and fluorescein signals to quantitate the amount of PEG lipid
incorporated.

Pseudouridine modified mRNA

mRNAs were transcribed as previously described,14 using
linearized plasmids encoding wild-type and codon-optimized murine EPO
(pTEVwtEPO and pTEVoptEPO), firefly luciferase (pTEVLuc), and rhe-
sus macaque EPO (pTEVrhEPO). The rhesus EPO and codon-­ ptimized
o
mouse EPO genes were synthesized by Entelechon (Bad Abbach, Germany).
Codons for the mouse EPO gene were selected based on Entelechon’s pro-
prietary algorithm and codon-optimized human EPO.18 The Megascript
T7 RNA polymerase kit (Ambion, Austin, TX) was used for transcription,
and UTP was replaced with pseudouridine triphosphate (TriLink, San
Diego, CA) to generate pseudouridine-containing mRNA. All mRNAs
were transcribed to contain 51-nt long poly(A) tails. Additional poly(A)
tail was added in a 3-hour incubation with yeast poly(A) polymerase
(USB, Cleveland OH). To obtain cap1, RNA was processed using the m7G
capping and 2′-O-methyltransferase kits (CellScript, Madison, WI). All
mRNAs were HPLC-purified as described.1
mRNAs were transcribed as previously described,14 using
linearized plasmids encoding wild-type and codon-optimized murine EPO
(pTEVwtEPO and pTEVoptEPO), firefly luciferase (pTEVLuc), and rhe-
sus macaque EPO (pTEVrhEPO). The rhesus EPO and codon-­ ptimized
o
mouse EPO genes were synthesized by Entelechon (Bad Abbach, Germany).
Codons for the mouse EPO gene were selected based on Entelechon’s pro-
prietary algorithm and codon-optimized human EPO.18 The Megascript
T7 RNA polymerase kit (Ambion, Austin, TX) was used for transcription,
and UTP was replaced with pseudouridine triphosphate (TriLink, San
Diego, CA) to generate pseudouridine-containing mRNA. All mRNAs
were transcribed to contain 51-nt long poly(A) tails. Additional poly(A)
tail was added in a 3-hour incubation with yeast poly(A) polymerase
(USB, Cleveland OH). To obtain cap1, RNA was processed using the m7G
capping and 2′-O-methyltransferase kits (CellScript, Madison, WI). All
mRNAs were HPLC-purified as described.1