Editing Cleancap mRNA Invitro Transcription (section)

== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Transfection</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Thaw mRNAs were thawed on ice and denatured at 65°C for 3min</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Incubation at room temperature for 45min.</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''1. Lipofectamine 3000'''</span>

*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">On day 0, HEK 293T cells are seeded at a concentration of&nbsp;5 × 10<sup>5 </sup>cells/well in a 6-well plate to ascertain 70–90&nbsp;% of confluency on the day of transfection (day 1).</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">For transfection, a ratio of 2 μl of Lipofectamine® reagent to 1 μg of RNA is used. Cells are transfected with 1 and 2 μg of&nbsp;mRNA. Transfection is performed in duplicates.</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">On the day of transfection, an appropriate volume of Lipofectamine® reagent is prepared in Opti-MEM® medium and incubated at room temperature for 5 min.</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">&nbsp;1 and 2 μg of mRNA are diluted in Opti-MEM® medium. If required, pre-dilute mRNA to a concentration of 0.5 μg/μl.</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Mix Lipofectamine® reagent and Opti-MEM®-diluted mRNA&nbsp;gently in a total volume of 500 μl at a 1:1 ratio (v/v) and incubated&nbsp;at room temperature for 20 min.</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Wash cells twice with 500 μl DMEM medium containing glutamine&nbsp;but without FBS. Add 2 ml of DMEM to the cells.</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Add 500 μl of mRNA–lipid complexes to each well. Mix gently.&nbsp;</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Incubate cells at 37 °C in a CO2-incubator for 4–6 h.</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">After the incubation, replace the medium with 2 ml of fresh DMEM medium containing 10&nbsp;% FBS.</span>
*

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''2. TT3 nanoparticles'''</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">lipid:DOPE:cholesterol:DMG-PEG2000 at a molar ratio of 20:30:40:0.75 ([https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869688/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869688/])</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">3. Protamine&nbsp;as an immunostimulatory component</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Protamine. Pharmaceutical Protamine is available at two&nbsp;concentrations: 1000 (1 ml neutralizes 1000 IU of heparin) or&nbsp;5000 (1 ml neutralizes 5000 IU of heparin). To generate particles&nbsp;of 130 nm, we use Protamine Ipex 5000 from MEDA&nbsp;Pharmaceuticals (Protamine hydrochloride 5000 IU/ml). The Protamine is stored at 4 °C&nbsp;</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Dissolve the protamine in Ringer lactate solution.<br/>&nbsp;While stirring, slowly add the protamine-Ringer lactate solution&nbsp;half of the mRNA until you reach a weight ratio of&nbsp;mRNA and protamine of 2:1.<br/>&nbsp;Stir the solution for another 10 min to ensure the formation of&nbsp;stable complexes.<br/>&nbsp;Add the remaining half of free mRNA and stir briefly.<br/>&nbsp;Adjust the final concentration of the vaccine with Ringer lactate&nbsp;solution.</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Carry out all procedures at room temperature unless otherwise&nbsp;specified.</span>

<br/><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">This step is performed under laminar flow using sterile&nbsp;equipment.</span>

*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Prepare a 0.5 mg/ml Protamine solution (see Note 5) by diluting&nbsp;10 μl of Protamine 5000 in 280 μl of water.&nbsp;</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Prepare a 0.5 mg/ml RNA solution (see Note 5) by diluting&nbsp;the RNA stock with the appropriate amount of water.</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Add an equal amount of Protamine solution to the RNA solution&nbsp;(see Note 6), and pipet quickly up and down at least ten&nbsp;times (see Note 7).</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Leave the solution at room temperature for 10 min (see Note 8).&nbsp;1. Dilute 40 μl of the Protamine–RNA formulation (containing&nbsp;10 μg of RNA and 10 μg of Protamine) with 1 ml of water (see&nbsp;Note 9).</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Set the viscosity of the analyzer to 0.89 (select within the software&nbsp;setting either measurement in water or a viscosity of&nbsp;0.89). As shown in Fig. 1, particles made in the conditions&nbsp;described above are approximately 130 nm in size with a polydispersity&nbsp;index (PDI) of less than 0.3 (see Note 10).</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Protamine sulfate, such as Valeant Protamine, gave identical results.<br/>Freezing Protamine solutions may jeopardize&nbsp;its functionality as far as production of immunostimulating&nbsp;RNA polyplex is concerned. Pharmaceutical Protamine should&nbsp;be stored at 4 °C as indicated on the package.<br/>Any RNA (oligonucleotide, long uncapped and/or non-polyadenylatedmRNA, or capped mRNA) can be used to generate&nbsp;the 130 nm particles</span>