<p>Fluorescence microscopy with the Leica DMI6000 B microscope at 20x magnification was by performed us on HEK293 cells transfected with LNPs containing pcDNA 3.1 eYFP DNA and mRNA. Minicircle DNA served as the positive control, while LNPs without cargo acted as the negative control. Cells were imaged at 24h, 48h, and 72h post-transfection.</p>
<p>Fluorescence microscopy with the Leica DMI6000 B microscope at 20x magnification was performed by us on HEK293 cells transfected with LNPs containing pcDNA 3.1 eYFP DNA and mRNA. Minicircle DNA served as the positive control, while LNPs without cargo acted as the negative control. Cells were imaged at 24h, 48h, and 72h post-transfection.</p>
<p>Retrospectively, after 72h none of the LNP-treated samples showed significant fluorescence, indicating a failure in transfection (Figure 21). The lack of fluorescence in all experimental groups, except lipofectamine and Minicircle DNA, suggests either insufficient uptake of the LNPs by the cells or a failure in expression of the YFP reporter, indicating that the Corden LNP may not suited as our delivery system. Also the deformed morphology and decreased growth are indicators for negative effects of the Cayman LNP on HEK293, probably reasoned in the employment of more cytotoxic mPEG-DSPE compared to DMG-PEG in the Cayman and SORT LNP.</p>
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<p>Test__In light of these results, we improved our experimental setup and performed additional validation experiments. Unfortunately, the repeated measurements yielded similar outcomes, confirming the absence of a significant difference between the two CFTR-expressing cell lines (Figure 38). This finding led us to consult with the research group at <aonClick={()=>goToPagesAndOpenTab('mattijsvisit','/human-practices')}>KU Leuven</a>, who established these cells lines. Although they had not conducted similar Patch Camp measurements, they suggested an alternative approach using Ussing Chamber measurements. This technique, unlike Patch Camp, does not rely on single-cell measurements but rather examines the ion currents across the entire cell monolayer, which may provide a more comprehensive view of CFTR functionality.</p>
<p>Fluorescence microscopy was performed using the Leica DMI6000 B microscope at 20x magnification to analyze CFBE4o- cells. These cells were initially pre-transfected with pPEAR_CFTR to establish a baseline for Prime Editing activity. Subsequently, transfection ofPE6cpegRNA4, delivered via the AirBuddy system, was carried out. LNPs without cargo acted as the negative control. Imaging was conducted at 24h, 48h, and 72h post-transfection to assess fluorescence intensity and evaluate editing efficiency over time.</p>
description="Microscopy of CFBE4o- 48h aftr transfection. Cells were previoulsy transfectet with pPEAR_CFTR and later tresnfected with PE6c+pegRNA4 via AirBuddy. BF = Brightfield "
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<p>The results show a sucsessful transfection of PE6c and pegRNA4 encapsulated in our LNPs was achieved following prior transfection with pPEAR_CFTR. The experimental setup demonstrated a significant increase in fluorescence intensity, indicative of enhanced Prime Editing efficiency under these conditions.
Moreover, the PreCyse results confirm the effectiveness of our strategy, showcasing the synergy between our proprietary PrimeGuide system and the AirBuddy platform. This combination has proven to be pivotal in optimizing the editing outcomes and advancing the precision of the Prime Editing process.
These findings underscore the robustness of our delivery approach and the innovative integration of the PrimeGuide and AirBuddy systems in driving Prime Editing to new levels of efficiency and accuracy.</p>
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<p>Following the recommendations from KU Leuven, we have also taken steps to expand our experimental approach. To further investigate the CFTR functionality, we have ordered <aonClick={()=>goToPageAndScroll ('Cell Culture','/materials-methods')}>CFBE41o-</a> as a new cell line from <aonClick={()=>goToPagesAndOpenTab('ignatova','/human-practices')}>Prof. Dr. Ignatova</a> in Hamburg. Our goal is to use these patient-derived cells to measure ion currents and further elucidate the impact of the mutation on chloride conductance. This will not only provide a more clinically relevant model but may also yield more distinct results in comparison to the previous experiments with the engineered HEK293T cells.</p>
