Delivering inhalable nanoparticles containing AI-optimized CFTR mRNA with a novel microfluidic device for treatment of CF

Project summary Cystic fibrosis (CF) is an autosomal genetic inherited disease caused by over 1,000 known mutations in the CFTR gene, which encodes an ion transporter. Gene therapy offers a single therapeutic solution to treat all CF patients, regardless of underlying mutation. Previous gene therapy approaches delivering CFTR DNA via viral or liposomal delivery have not proven clinically viable. Airway administration of CFTR mRNA packaged in lipid nanoparticles (LNPs) offers a promising therapeutic approach for CF patients that is non-invasive, repeatable, and safe. A recent Phase 1/2 clinical trial of CFTR mRNA delivery using repeated administration of inhaled CFTR mRNA-LNPs demonstrated clinical safety but did not adequately improve CF endpoints. Advances have been made in LNP material design and formulation for mRNA delivery to the lungs but clinical feasibility for CF treatment requires additional therapeutic improvements. One neglected area of innovation is modification and optimization of the delivered CFTR mRNA cargo. Current therapies use wild-type mRNA or basic codon optimization to increase translation efficiency of the CFTR mRNA. However, recent advances in AI-based design provide a significantly better method to optimize both protein translation levels and mRNA half-life. This optimization would improve the efficacy of CFTR mRNA therapy and potentially reduce dosage and administration frequency compared to current mRNA cargoes. Another technical limitation is the current nebulizer technology for aerosolization of LNPs, which physically disrupts nanoparticles, reducing their effectiveness and necessitating reformulation of LNPs that can diminish their efficacy and provoke immune responses. New nebulizer technology with reduced LNP disruption would enhance mRNA- LNP delivery and remove the need for deleterious reformulations. We propose to address the efficacy gap of inhaled mRNA-LNP therapies by completing the following objectives: 1) employ AI-based mRNA design with rapid in vitro screening to generate a therapeutic CFTR mRNA optimized for protein expression, half-life, and ion channel function; 2) utilize mRNA barcoding for rapid in vivo screening of longevity of these novel CFTR mRNA sequences delivered by nebulizer; and 3) evaluate therapeutic efficacy and safety of repeated inhaled administration of CFTR mRNA-LNPs using a novel microfluidic nebulizer device in a rat model of CF. These studies will advance inhalable mRNA-LNP therapies by applying new nebulizer and computational technologies to address overlooked aspects of airway mRNA-LNP delivery. This work evaluates new strategies in vitro and in vivo to improve clinical viability of inhalable LNP delivery of mRNA that provides a noninvasive and repeatable treatment for CF and other pulmonary diseases.