<Subesctiontitle="Patch Clamp: A Key Tool in Electrophysiology"id="Patch Clamp1">
<p>The patch clamp technique is a highly sensitive method for measuring ionic currents through individual ion channels in cells, making it a cornerstone of electrophysiological research. Initially developed by Erwin Neher and Bert Sakmann in the 1970s [1], this technique has evolved into various configurations, including the Whole-Cell and Single-Channel recordings [2], which provide critical insights into the functional properties of ion channels. </p>
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<Subesctiontitle="Principles of the patch clamp technique"id="Patch Clamp2">
<Subesctiontitle="Principles of the Patch Clamp Technique"id="Patch Clamp2">
<p>Patch clamp recording involves the use of a glass micropipette which is manufactured from a glass capillary through the use of a Micropipette Puller. The micropipette is then filled with an electrolyte solution, which is subsequently brought into contact with the cell membrane. By applying gentle suction, a high-resistance seal called giga seal is formed between the pipette tip and the membrane patch. This enables the measurement of ionic currents with minimal noise interference [3]. <strong>Whole-Cell Configuration</strong> records currents from the entire cell by rupturing the membrane patch, accessing the intracellular environment, and is useful for analysing overall ion channel activity and cellular responses. <strong>Single-Channel Recording</strong> measures currents through individual ion channels without rupturing the membrane, enabling high-resolution study of channel conductance, gating, and selectivity [2].</p>
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<p>The success of patch clamp experiments heavily depends on the composition of the solutions used. Typically, two main types of solutions are employed: The <strong>Pipette Solution</strong> in the micropipette mimics the intracellular environments, while the <strong>Bath Solution</strong> surrounds the cell and usually contains components that replicate the extracellular environment. Both solutions are meticulously designed to reflect the physiological conditions under which the cells operate, thereby ensuring that the measurements accurately reflect ion channel activity in a natural setting [2].</p>
<Subesctiontitle="Application in CFTR gene prime editing validation"id="Patch Clamp3">
<Subesctiontitle="Application in CFTR gene Prime Editing validation"id="Patch Clamp3">
<p>In our ongoing research project focusing on the treatment of Cystic Fibrosis, our patch clamp measurements, performed in collaboration with Dr. Oliver Dräger from the Cellular Neurophysiology working group at Bielefeld University, serve as a powerful validation tool for the assessment of the functional correction of the CFTR gene, particularly the common F508del mutation, via prime editing. The patch clamp technique can be employed in this context to measure the resulting chloride ion channel activity which is altered by the mutation [4]. Whole-Cell recordings were performed to assess whether the corrected CFTR channels function similarly to those in healthy cells. If the chloride ion currents in the edited cells approach levels of healthy cells, this would strongly suggest successful gene editing and validate the functionality of our therapeutic approach.</p>
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<p>For testing our prime editing approach, we needed an easy-to-handle cell line with a measurable high expression of CFTR and the CFTR F508del mutation. When talking to Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven, he recommended to use HEK293T cell lines overexpressing CFTR they had used. HEK293 cells are a very common immortalized human cell line derived from the kidneys of a female embryo. They are particularly suited to research due to their convenient handling and transfection properties. Basic HEK293 cells were provided to us by the Cellular and Molecular Biotechnology working group at Bielefeld University led by Prof. Dr. Kristian Müller, who is also one of the Principal Investigators of our team. HEK293T cells express an additional tsA1609 allele of the SV40 large T-antigen, allowing for replication of vectors containing the SV40 origin of replication[5]. Besides the native CFTR gene, which is not expressed in HEK cells, the HEK293T cell lines used in Leuven carry another copy of the gene embedded in an expression cassette. The cassette includes a CMV promoter, which is a standard promoter used for gene overexpression in human cells derived from the human Cytomegalovirus[6], as well as a puromycin resistance co-expressed with the CFTR allowing for continuous selection of CFTR expressing cells. The whole construct was stably inserted into the genome using lentiviral transduction[7][8]. </p>
alt1="Phase contrast image of HEK293T at 20x magnification"
description="Phase contrast image of HEK293T at 20x magnification"
alt1="Phase contrast image of HEK293T at 20x magnification."
description="Phase contrast image of HEK293T at 20x magnification."
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<b>Figure 4:</b> ALI cultures of hNECs: The active cilia beat frequency of differentiated human nasal epithelial cells (hNECs) in air-liquid interface (ALI) culture is visible. This ciliary movement is crucial for mucociliary transport, which contributes to the clearance of particles and pathogens in the respiratory tract.
<b>Figure 4.</b> ALI cultures of hNECs: The active cilia beat frequency of differentiated human nasal epithelial cells (hNECs) in air-liquid interface (ALI) culture is visible. This ciliary movement is crucial for mucociliary transport, which contributes to the clearance of particles and pathogens in the respiratory tract.
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<b>Figure 5:</b> Apical-Out Airway Organoid (AOAO) culture: Visible apical-out airway organoids in action. These 3D structures, which mimic the airway epithelium, allow detailed study of cellular processes such as mucociliary transport and secretory activities, in which cilia and vesicles play a key role..
<b>Figure 5.</b> Apical-Out Airway Organoid (AOAO) culture: Visible apical-out airway organoids in action. These 3D structures, which mimic the airway epithelium, allow detailed study of cellular processes such as mucociliary transport and secretory activities, in which cilia and vesicles play a key role.
description="MTT Assay: Formation of purple formazan crystals by living cells"
description="MTT Assay: Formation of purple formazan crystals by living cells."
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<H4text="Proliferation Assay to Monitor Long-Term Safety"></H4>
<p>In addition to assessing immediate cytotoxicity, we also evaluated the long-term safety of the LNPs by conducting a proliferation assay. This assay tracked cell division and growth over time to determine whether the LNPs impacted cellular function. Our results showed that LNP-treated cells had similar growth rates to untreated controls, indicating that the LNPs do not interfere with normal cell processes. This further confirms their biocompatibility and suitability for use in biological systems.</p>
<p>To assess the transfection efficiency of our LNPs, we used flow cytometry. This method involved tagging the LNPs with fluorescent markers and measuring their ability to deliver genetic material into target cells. The flow cytometry results provided quantitative insights into how effectively the LNPs transfected cells, helping us optimize their design for gene therapy applications. </p>
