Toward Effective Cystic Fibrosis Gene Therapy by Optimizing Prime Editing and Pulmonary-Targeted LNPs

Kaya Sophie Lange^1,2Lisa Marie Wiesner¹Kathleen Susat¹Vera Köhler¹Malte Lenger¹Christian Alexander Michalek^1,3Philip Frederic Mundt¹Anna-Lena Baack¹Kai Kanthak¹Isabell Alexandra Guckes¹Liliana Sanfilippo¹Lucas Haverkamp²Utkarsh Anil Mahajan^1,4Felicitas Helena Zimmer¹Sinan Zimmermann¹Levin Joe Klages¹Jörn Kalinowski¹Kristian Mark Müller^2*

• ¹Center for Biotechnology, Bielefeld University, Bielefeld, Germany
• ²Bielefeld University, Cellular and Molecular Biotechnology, Bielefeld, North Rhine-Westphalia, Germany
• ³Faculty of Chemistry, University of Bielefeld, Bielefeld, North Rhine-Westphalia, Germany
• ⁴Faculty of Technology, Bielefeld University, Bielefeld, North Rhine-Westphalia, Germany

Cystic fibrosis (CF) is the most prevalent inherited disease, resulting from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. This leads to the accumulation of viscous mucus and subsequent respiratory complications. This study optimized a prime editing approach to correct CFTR mutations, with a particular focus on the F508del mutation. Prime editing allows for precise DNA modifications to be made without the introduction of double-strand breaks. This is achieved by using a Cas9-nickase and reverse transcriptase fusion protein, which is guided by novel prime editing guide RNA (pegRNA), thus minimizing off-target effects.To ensure the effective delivery of the prime editing complex to the lungs, we have utilized a lungspecific lipid nanoparticle (LNP) system, which has been optimized for stability and targeted delivery to lung cells. Into the bronchial epithelial cell line, the LNP successfully delivered the prime editing complex, achieving precise gene correction of the reporter plasmid pPEAR_CFTR as verified by flow cytometry. The custom-designed fluorescence reporter system, pPEAR_CFTR, was developed mimicking F508del to validate the efficacy of our designed pegRNAs. Self-designed pegRNAs were compared using our custom-designed reporter system, pPEAR_CFTR. This system was also used to measure the prime editing efficiency of LNP delivered prime editing as RNA-chitosan-complexation, resulting in higher prime editing efficiencies. The 5 pegRNAs resulting in the highest prime editing efficiency were selected and used for genomic CFTR correction in CF bronchial cell line cells. Following genomic DNA isolation and sequencing 5 % edited reads were observed via Nanopore sequencing.Ongoing optimization and testing in primary cells and in vivo models are expected to further increase editing efficiency and therapeutic potential. These results highlight the promise of this prime editing-LNP system for precise and lung-specific gene correction, paving the way for novel therapies in cystic fibrosis and other pulmonary genetic disorders.