Results Cell Culture Assays

Analysis of eGFP expression pattern

To assess the expression capabilities of our promoter, we monitored eGFP expression over the course of three days as described in the methods part. Figure 1 visualizes the eGFP expression. Logistic course of eGFP expression can be observed with already continuous eGFP expression before the cells were placed in the IncuCyte. Red bars indicate the value and time stamp of maximum eGFP expression. Blue line indicates the half maximum time stamp of eGFP expression.

Visual representation of the progression of eGFP expression can be seen in Figure 2. During expression measurement pictures were taken at 10h, 24h, 34h, 48h and 58h post transfection. An increase in fluorescence can be observed.

Analysis of eGFP knockdown

In the following experiment, we wanted to determine the knockdown capabilities of our liposomal formulation (vehicle). We achieved this by using the same method as for the eGFP expression pattern. The delivery of the anti eGFP-siRNA in a commercially available transfection reagent served as a comparison. Figure 3 visualizes the eGFP expression after treatment over time. Green line indicates the eGFP control, which received no treatment. The blue line indicates cells treated with the empty vehicle and the red line shows cells treated with the anti eGFP-siRNA encapsulated in our vehicle. The black line is the positive control, therefore anti eGFP-siRNA in transfection reagent. Whilst green and blue increase over time, red reaches a high and maintains this level, black line shows a deep decrease in eGFP fluorescence.

Analysis of knockdown assay results

In our cell culture assay we first tried to knockdown eGFP expression as well as UL19 fragment expression in HeLa-cells by delivering siRNA against these two genes either encapsulated in liposomes or with a commercially available transfection reagent in order to evaluate the knockdown effectiveness. Afterwards, we assessed the success of our knockdown via RT-qPCR, Western Blot and bulkRNA sequencing.

RNA isolation and quality of isolated RNAs

After transfection with the plasmids either encoding for EGFP or UL19 fragment and treatment with different siRNA formulations, we wanted to determine RNA expression levels. For this reason RNA was isolated and the quality of the RNA had to be assessed.

Quality of RNA

We obtained values between 0.033 and 1.929 for the A260/230 ratio. The Quotient of 260/230 is an indicator of how free the sample is of salts and organic compounds such as isopropanol, ethanol and guanidine thiocyanate. These values should be greater 2. Therefore our isolated samples show a high level of salt contaminants. For the A260/280 ratio, an indicator for contaminations with proteins, we obtained values between 0.879 and 2.1 with the majority of values ranging between 1.8 and 2.1. Ideally obtained values range from 1.8 to 2.2 so the majority of our samples are free of protein contaminants. Sample 2A has high protein and salt impurities.

We further analysed the integrity of our samples used for RNA sequencing by determining the RNA integrity number with an Bioanalyzer. This procedure was carried out before RNAseq and obtained RIN values and chromatograms of 18S 28S rRNA are shown in Figure 4. RIN Numbers between 10 and 7 indicate low RNA degradation, values between 7 and 5 indicate moderate RNA degradation. Values between 5 and 0 high degradation.

All samples except A2 (HeLa + eGFP 3, RIN = 2.5) and E3 (HeLa + pRK5 + pro-siRNA PEI 1, RIN = NA) show RIN values above 7. The low RIN number for A2 is congruent with the low values obtained for 260/230 and 260/280 ratio for this sample.

Since bulkRNA Sequencing is time intensive we expect to present our results at the Grand Jamboree in Paris.

qPCR Primer evaluation

Our next goal was to find the best performing primer for our further experiments, to ensure an efficient workflow. Three different Primer sets were evaluated concerning their capabilities to quantify eGFP or UL19 enscripts. For this purpose Primers were first evaluated in a temperature gradient PCR since the used kit needs two different annealing temperatures, one for reverse transcription and one for amplification for the target gene by conventional PCR. The used gradient ranged from 53.8 °C to 64 °C. Results are visualized in Figure 5.

Furthermore, all three primers were evaluated for each target in a plasmid titration assay. Aim of the assay is to determine cut-of values for kit and primer per combination and determine primer efficiency. Titration curves for each primer set and target are shown in Figure 6. Figures 6G and 6H show the calculated primer efficiency based on the obtained slope of the titration regression model.

RT-qPCR

We conducted RT-qPCR on our UL19 siRNA treated samples in duplicates each to measure RNA expression and therefore investigate knockdown 2 -Fold expression is visualized in Figure 7. For treatment with dsRNA and siRNA encapsulated in our vehicle, no change in expression can be detected. For treatment with siRNA delivered by a commercial transfection reagent, a reduction of nearly 50% is seen.

Figures 7 displays the normalized intensity values per sample measured. Here, the UL19 baseline sample with dsRNA PEI clearly shows the highest normalized intensity value, followed by the pro-siRNA vehicle sample and the baseline UL19. The lowest intensity value was measured for the UL19 baseline with pro-siRNA PEI.

Western Blot analysis

To calculate the total protein amount per combined treatment condition, a Bradford assay was conducted as described earlier. Bradford standard curve is visualized in Figure 8.

30 µg of each sample were loaded on a 12% SDS PAGE gel (see: methods). Obtained Western Blots are shown in Figure 9. Figure 9A shows an eGFP Western Blot where multiple stained bands for eGFP are visible, which indicates oligomerization around 32kDa. Housekeeping gene and control tubulin are also visible as a stained band above 48kDa. Proposed size of eGFP is 32kDa and alpha Tubulin is 55kDa. Obtained results are therefore congruent with theoretical values. No significant change in eGFP can be determined with bare eye. Figure 9B shows the western Blot of UL19 samples for the sample treated with dsRNA. No tubulin band can be observed. In all samples the expected protein, which should be marked with the Histag antibody around 38kDa is missing (actual size 36kDa).

Discussion

To find an appropriate time for treating the cells with our siRNA-liposome formulation, we first analyzed the expression pattern of GFP in the cells. The aim was to determine the time point before eGFP expression reaches 50%, since the corresponding mRNA is already present at this point. A treatment with our formulation at this time point would therefore lead to an effective mRNA degradation, whereby the affected protein can no longer be synthesized. The reason why mRNA levels are that high after a short time frame after transfection is found in the continuous CMV promoter driven transcription of said mRNA, which is further enhanced by the CMV enhancer element present on the eGFP plasmid (Norman et al., 2010). These findings are transferable onto the pRK5 UL19 expressing plasmid since both plasmids carry identical promoter, enhancer, terminator and kozak sequences. A disadvantage of the chosen plasmid system is that without selection pressure cells will lose their plasmid upon division because of HeLa cells lacking large T-antigen of SV40 Virus for replication of the plasmid. The transfection is therefore only transient. This has to be considered in the evaluation of all time dependent results (Condreay et al., 1999).

Given the large difference in fluorescence upon measuring eGFP expression as shown in Figure 3 on the Western Blot, nearly no eGFP was expected to be detected after immunostaining for the sample treated with siRNA delivered by the transfection agent. For our liposomal formulation, a slightly lower intensity of the stained band was expected. For the treatment with the empty vehicle, no difference was expected. Contrary to these expectations, the Western Blot shows multiple clear bands around the expected size of eGFP around 32kDa. This observation can be explained by eGFP oligomerization and thus suggests that the protein denaturation process has not worked properly (Costantini et al., 2012). Since alpha tubulin is visible in all discussed samples, a full transfer and functionality of the used staining protocol can be anticipated. This issue of contradictory results will be resolved upon analysis of the yet to receive bulkRNA Seq data.

From the decrease in fluorescence that can be observed in our eGFP measurement experiment we conclude that the siRNA delivery with our vehicle worked.

The evaluation of our qPCR primers shows that three primer sets for both targets are functional at the proposed temperature (55°C) for reverse transcription, as well as for amplification by PCR at 60°C. Titration curves for eGFP primer set 1 and 2 show overfitting for the target due to their R² of 0.9994 and 0.999 for primer set 1 and 2 respectively. Primer set 3 shows a R² of 0.9988 and therefore no over- or underfitting. In case of primer efficiency, primer setb1 shows an efficiency of 96.18%, primer set 2 of 96.86% and primer set 3 of 80.84%. Therefore, considering the overfitting and calculated primer efficiency, we carried out the RT-qPCR or qPCR for eGFP transcripts derived from pEGFP C1 plasmid with primer set 2 (highest efficiency and a smaller overfitting than primer set 1).

For the UL19, primer set 1 shows a R² of 0.9956 and an efficiency of 104.91%. Primer set 2 shows a R² of 0.9995 and an efficiency of 99.74%. Primer set 3 a R² 0.9998 and efficiency of 100.20%.
Primer set 2 and 3 show overfitting and high efficiency, while primer set 1 shows no overfitting at very high efficiency. Following the same principle, we identified that the RT-qPCR or qPCR for UL19 transcripts derived from pRK5-HSV UL19 plasmid should be carried out with primer set 1.

To assure our generated data sets used for analysis of gene expression of UL19 have a high quality and therefore data integrity, we anticipated recommendations made by Fleigel & Pfaffl regarding RNA quality for RT-qPCR: free of impurities of proteins, salts and a high RNA integrity (Fleigel & Pfaffl, 2006). The high RIN values (>7) observed suggest good quality of the purified RNA. The obtained results by RNA sequencing and qRT-PCR can therefore be regarded as valid from the point of input material quality.

RT-qPCR was carried out for UL19 transcripts with the aforementioned recommended primer set. Due to non comparability to the C_q-Threshold cycles obtained for different amounts of target gene copies by qPCR and the C_q-Threshold cycles obtained by RT-qPCR only a relative comparison of the expression levels with GAPDH as housekeeping gene reference with the ΔΔC_q method is possible (Soga et al., 2022). ΔΔC_q calculation for all treated samples revealed a 50 % expression loss in samples treated with pro-siRNA mixture delivered by transfection reagent. For dsRNA and pro-siRNAs encapsulated in liposomes no decrease could be detected. However, more replicates of this experiment need to be conducted in order to ensure statistical significance. Possible reasons for missing knockdown with dsRNA could be fast degradation of dsRNA by cellular RNases originating from the pattern-recognition-receptor mediated inflammatory response (Gradzka-Boberda et al., 2022).

Another important key factor for efficiency of liposomal formulations is nanoparticle size. Thus, most efficient cellular uptake can be seen for small liposomes around 50 nm diameter (Frey, F., Ziebert, F., & Schwarz, U. S., 2019). The liposomes that we used had a diameter of ~600 nm, and were therefore too large to mediate efficient siRNA delivery. The large size of the liposomes could be explained by the incorporation of a 50 kDa large recombinant RNase inhibitor which was used to protect the pro-siRNAs from degradation (Feng et al., 2013).

The described RT-qPCR results are contradicted by the findings obtained by qualitative Western Blot analysis. On Western blot no recombinant UL19 fragment could be detected for either control group nor treated group. Moreover, for the group treated with dsRNA complete abundance of alpha tubulin on plot was also detected. The missing protein signal at 38 kDa can be explained by Anti-His mAb depletion through unselective binding to proteins which carry similar structural elements such as His-Shuttles. For further evaluation and repetition of the experiment the used marker tag should be changed to a more convenient and larger one such as FLAG or Haemagglutinin. Additionally, we did not yet check for efficiency of the inserted Kozak sequence and only anticipated results obtained by eGFP expression measurement as comparable due to their promoter, terminator similarity. It is to conclude that the Western Blot is not suitable to serve as a reference for qualitative assessment of gene knockdown due to the missing protein signal in the control group at 38kDa and the mentioned disadvantages of the His-tag detection. Therefore, results obtained by RT-qPCR are more valid and have to be regarded as more precise.

Despite these contradictory results we are confident that Bulk-RNA seq. will verify results obtained by RT-qPCR. Our chosen model for proof-of-concept has yet to be refined and validated with more samples. Nevertheless, these findings suggest our novel approach for targeting viral diseases holds some potential. In several different experimental setups we validated the possibilities of our chosen concept and have proven that the chosen vehicle formulation is capable of delivering siRNAs in-vitro as well as that pro-siRNAs are able to mediate gene knockdown of a recombinant protein in-vitro.

Further experiments could deliver well needed insights, especially regarding the connection between knockdown efficiency and liposome size. Here the next step would be to establish a production protocol for liposomes or LNPs (lipid nanoparticles) of 100 nm in diameter and below, for example using the microfluidic devices designed by us. Testing different production techniques and compositions of the vehicles could unveil the true potential of our siRNA knockdown method.

Final Remarks

Viral encephalitis, especially herpes simplex encephalitis, is the cause for numerous deaths all over the world. The available treatments have been plagued by problems of severe side effects and lacking specificity. This is understandable keeping in mind that the therapeutics were restrained by the blood brain barrier. Therefore, the most successful therapeutic agent to date, acyclovir, is stained with being nephrotoxic and additionally counting many existing resistant strains already. Furthermore, the dosage scheme for acyclovir demands administration up to five times a day.
As the mechanism of acyclovir is not virus specific, the viral genome and exome displays an attractive target. Likewise, changing the focus to facing delivery routes off the blood to brain barrier seem promising. We combined those approaches by implementing herpes simplex specific siRNA in liposomes and using the nose-to-brain route as an innovative way to reach the center of infection. Moreover, our concept can be applied to several different viral encephalitides depicting a safe and effective solution to the present problem.
In addition, the theoretical construction of a microfluidic device contains great potential for upcoming biotechnological engineers. In this context, the reduction of resources needed for the testing of siRNA loaded liposomes might prove possible. Keeping our interviews with experts in industry as well as in academia in mind, this would illustrate an enormous improvement in drug delivery.
In total, the main focus should be to improve the condition of the people suffering from viral encephalitis. Through our novel application approach, we aim to increase the drug density reaching the infected brain area thereby reducing the application frequency and alleviate the treatment of patients.
Since many evaluations and testing remain open, we long for future scientists to hold on to this new track. The only way to achieve the success of this concept is to continue developing, producing, and improving the affinity of virus specific siRNA, the delivery routes and liposomes as well as the microfluidics device. Further steps would also include animal testing of the nose-to-brain delivery.

The iGEM team Heidelberg 2022 is thankful for all advice, information and resources we received during our journey and appeals to the next iGEM generations to pursue unusual yet fascinating routes as we did through the nose-to-brain.