<p>For the prime editing of cystic fibrosis (CF), we on the one hand optimized a prime editing complex and on the other hand developed an efficient delivery system. For testing, we set up cell culture with model cell lines as well as primary cells taken from team members and a patient.
For editing, we first compared different existing prime editors (pCMV-PE2, pLV-PE_CO-Mini, pCMV-PE6c) and constructed a reporter plasmid simulating the CFTR context. In addition and to further enhance the editing process, we designed various pegRNAs tailored to our construct incorporating features such as silent edits, for a lower mismatch repair, and a 3′ stabilizing stem loop (tevropQ1). The aim was to identify the most effective pegRNA for our specific target, which is why pegRNA especially for CFTR F508del mutation were designed.
As proof of concept, we transfected these constructs in HEK-293 and CFTR mutated CFB41o- cells and observed significant prime editing of our reporter via fluorescence microscopy. We identified the PE6c editor and our pegRNA variant 4 as optimal. This resulted in our Best New Basic Part, PEAR_CFTR. Furthermore, we extended our approach to primary human nasal epithelial cells generated from our own nasal epithelial cells through nasal swabs. By cultivating them in Air Liquid Culture (ALI) and Apical-Our Organoids, we successfully tested our technologies in vitro, mimicking the in vivo situation.
Furthermore, we successfully designed and cloned novel nickases of Fanzor, which is special because of its smaller size and eukaryotic origin. This serves as valuable tool for future genome editing applications.
For delivery, lipid nanoparticles (LNPs) are a highly effective and versatile delivery system, valued for their larger cargo capacity, biocompatibility, and ability to protect RNA from degradation. To deliver our Prime Editing construct as mRNA, we optimized a Selective ORgan Targeting (SORT) LNP for targeted delivery to the lungs by using the cationic helper lipid DOTAP and encapsulating a stable Chitosan-RNA complex, achieving significant breakthroughs in transfection of in vitro lung epithelial cells.
We began by testing three different LNP formulations, starting with the Cayman LipidLaunc LNP-102 Exploration Kit. We confirmed by fluorescence microscopy, where Minicircle DNA effectively transfected HEK293 cells. Further experiments with the Corden LNP Stater Kit #2 failed to achieve successful transfection, likely due to increased cytotoxicity from a more cytotoxic PEG component.
Our successful formulation was a lung-specific SORT LNP, which demonstrated excellent stability, as confirmed by zeta potential measurements. Dynamic light scattering (DLS) analysis revealed an optimal particle size of 200 nm, aligning with literature and supporting the ability of the LNPs to penetrate deep lung regions via inhalation. Flow cytometry analysis showed that the SORT LNP had 14 times higher transfection efficiency compared to control formulations. Moreover, an MTT cytotoxicity assay revealed that the SORT LNP, along with Cayman LNPs, exhibited the lowest cytotoxicity, thanks to the use of low-molecular-weight PEG components.
To further enhance the stability and sustainability of the LNPs for inhalation, we incorporated chitosan-RNA complexes, 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. 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>
