Cystic fibrosis (CF) is conferred by any of ?1200 known mutations in the gene encoding the CFTR, an ion transporter protein, but in principle, a single gene therapy agent that delivers expression of functional CFTR could treat. Prior attempts to do so have used adeno-associated viral or liposomal delivery systems to deliver CFTR DNA. Although some of these reached Phase I-IIB clinical trials, they failed to produce consistent, impactful improvement of patient outcomes. We developed a novel lipid nanoparticle (LNP)-based system to deliver mRNA, and found it successfully restored up to 55% of normal CFTR-mediated chloride efflux in the nasal epithelium of CFTR-deficient mice. To exploit and apply these novel discoveries, the long-term goal of this project is to overcome two key remaining biological barriers that limit entry of all gene therapy vectors into the lung: 1) the thick, sticky airway/lung epithelial mucus of CF that impedes gene carriers from reaching airway cells, and 2) inadequate cytosolic bioavailability of genetic material after uptake by cells, due to endosomal entrapment. Overcoming these two obstacles will require opposing particle characteristics: mucosal penetration is achievable primarily by stabilizing particles with a muco-inert polymer, whereas intracellular bioavailability of genetic payloads relies on particle destabilization. There is urgent need to develop nanoparticles stable enough to cross CF mucus and reach airway cells, yet labile enough to facilitate endosomal escape of genetic material following cellular uptake. We will meet these criteria, by altering the stability of the LNP core, while maintaining a muco-inert LNP surface. We found that replacing cholesterol with its naturally-occurring analogues improves intracellular gene delivery by 200-fold, vs. that seen with a clinically successful cholesterol-containing LNP. We posit that this major improvement in gene transfer occur via modifications in the enhanced LNP (eLNPs) core structure and trafficking. Modifications in eLNPs' core structure as we propose will enable disassembly, endosomal escape and cytosolic delivery of mRNA, while maintaining their muco-inert surface properties. Our translational project's goal is to analyze LNP structure and intracellular trafficking within the CF lung. Our objectives are thus 1) elucidate and optimize the structural features of nanocarriers that drive endosomal trafficking of eLNPs and enhanced gene delivery, 2) test and optimize the ability of altered core structures to enhance mucopenetration across human CF sputum and state of art mice models, and 3) assess safety of sustained transmucosal transfection of CFTR mRNA via repeated aerosolization in CF rats, including toxicological analyses. These studies will significantly advance translational therapy, enabling long-term restoration of CFTR function to halt or reverse model CF disease progression. Our novel approach alters internal particle stability via small structural modifications of cholesterol, rather modifying LNP surface coating or cationic lipid as in prior studies. Our discoveries will propel toward translation new non-viral vector carriers that can traverse the thick sticky mucus and endosomal barriers to deliver genes for efficacious CF treatment.
Cystic fibrosis (CF) can potentially be corrected by gene therapy, regardless of patient genotype. Such gene delivery is currently impeded by inability of DNA/RNA to escape cellular degradation systems, preventing therapeutic effects, and any CF gene therapies must be able to traverse the tough, sticky mucus barrier in CF patients' lungs. Our project will overcome these barriers, by engineering and testing nanoparticles that 1) carry a cargo of mRNA that directs production of the chloride-transporter protein whose deficiency causes CF, 2) escape degradation for release inside cells after traversing mucus, and 3) can be easily administered as an aerosol, for repeated effective dosing directly to patients' lungs.