Experimental Design
Our project consists of 2 main parts: the LNP synthesis and the mRNA production of factor VIII. Furthermore, the experiments involved GFP expression as an assessment method for LNP’s in HEK cells. Lastly, the mRNA expression in HEK cells together with the insertion of the mRNA into the vesicles, along with the detection method and the evaluation of the engineered product towards the expression of factor VIII(Fig.1). You can refer to our protocols for the detailed experimental procedure.
Figure 1. Overview of the process to reach the required protein Factor VIII.
The process timeline starts with transfecting pCDNA4 plasmids into E.coli bacteria strain where they are amplified. The linearization is carried out via XhoI restriction enzyme cleavage. The pcDNA is transcribed into an mRNA sequence which undergoes 5’capping and 3’ poly-A tailing. The modified mRNA is encapsulated in our produced lipid nanoparticles. These nanoparticles are introduced in HEK cell line where the the full FVIII protein is translated.
DNA FVIII production
We made the DNA of FVIII by transfecting Top 10 and XL 1 blue E.Coli bacterial suspensions, with pCDNA4 plasmid in a medium containing ampicillin. The pc DNA from both bacterial colonies was extracted using EcoRI and XhoI and was analyzed for size with gel electrophoresis. We determined the concentration of the isolated plasmids and the XL1 blue had a higher concentration, thus we used it for mRNA production. The XL1 blue was linearised using Xhol digestion following the protocol: “Enzyme digestion”.
GFP Production
We wanted to amplify eGFP DNA, which would serve as control mRNA for the lipid encapsulation. The eGFP DNA was amplified by PCR using the following protocol: “PCR procedure Radboud University team” and PUC-18 GFP template. After a sufficient amount of GFP DNA was received, we continued with the purification of the DNA. For the PCR purification, we used QIAquick PCR Purification Kit and the protocol QIA PCR purification was followed (“QIAquick® PCR Purification Kit”).
mRNA Production
The FVIII and GFP mRNA were produced by T7 RNA polymerase from the cp FVIII DNA and the GFP DNA using the protocol listed as “In vitro synthesis of RNA using T7 RNA polymerase”. RNAase inhibitor was used to prevent the RNA from degrading. This step was repeated several times with several adjustments in the protocol for a high yield of mRNA. The concentration was assessed using the nanodrop Uv-vis spectrometer. After the production of a sufficient amount of RNA, the next step was its purification using the Phenol-Chloroform solution and extraction method using the protocol “RNA cleanup by phenol-chloroform” After the extraction, the mRNA had to be modified to prevent its degradation, thus it was capped and tailed. The mRNA was capped following the protocol Cap-0 synthesis using Vaccinia Capping enzyme and the Vaccinia (NEB#2080) enzyme to form the complete Cap-0 structure. Later the mRNA was tailed following the protocol “Poly(A) Tailing of RNA using E.coli Poly(A) polymerase” using E. coli Poly(A) Polymerase (NEB #M0276).
LNP production and evalution
For the LNP production, ionizable lipids 246 were synthesized using the following protocol: “Ionizable Lipid synthesis”. After the synthesis of our lipids, we continued with the LNP preparation using the protocol: “LNP procedure” with torula yeast mRNA as a trial attempt, which involved the mixing of the lipids (ionizable lipids 246, DOPE, cholesterol, DSPE PEG-2000, ethanol) and the encapsulation of the yeast mRNA using the ethanol injection method. The size of the LNPs was assessed with DLS (Dynamic Light Scattering). Then we continued with making LNPs encapsulating the GFP and FVIII mRNA made previously. The LNPs were checked again using DLS and the encapsulation was assessed using TEM (Transmission Electron Microscopy) and the protocol: “LNP encapsulation efficiency”.
mRNA evaluation
The produced mRNA’s of FVIII and GFP had to be evaluated for their translative abilities. This is why we performed experiments using Hek 293 cell lines. We encapsulated the mRNA into our LNPs and transfected the HEK cells using the lipotransfection method with the Jet messenger transfection reagent. For the GFP mRNA transfection, we used the following protocol: “Polyplus Short-protocol- jetMESSENGER-mRNA” and incubated the cells overnight. After the incubation, the cells were evaluated for any GFP production using the EVOS M5000 fluorescence microscope.