Editing basal cells for durable gene therapy of cystic fibrosis

Cystic fibrosis (CF) patients with nonsense mutations of the disease-causing CF transmembrane conductance regulator gene (CFTR) cannot benefit from existing small molecule therapies and need gene therapy of the CFTR mutation to restore any function and ultimately, for a cure. Since 95% of CF-associated comorbidities affect the respiratory tract, pulmonary administration of gene therapies would be advantageous, as it would increase the therapeutic index by significantly increasing dosing 10- to 20-fold without incurring the off-tissue toxicity seen with systemic administration. For inhalation gene therapy, the gene delivery systems must possess the physicochemical properties to overcome the transport barriers, such as mucus and reach the target cells in the airways at therapeutic concentrations. However, current approaches in the clinic would need repeated dosing since they do not target the basal cells, or the airway stem cells, for one-time, permanent correction. Also, gene therapy approaches currently in clinical trials do not fix the mutation but provide a corrected CFTR copy, whereby the therapeutic benefit is temporary. To address these challenges, it would be desirable to develop nucleic acid delivery systems that can reach the airway basal cells and fix the CFTR mutation towards permanent correction of lung CF disease. Here, we will develop peptide surface-functionalized lipid nanoparticles (pepLNPs) for pulmonary delivery that can reach the basal cells in vivo and deliver base editors in relevant cell culture and animal models to precisely edit and correct specific G542X and G553X nonsense mutations present in CF. Previously, we have identified peptide ligands that can penetrate through the mucus barrier present in CF and in preliminary evidence, demonstrate that these pepLNPs, can reach and achieve editing in the basal cells. Leveraging our platform technology, we propose to make pepLNP formulations that can stably encapsulate, protect, and deliver mRNA encoding base editing components, to reach, and edit basal cells in vitro and in vivo. Importantly, we will elucidate how these pepLNPs penetrate through the mucus barrier and are taken up by basal cells. Finally, we will validate that upon delivery to the basal cells, pepLNPs will deliver adenine base editors to correct the genotype and restore functional phenotype of cells that had possessed the previously undruggable nonsense CFTR mutations. This proposed work can have a transformative impact, as it aims to achieve one- time, “permanent” correction for durable gene therapy of CF by targeting the basal cells and specifically fixing the inherent CFTR mutation. While this strategy focuses on CF, in the long-term, this strategy can transform treatment of many genetic diseases needing permanent correction. Project Number: 1R01HL177349-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: Debadyuti Ghosh | Institution: UNIVERSITY OF TEXAS AT AUSTIN, AUSTIN, TX | Award Amount: $564,121 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 BBBT-X (81)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01HL17734901A1