Results

Results mRNA

Summary

Our project consisted of three main parts: mRNA production, lipid nanoparticle production and cell line transfection (Figure 1). In result we did not manage to produce a full line of living cells with expression FVIII protein after transfecting them with LNPs containing this mRNA. However, there are multiple points throughout our project that can be considered as a success and basis for possible future research. For example, the cells transfected with our produced nanoparticles were seen to be alive which gave an indication of the LNP safety. The BioBrick of our project was successfully produced in a form of functional cpDNA - BBa_K5342000.

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 the HEK cell line where the full FVIII protein is translated.

Main points of mRNA results

PCR of FVIII and GFP cDNA

As the first part of our project, polymerase chain reaction (PCR) was carried out on FVIII, GFP and eGFP cDNA to obtain higher concentration of DNA which was then further transcribed via IVT (In-Vitro transcription). Polymerase chain reaction is a well known nucleic acid chain elongation procedure that can be used for both DNA and RNA sequences. It consists of denaturation, renaturation, elongation steps [3]. For our project we implemented the use of Pfu polymerase. The reaction efficiency was then analysed by agarose gel electrophoresis to detect the amplified DNA and any impurities. A smearing in the sample rows could be seen frequently, the band size of F8 cDNA was faint or the purity of acquired samples was not optimal (Figures 2-5). On 03/07 engineered GFP was produced via PCR (Figures 6 and 7). The result was assessed on agarose gel on 04/07. A clear band of GFP cDNA was detected on the gel and a concentration of 60.2 ng/ul was acquired via nanodrop (Figures 8, 9).

Figure 2. Agarose gel electrophoresis of amplified cDNA via PCR. (24/05/24)

Indication of rows: 1st DNA ladder, 2nd positive control, 3rd FVIII template, 4th negative control. Visible bands after amplifying the Factor 8 cDNA via polymerase chain reaction. Correct FVIII DNA band visible at around 7.5 kb. A smearing is detected. A decision is made to extract the DNA from this gel, purify and amplify it further to acquire a larger quantity of the DNA.

Figure 3. Agarose gel electrophoresis of amplified cDNA with a different set of primers in PCR. (03/06/24)

Indication of rows: 1st DNA ladder, 2nd negative control, 3rd positive, 4th PCR reaction with a new set of primers, 5th eluted DNA, 6th another DNA ladder. The purified DNA is visible as a clear band at the expected height (5th row), the vagueness of the band is due to the sample not being amplified. The negative control shows a smear, the positive control is not detectable.

Figure 4. Agarose gel electrophoresis of amplified cDNA via PCR. (28/06/24)

Indication of 4 rows (from left to right): gene ladder, negative control (using 2nd set of primers), positive control, cDNA with primers (using 1st set of primers). The 4th slot contains a very slight band indicating the amount of amplified DNA is very scarce. A slight band at around 7.5 kB can be seen. The amplification still results in big smears.

Figure 5. Agarose gel electrophoresis of amplified cDNA via PCR. (01/07/2024)

Indication of rows: 1st DNA ladder, 2nd – 6th FVIII, 7th GFP, 8th DNA ladder. Very clear smears visible after PCR of F8 cDNA with another set of new primers and GFP with the standard GFP primers. 5th row is blank due to loss of the sample. In row 7 very vague bands are seen - low in the bottom of the gel and at the top. The size of GFP cDNA is 730 bp.

Figure 6. Agarose gel electrophoresis of amplified cDNA via PCR. (03/07/2024)

Indication of rows: 1st DNA ladder, 2nd -4th eGFP cDNA, 5th DNA ladder. An eGFP cDNA correctly received at around the 1000bp region (see the ladder). Bands are very dim due to low concentration.

Figure 7. Agarose gel electrophoresis of amplified eGFP cDNA via PCR. (04/07/2024)

Indication of rows: 1st DNA ladder, 2nd to 5th eGFP cDNA. All samples show a clear band at ~1000kb.

Figure 8. Agarose gel of purified eGFP via PCR (done on 03/07/2024)

Indication of rows: 1st DNA ladder, 2nd purified eGFP cDNA. A little smear at the top. The band is detected at around 1000 bp, the expected eGFP size.

Figure 9. Analysis of eGFP cDNA by nanodrop. (04/07/2024)

Measured samples of the GFP cDNA show a concentration of 60.2 ng/ul. The ratio of A260/280 is 1.92. This cDNA is further used for GFP mRNA production.

Analysis and conclusion:

The smearing during PCR of the F8 and GFP DNA might have resulted from primers not being optimally designed for the amplification, or from cross-contamination during pipetting and other steps of the experiment. The generally low amplification of F8 cDNA may have been due to the large size of the DNA chain and poor primer design which led to improper primer annealing. Although, in experiments where the primers would have worked well normally, the yield of mRNA was very poor or highly faulty. A deeper insight has to be done to assess the optimal amount of cycles during PCR to obtain a large quantity of cDNA. This can be very time consuming as even the previously calculated conditions did not meet the resultative expectations.

Amplification of pcDNA via bacterial plasmids

It was suggested by our instructor to try out pcDNA amplification. From 03/07 till 08/07 pcDNA amplification via bacterial plasmids was executed out with successful results. The bacterial cultures used in this part were Top10 and XL1Blue. The plasmids we used had been retrieved from DH5alpha bacteria strain with ampicillin resistance (Figure 12) [1]. The plasmids we used were a mammalian expression vector with 5021 bp backbone and an insert size of 7065 bp. The 5' cloning site was BsiWI and 3' cloning site NotI. A H. Sapiens FVIII gene had been inserted in the specified cloning site. [1] These plasmids were transfected into bacteria cultures Top10 and XL1 Blue. The isolation of plasmids showcased better results from XL1 Blue bacteria colonies, thus, these were used further. The plasmids were digested by the means of XhoI and EcoRI restriction enzymes. The XhoI cleavage sequence is C'TCGAG at position 7126 of the plasmid (Figures 11, 12) [1]. This sequence is found once in the plasmid. In result, a 12.1 kbp linearized plasmid was received. The EcoRI cleavage was used to assess the accuracy of plasmid construct parts. An agarose gel electrophoresis was prepared to assess the purification and digestion of FVIII pcDNA plasmids (Figure 10). The digested plasmids displayed clear bands at the expected length of the vector – 12086 base pairs. [1] This attested the capability of these samples for further run-off IVT.

Figure 10. Agarose gel analysis of digested F8 pcDNA plasmids. (08/07/2024)

Indication of rows: 1st DNA ladder, 2nd unpurified digested plasmids, 3rd-4th remnants of sample 7th – 10th empty wells, 11th-12th purified digested plasmids, 13th DNA ladder, 14th-15th purified digested plasmids. Digestion was done via XhoI restriction enzyme. The plasmids show clear bands at the expected length of 12086 bp.

Figure 11. pCDNA4/Full length FVIII sequence[1].

The length of F8 is noted as a yellow arrow. The restriction enzyme used is XhoI which cleaves in sequence C'TCGAG.

Figure 12. Full sequence map of pCDNA4/Full length FVIII [1].

The length of F8 is noted as a yellow arrow.

Conclusion

pcDNA amplification resulted in much better yield of DNA as can be seen in figure 10. The purified digested plasmids of 12086 base pairs were visualised as bright bands above the 10000 bp mark. This proved the viability of these samples to be used for further run-off IVT.

IVT of FVIII pcDNA and eGFP cDNA.

From 09/07 till 30/08 In-Vitro transcription was carried out for F8 pcDNA and eGFP cDNA to obtain functional mRNA. A nanodrop assessment on 28/08 revealed high concentrations of mRNA samples. The average concentration of F8 and GFP mRNA was calculated to be 2480,41 ng/ul and 1845,70 ng/ul, correspondingly. An average A260/280 ratio of F8 and GFP mRNA was calculated to be 2,10 and 2,14, respectively. And, an average A260/230 ratio for GFP mRNA was calculated to be 2,46 (Table 1). On 29/08 the transcribed samples were analysed via agarose gel electrophoresis. A clear, but vague band was seen, indicating the presence of GFP mRNA. The rest of the samples were not observable, including a positive control sample (Figure 13). On 30/08 a new agarose gel electrophoresis was carried out for the purpose of analysing the samples from 28/08 again. A bright band of GFP mRNA was noticed. The positive control appeared in the form of a vague band. The GFP sample was further used for capping, tailing and analysis.

Table 1. Nanodrop analysis of transcribed F8 and GFP mRNA.

The A260/280 ratio detects protein contamination in samples. The A260/230 ratio detects salt contamination, data for samples H, I, J were lost during notebook keeping. The average concentration (ng/ul) is 2480 and 1847 for F8 and GFP, respectively. The average A260/280 ratio is 2.1 for both mRNA types. The average GFP A260/230 ratio is 2.5.

Figure 13. Agarose gel analysis of transcribed samples via IVT. (29/08/2024)

Indication of rows from left to right: A-B-C-D-E-F-G-H-I-J-F8(1) -F8(2)-GFP-Positive sample (see, Table 1). A clear, vague band on row 3 (sample C – GFP) and 13 (modified GFP). The rest of the samples are not visually detectable. F8(1), F8(2), and GFP indicate capped and tailed (modified) mRNA samples acquired from previous experiments.

Figure 14. Agarose gel analysis of IVT transcribed samples. (30/08/2024)

Indication of rows from left to right: B-D-E-F-G-H-I-J-F8(2)-Positive sample (processed samples) (see Table 1). On the 5th row a clear band can be seen with a smear. The supposed sample is GFP mRNA. A clear band is visible in the 10th row as the positive sample. F8(2) indicates the capped and tailed mRNA sample acquired from previous experiments.

Analysis and conclusion

Throughout the IVT experiments no clear products were obtained. Frequent cases with large smearing were observed or even no viable mRNA could be found when assessing the gel. Multiple reasons were listed as causation of these results. For example, due to the large size of F8 cDNA, a larger volume of T7 polymerase was required. As can be seen in Figure 19, the samples, although with smears, showed higher translational efficiency when using 4 µl T7 polymerase instead of 2 µl as requested in the protocol. Even with the samples of lower efficiency a phenomenon of uneven band strength was observed. A reason might have been contamination and RNA degradation prior to analysis. Additionally, The FVIII RNA was visible at 7000 bp, but the expected is at 1200 pb. This could also have resulted from RNA degradation, self-splicing, due to the properties of the gel and the non-linearity of the RNA. As can be seen in Figure 21 the GFP RNA did not show clear bands in both sample rows. Likely reason was the template. The calculations on the amount required might have been incorrect. Further trials were tested including RNAse inhibitors. This showed partially promising results. Some samples were seen with a clear band, others – smears. Furthermore, an empty row was noticed. The cause was concluded to be a blunder – the falling of pipette tip from the pipette (Figure 20). Further discussion can be made to assess whether such blunders of improper pipetting could have caused absent results and contamination not only in this particular experiment but also throughout the project. Lastly, in Figure 13 no positive control sample band was detected. This requires a deeper insight whether different properties are required for optimal agarose gel analysis. One of the IVT experiments did show promising results when analysing samples via nanodrop. The concentrations of both GFP and F8 were found to be high, the A260/280 and A260/230 ratio gave a good indication of purity. These ratios can estimate whether the sample is contaminated with protein structures and salts, respectively (Table 1). When analysing these samples on gel, no band presence was visualised. Possible cause thought to be mRNA splicing which would not have been detected on the 0.7 % agarose gel. All small RNA samples would have ‘run out’ during the electrophoresis process (Figure 15).

Figure 20. Agarose gel of eGFP with wild type T7 polymerase and RNAse inhibitor. (10/07/24)

Indication of rows: 1st – 5th and 7th – 11th eGFP mRNA, 6th DNA ladder. Row 3 sample is absent (due to blunder error). Samples of rows 4 and 5 are visible with smearing. Samples 2 and 7 have a significant smear.

Figure 21. Agarose gel electrophoresis of IVT samples. (09/07/24) (To prevent waste, the same agarose gel is used for multiple experiments. The crossed out lanes are from a previous experiment.)

Indication of rows (excluding crossed out ones): 1st F8 mRNA, 2nd- 4th F8 mRNA, 5th GFP mRNA, 6th DNA ladder, 7th – 8th F8 mRNA, 9th GFP mRNA. 1st, 2nd and 4th F8 mRNA samples with 2 µL T7 polymerase does not show high transcriptional efficiency while the 3rd seems to have been transcribed well. Samples of rows 7 - 9 show a possible band but with significant smearing.

Modification of mRNA samples

The modification of the mRNA samples was carried out from 26/08 till 13/09. The samples were capped with 3’ poly-A tail sequence and tailed with 5’ cap, with purification included in-between both steps. The 5’ cap is a modified guanosine which consists of a methyl group on the 7-position. This process is vital for the maturity of the mRNA and translational competence. The poly-A tail ensures protection from enzymatic degradation and ensures the translational process [2]. Multiple sets of samples were assessed on the gel which included normal, unprocessed transcribed samples as can be seen in figures 13 and 14. This section of our research did not bear a highly positive outcome. Our instructor provided several samples of mRNA transcribed via his methods. All present samples were shown on an agarose gel predominantly as smears, vague bands or blank rows (Figures 13,14, 15). Additionally, low concentration of mRNA was assessed via nanodrop after the modification (Table 2). Due to time constraint, further transfection was carried out with the samples we had acquired.

Figure 15. 0.7% Agarose gel electrophoresis of denatured processed mRNA samples. (13/09/2024)

Indication of Rows: 1st-4th F8 mRNA, 5th – 8th GFP mRNA, 10th – 11th F8 mRNA (given by our instructor), 12th GFP mRNA (given by our instructor). A smear in 1st - 3rd and 9th row (F8), a band (GFP received form instructor) in 11th row. Other wells indicate little to no mRNA presence.

Table 2. Nanodrop analysis of modified F8 and GFP mRNA.

The samples were processed via addition of 5’cap and poly-A tail. The numbering of 1-4 indicates the sample number.

Analysis and conclusion

The acquired samples were further modified with a poly-A tail and 5’ cap to ensure the stability and translational efficiency in-vitro. The protocols used were suggested to be reliable by our instructor. But, as can be seen in Figures 14 and 15, vague band visibility was detected. Large blunders might have occured during the purification and extraction in-between the capping and tailing phases. As was noted in many experiments, Phenol:Chloroform:Iso-amyl alcohol extraction was not very reliable in the sense of acquiring a good yield of pure mRNA. There were instances when a pellet of mRNA was received after purification of 5' capped samples but none after proceeding with poly-A tailing. For future experiments, it would be advised to review the protocols for this exact purification and assess if another purification method could be more optimal. As explained previously, the modification of mRNA is crucial for further translational experiments.

Results - LNPs

Ionizable lipid synthesis Our team synthesized the ionizable lipids needed for lipid nanoparticle formation. We recreated the ionizable lipids produced by Kim et al [1], who first synthesized this lipid by an epoxide ring-opening reaction with amines. To confirm that the product was correct and pure, the lipids were analyzed with nuclear magnetic resonance (NMR) spectroscopy.

Figure 1. 1H-NMR of ionizable lipids in CDCl3 at RT.

Figure 2. Reference H-NMR spectra by Kim et al. [1].

In NMR, each nucleus of a specific type (for example H1 or C13) produces an electromagnetic signal when in the presence of an oscillating magnetic field. These signals can shift based on their chemical environment so that every nucleus in a unique environment produces a unique signal which allows chemists to elucidate a molecule's structure. In this case we are looking at a 1H-NMR which shows all the unique hydrogen nuclei. This lipid is a very complex molecule which makes it impossible to assign every single peak. However, when comparing with the spectrum from the publication by Kim (figure 2), the same peak groups can be observed. We see the same tall peak around 0.88 ppm, the high peak at 1.27 ppm and the low broad series of peaks at 1.44 and 2.5 ppm as well as the three small peaks at the end.

Synthesis results: Yield: crude yellow oil, 1.018 g (pure), 4.224 g (total - not pure), 0.00451 mol, 94.5%; 1H-NMR (300; CDCl3, RT), ppm: 0.9 (A, CH3, m), 1.3 (B, CH2, m), 2.15 (acetone), 2.5 (C, NC, m), 3.5 (D, COH, m), 5.25 (DCM), 7.3 (CDCl3); 13C-NMR (300; CDCl3, RT), ppm: 15 (A, CH3, s), 25-35 (B, CH2, many s), 30 (acetone), 50-60 (C, CN, many s), 60-70 (D, COH, many s), 78 (CDCl3); COSY-NMR (300; CDCl3, RT), ppm: 1 to 1.5, 2.5 to 3.5, 1.5 to 3.5; HSQC-NMR (300; CDCl3, RT), ppm: 3.75 (H) - 70 (C); 2.5 (H) - 60 (C); 1.5 (H) - 25~35 (C); 1(H) - 10 (C).

LNPs The ionizable lipids were used to form ionizable lipid nanoparticles. LNPs were analyzed for their size, zeta potential and encapsulation efficiency. Size and Z-potential can be acquired through dynamic light scattering (DLS), while the encapsulation efficiency is measured with a fluorescence assay. The LNPs described by Kim [1] had a size of 60-100nm. Our LNPs had a size of just over 100nm (figure 3a-c), which is a bit larger. This can be attributed to our mixing method, which was less precise than their microfluidics. Batch 2 (figure 3b) shows an increase in LNPs with a diameter of 10,000nm. This is the result of LNP aggregation, which happens if LNPs are not sonicated before analysis. The zeta potential described in the article by Kim was -0.997 mV. We measured a different, highly positive zeta potential. Zeta potential is a measure for the dispersion of the particles in a solution. a high (positive or negative) zeta potential means that the particles are dispersed in solution while a potential close to zero signifies that particles are likely to aggregate. A zeta potential that deviates from literature would mean that our LNPs have different interactions with each other which could also change how they interact with cells.

Figure 3. DLS analysis of the first 3 batches of trial LNPs containing torula yeast RNA. Size distribution of batch 1 (a) and batch 2 (b) in PEG2000 material mode; Dispersant PBS: Average; 3 single measurements. (c) Overview of average Z-potential PEG2000 material mode; Dispersant PBS. To address the unusual zeta-potential, the fourth batch of LNPs was made in 1x PBS buffer instead of 10X PBS. This time the LNPs were sonicated before analysis so there is no aggregation. The average zeta potential was measured at 7.156 mV in Liposome material mode; Dispersant PBS. It was concluded that a change in buffer does not impact the zeta potential. It was concluded that the difference in zeta potential from the source was due to the component substitutions we made.

figure 4. DLS analysis of batch 3 (LNP204) of trial LNPs containing torula yeast RNA with 1x PBS buffer. Size measurement in Liposome material mode; Dispersant PBS To test the transfection properties of our ionizable LNPs in vitro, LNPs were produced containing mRNA coding for GFP. DLS revealed that the size of the LNPs containing GFP mRNA was 1000nm, way higher than it should be (figure 5a). The negative control LNPs without RNA had a slightly larger size, again a result of aggregation since the sample had not been sonicated before analysis (figure 5b). Analysis with transmission electron microscopy (TEM) showed that the lipids formed clusters in the aqueous phase without incorporating any of the mRNA as the result of incorrect mixing (figure 5c). It is important to note that lipids should always be added to the aqueous phase during mixing to prevent this. It is also recommended to use other mixing methods like microfluidics that are better suited for smaller volumes.

Figure 5. Analysis of LNPs produced for transfection. (a) DLS size measurement of LNPs205 LNPs with GFP mRNA, Liposome material mode; Dispersant PBS. (b) DLS size measurement of LNPs205 LNPs with no mRNA, Liposome material mode; Dispersant PBS. (c) TEM pictures of LNPs205 LNPs with GFP mRNA show that no encapsulation occurred. (d) TEM pictures of LNPs205 LNPs with no mRNA show LNP formation. (e) DLS Z-potential measurement of LNPs205 LNPs with no mRNA and with GFP mRNA, Liposome material mode; Dispersant PBS. On 23/09 a second batch of LNPs was prepared for the purpose of transfection. LNPs were formulated with previously prepared mRNA: GFP1, GFP2, F8 1, 5xFV F8, GFP control. Control GFP samples will be borrowed from Martin Emmaneel. DLS size analysis showed that the prepared LNPs did not have the desired size, most had a size between 1 and 10 nm or a size between 100 and 1000 nm (figure 6).

figure 6. DLS size measurement, Liposome material mode; Dispersant PBS. (a) negative control LNPs, no mRNA. (b) positive control LNPs, control GFP mRNA. (c) GFP2 LNPs. (d) GFP1 LNPs. (e) 5xVF8-2 LNPs. (f) F8-1 LNPs. With TEM, some LNPs could be seen, although in extremely low abundance - in total 6-10 LNPs were observed in 3 samples.

figure 7. TEM pictures of F8 1 mRNA LNP With this, we conclude that the formulation method is not efficient. LNPs almost do not form with the ethanol injection method when using low lipid concentrations. It is recommended to use higher concentration of lipids and implement a pulsification method or microfluidic mixing. Encapsulation efficiency of the last batch of LNPs was measured by making a dilution series with known concentrations of the same RNA that is encapsulated in the LNPs. By lysing the LNPs and measuring the fluorescence with SYBR® Gold Nucleic Acid Gel Stain, the concentration of mRNA encapsulated in the LNP could be measured. The encapsulation efficiency measurement of the last batch was not effective since the LNPs did not form correctly.

Figure 8. Encapsulation efficiency. Standard curve of GFP mRNA In Figure 8, It can be seen that the intensity of mRNAs in the standard curve is not correlated to the decreasing concentration values of mRNA which proved to us the SYBR Gold dye in the end was not applicable for this analysis. It is important to note that the SYBR Gold dye is also usually used for analysis of DNA and not RNA [2], and was used in this case due to the lack of budget and materials.

M. Kim et al. ,Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver.Sci. Adv.7,eabf4398(2021).DOI:10.1126/sciadv.abf4398

Fisher, Thermo. “SYBRTM Gold Nucleic Acid Gel Stain (10,000X Concentrate in DMSO).” Thermofisher.com, 2022, www.thermofisher.com/order/catalog/product/S11494.

HEK293 cell transfection

Cell transfection into HEK293T/17 cell (Human Embryonic Kidney) culture with mRNA and lipid nanoparticles (LNPs) was carried out from 19/09 till 24/09. This is a reliable cell strain which is frequently used in therapeutic research. The results were assessed via fluorescence microscopy. The positive control exhibited fluorescence of GFP mRNA without presence of lipid nanoparticles (Figure 18). The synthesised eGFP mRNA was seen being expressed in the cells in low concentration. This batch of samples did not contain mRNA inserted in LNPs (Figure 19, Table 3). A new batch of HEK cells was transfected with our synthesised F8 and GFP mRNA in LNPs on 25/09. The fluorescence microscopy results indicated no evident fluorescence but the cells were seen to have survived after transfection with LNPs (Figures 16 and 17; Table 3).

Figure 1. Expression of positive control GFP mRNA via fluorescence microscopy in HEK cells.(24/09/2024)

Fluorescence detected after transfecting HEK293 cells with positive control GFP mRNA.

Figure 2. The expression of synthesised eGFP mRNA via fluorescence microscopy in HEK cells. (19/09/2024)

The cells expressing GFP are indicated by the red arrow. Translational efficiency is low.

Figure 3. Radboud iGEM teams’ mRNA samples and LNPs with F8 and eGFP mRNA (including control GFP). (24/09/2024)

No fluorescence detected in all HEK cells.

Figure 4. HEK cells without fluorescence. (24/09/2024)

Live cells after transfection of LNPs.

Overview of results from experiment (done on 24/09/2024)

No fluorescence observed in cell cultures transfected with F8, GFP mRNA and lipid nanoparticles. Cells with empty transfected LNPs show signs of survival (see Figure 18 and 19).

Analysis and conclusion

The transfection of the acquired GFP and F8 mRNA was preceded in HEK cells. The fluorescence of eGFP was observed to not be optimal indicating low viable concentration of the mRNA. Moreover, the cells transfected with F8 and eGFP mRNA with LNP included did not show any visible fluorescence. The latter could have resulted from low concentration of lipid nanoparticles produced prior to transfection. The cells that got transfected with these LNPs did show signs of survival which proved the safety and non-toxicity of the produced nanoparticles. It has to be taken into account that the cell line used was not the originally planned HELA cell strain. This was due to cost restrictions and availability of usable HELA cells.

Future plans

For now, if we would consider continuing our project, we would implement changes in LNP production to acquire better yield and functionality of the vesicles and change the structure of mRNA production. We observed that methods which were normally used for experiments such as amplification did not work well. We would delve deeper in research about our protein and its characteristics to find the optimal way of its production. Also, in the final step we would like to implement the nasal spray delivery system of our mRNA-LNP product. This would be the groundbreaking stage of our research for a safer and cheaper alternative of Hemophilia A therapy.

Project achievements

All experiments were listed with 3 letter abbreviation RAD (Radboud University) with the number of the experiment corresponding to the project part and the execution order.

For example, RAD302 = second experiment done in the third part of our project, specifically, HEK cell transfection.

A list of successful results during our project from our Radboud University Team Lab Notebook :

• RAD105
• RAD108
• RAD107
• RAD109
• RAD110
• RAD111
• RAD113
• RAD114
• RAD115
• RAD120
• RAD124
• RAD127
• RAD128
• RAD129
• RAD130
• RAD201
• RAD204
• RAD205
• RAD206
• RAD301
• RAD302

A list of unsuccessful results during our project from our Radboud University Team Lab Notebook :

• RAD101
• RAD102
• RAD103
• RAD104
• RAD106
• RAD112
• RAD116
• RAD117
• RAD118
• RAD119
• RAD121
• RAD122
• RAD123
• RAD125
• RAD126
• RAD131
• RAD132
• RAD133
• RAD202
• RAD203
• RAD302

Project achievements

1. Biochemical and functional characterization of a recombinant monomeric Factor VIII-Fc fusion protein. Peters RT, Toby G, Lu Q, Liu T, Kulman JD, Low SC, Bitonti AJ, Pierce GF. J Thromb Haemost. 2012 Dec 4. doi: 10.1111/jth.12076. 10.1111/jth.12076 PubMed 23205847
2. Passmore, L. A., & Coller, J. (2021). Roles of mRNA poly(A) tails in regulation of eukaryotic gene expression. Nature Reviews. Molecular Cell Biology, 23(2), 93–106. https://doi.org/10.1038/s41580-021-00417-y
3. Passmore, L. A., & Coller, J. (2021). Roles of mRNA poly(A) tails in regulation of eukaryotic gene expression. Nature Reviews. Molecular Cell Biology, 23(2), 93–106. https://doi.org/10.1038/s41580-021-00417-y