<p>To further enhance the stability of the LNPs for inhalation, we incorporated <strong>chitosan-RNA complexes</strong>, which provide thermal stability and protect RNA from degradation by RNases. Integration of these complexes into the SORT LNP resulted in a lung-specific delivery platform with superior stability. Using this system, we achieved highly efficient transfection of a bronchial cell line from a cystic fibrosis patient (CFBE41o- with F508del mutation), demonstrating the potential of this approach for targeted gene delivery to lung epithelial cells. These results highlight the remarkable efficiency, stability and specificity of our optimized SORT LNP formulation, positioning it as a promising platform for lung-specific genetic therapies. </p>
<p>To further enhance the stability of the LNPs for inhalation, we incorporated <strong>chitosan-RNA complexes</strong>, which provide thermal stability and protect RNA from degradation by RNases. Integration of these complexes into the SORT LNP resulted in a lung-specific delivery platform with superior stability. Using this system, we achieved highly efficient transfection of a bronchial cell line from a cystic fibrosis patient (CFBE41o- with F508del mutation), demonstrating the potential of this approach for targeted gene delivery to lung epithelial cells. These results highlight the remarkable efficiency, stability and specificity of our optimized SORT LNP formulation, positioning it as a promising platform for lung-specific genetic therapies. </p>
<Subesctiontitle="Proof of Concept"id="Experimental Design1">
<H4text="Proof-of-concept"/>
<H5text="Workflow"/>
<H5text="Workflow"/>
<p>The prepared pDAS12124-preedited plasmid serves as a positive control to validate the success of the experiment. A technical control with the pZMB938 plasmid confirms successful transfection of the cells. In the main part of the experiment, pDAS12489-2in1 and pCMV-PE2 are co-transfected. Successful transfection is visualised by GFP signals.</p>
<p>The prepared pDAS12124-preedited plasmid serves as a positive control to validate the success of the experiment. A technical control with the pZMB938 plasmid confirms successful transfection of the cells. In the main part of the experiment, pDAS12489-2in1 and pCMV-PE2 are co-transfected. Successful transfection is visualised by GFP signals.</p>
<p>The results demonstrate that chitosan-RNA complexes, when packaged with SORT LNPs, can efficiently deliver mRNA into CFBE41o- cells and facilitate the expression of a YFP reporter gene. Furthermore, the absence of fluorescence in the control samples validates the specificity of the system, ensuring that the fluorescence signal is solely due to the delivered mRNA and not from other components of the system. Additional testing, such as flow cytometry analysis, is planned to provide a more quantitative assessment of transfection efficiency. This would allow us to accurately determine whether there are subtle differences between the two concentrations that were not detectable by microscopy alone.</p>
<p>The results demonstrate that chitosan-RNA complexes, when packaged with SORT LNPs, can efficiently deliver mRNA into CFBE41o- cells and facilitate the expression of a YFP reporter gene. Furthermore, the absence of fluorescence in the control samples validates the specificity of the system, ensuring that the fluorescence signal is solely due to the delivered mRNA and not from other components of the system. Additional testing, such as flow cytometry analysis, is planned to provide a more quantitative assessment of transfection efficiency. This would allow us to accurately determine whether there are subtle differences between the two concentrations that were not detectable by microscopy alone.</p>
<p>Overall, this experiment highlights the potential of chitosan-RNA complexes in combination with SORT LNPs as a promising platform for mRNA delivery and gene expression in airway epithelial cells. Further investigation could focus on optimizing the mRNA dose to maximize expression levels and transfection efficiency.</p>
<p>Overall, this experiment highlights the potential of chitosan-RNA complexes in combination with SORT LNPs as a promising platform for mRNA delivery and gene expression in airway epithelial cells. Further investigation could focus on optimizing the mRNA dose to maximize expression levels and transfection efficiency.</p>
<p>To validate our gene editing approach by prime editing of CFTR F508del delivered to lung cells via SORT LNPs, we planned to use <aonClick={()=>goToPageAndScroll ('Patch Clamp','/materials-methods')}>Patch Clamp</a> as a downstream method. Our goal was to detect the restored conductance of the repaired CFTR by this electrophysiological method. This was made possible through the assistance of the <aonClick={()=>goToPagesAndOpenTab('patchclamp','/human-practices')}>Cellular Neurophysiology research group</a> at our university.</p>
<p>To validate our gene editing approach by prime editing of CFTR F508del delivered to lung cells via SORT LNPs, we planned to use <aonClick={()=>goToPageAndScroll ('Patch Clamp','/materials-methods')}>Patch Clamp</a> as a downstream method. Our goal was to detect the restored conductance of the repaired CFTR by this electrophysiological method. This was made possible through the assistance of the <aonClick={()=>goToPagesAndOpenTab('patchclamp','/human-practices')}>Cellular Neurophysiology research group</a> at our university.</p>
