mRNA synthesis and formulation

1.      Design plasmid DNA sequence to include:

a.      Origin of replication, antibiotic resistance, restriction enzyme site, T7 promoter, 5’UTR, ORF, 3’UTR, polyA tail, restriction enzyme site.

2.      Purchase plasmid DNA from preferred vendor with or without polyA tail encoded.

a.      E. coli resists plasmids with long stretches of purines, so be prepared to sequence many colonies to find a correct clone.

b.      If there is continued failures around tail heterogeneity, we recommend PCR to incorporate the polyA tail using a reverse primer containing the desired tail length. The high purity primer can be obtained from Oligo Factory, Holliston, MA.

3.      Transform the plasmid into a desired E. coli strain and use Sanger sequencing to find a correct clone.

4.      Inoculate bacterial culture media with the appropriate selection antibiotic and grow to desired optical density.

5.      Perform giga prep to recover plasmid DNA.  Yields depend on the plasmid sequence and the E. coli strain (0.1 – 5 mg for giga prep is expected).

6.      Digest your circular plasmid DNA with the restriction enzymes to linearize it and clean it up by tangential flow filtration (TFF). Plasmid DNA can be frozen for years.

7.      To perform in vitro transcription; add NTP mix, buffer, pyrophosphatase, RNase inhibitor (optional), and T7 RNA polymerase to a tube and bring to 37 °C.

a.      Temperature control is critical.

b.      Follow T7 RNA polymerase standard reaction conditions.

c.       Detailed concentrations of the reaction components can be found in U.S. patent number 10,653,712, Table 1.

8.      Add linearized DNA template also pre-heated to 37 °C.

9.      Incubate for 2 hours with mixing.

10.  Quench the reaction with the addition of EDTA to a final concentration of twice the magnesium concentration in the reaction.

11.  Clean up the mRNA using column purification (e.g., oligo dT, ThermoFisher A48605 and reversed-phase HPLC described in DOI 10.1093/nar/gkr695).

12.  Store the mRNA frozen in slightly acidic buffer.  We recommend 2 mM sodium citrate, pH 6.5.

a.      The mRNA is stable for years at -20 °C.

13.  To formulate mRNA in lipid nanoparticles (LNP); prepare lipid stocks in neat ethanol in the ratios desired.

14.  Prepare mRNA stock by acidifying to a final concentration of 37.5 mM sodium acetate, pH 5.

15.  Using a micro T (Signa 51283-U) and peristaltic pumps, set to a total of 12 mL/min (3:1 aq:org), mix lipids and mRNA with an ionizable:phosphate ratio of 5.67 and a total lipid to mRNA ratio of 20:1.

16.  After a brief wait (approximately 5 minutes), neutralize the mix with a desired final buffer. We prefer using 1M Tris, pH 8.0 to reach a final ethanol concentration less than 10 % (v/v)

17.  Place in dialysis immediately (ThermoFisher 66333) and leave overnight in desired storage buffer (e.g., PBS or Tris-Sucrose).

18.  Filter through 0.22 um filter disc (Pall 66234).

19.  Measure mRNA encapsulation (ThermoFisher R11490) and LNP particle size (Wyatt DynaPro NanoStar)

a.      Encapsulation should be above 80 %, and particle size should be 80-120 nm by DLS with a PDI under 0.5.

20.  Store either frozen or at 5 °C.

a.      Stable for approximately 1 month at 5 °C, and approximately 1 year at -70 °C.