Proof of concept

We developed two different proof of concept strategies: To prove that our liposomal formulation is able to deliver siRNAs into cells in vitro we encapsulated a published siRNA against eGFP and treated eGFP-expressing HeLa-cells. Knock-down efficiency was evaluated by measuring the eGFP-expression as fluorescence values over time. For comparison we transfected the same siRNA with a commercially available transfection reagent. We treated HeLa cells expressing a gene fragment of the UL19 gene proving that our procaryotic siRNA production method yields functional siRNAs against HSV1 UL19 . Evaluation was conducted with RT-qPCR, RNAseq and Western Blot.

Analysis of eGFP knockdown

In the same manner as the eGFP expression pattern we evaluated the knockdown capabilities of our liposomal formulation (vehicle). As comparison served delivery of the anti eGFP siRNA in a commercially available transfection reagent. 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 of eGFP fluorescence.

**Figure 1: Visualization of GFP knockdown efficiency.** Green line indicates the eGFP control, which received no treatment,blue line: cells treated with the empty vehicle, red line: cells treated with the anti eGFP- siRNA encapsulated in our vehicle, black line: positive control, therefore anti eGFP siRNA in transfection reagent. Whilst green and blue increase over time, red reaches a peak and maintains this level, black line shows a deep decrease of eGFP fluorescence.

RT-qPCR

We conducted RT-qPCR on our UL19 siRNA treated samples in duplicates each. Obtained mean Cq values for UL19 transcript and GAPDH housekeeping gene transcript, ΔCq, ΔΔCq and the 2-Fold are listed in Table 2. 2-Fold expression is also 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.

Table 1: Calculation of ΔΔC_q

Sample	C_q (UL19)	C_q(GAPDH)	ΔC_q	ΔΔC_q	2x Expression
UL19 baseline	12.95	14.49	-1.54	0.00	1
UL19 baseline + pro-siRNA PEI	11.98	12.59	-0.61	0.93	0.5248583418
UL19 baseline + pro-siRNA vehicle	10.60	12.27	-1.68	-0.14	1.101905116
eGFP baseline + dsRNA PEI	10.51	12.37	-1.86	-0.32	1.248330549

**Figure 2: 2-Fold expression.** Values were calculated with the obtained ΔΔCq Values for each condition as stated in **Table 1**. 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.

30 µg of each sample were loaded on a 12% SDS PAGE gel (see: Experiments). Obtained Western Blots are shown in Figure 3. Figure 3A shows 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 3B 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).

**Figure 3: Western Blot of treated cells with different conditions.** A: eGFP Western Blot where multiple stained bands for eGFP are visible, which indicates oligomerization around 32kDa. Housekeeping gene and control alpha tubulin are also visible as a stained band above 48kDa. Proposed size of eGFP is 32kDa and alpha Tubulin 55kDa. Obtained results are therefore congruent with theoretical values. No significant change in eGFP can be determined with bare eyes. B: 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 His-Tag antibody around 38kDa is missing (actual size 36 kDa).

eGFP expression was assesed as described in our methods. We were able to proof that our liposomal formulation is capable of siRNA-delivery into HeLa-cells. Therefore, decreasing eGFP expression in comparison to an untreated control. This system was then tested with our pro-siRNA which were delivered as either encapsulated in our liposomal formulation or in a conventional transfection reagent. We showed by RT-qPCR that our pro-siRNA are capable of decreasing the RNA-expression of our target gene by 50 percent. The significance of the obtained results are yet to be debated because RT-qPCR and Westernblot delivered contradictionary results. RT-qPCR showed a decrease in RNA expression level in comparison to the untreated control Fig Liza. But in Westernblot no recombinant protein was detectable for the treated groups as well as for the control group. Furthermore, the low amount of samples in the RT-qPCR lower the weight of the obtained results. It is to note that we obtained high RIN-values in ou RNA-isolation. Therefore, we are confident that our Bulk-RNA sequencing will back up the results of RT-qPCR. Because Westernblot and staining with His-taged monoclonal antibody on a whole cell lysate of eucaryotic cells is not optimal and does not allow a good qualitative of quantitative evaluation. If the experiment is to be repeated the His-tag should be exchanged to a FLAG-tag.