Targeted correction of the human CFTR gene



 DESCRIPTION (provided by applicant): Cystic fibrosis affects 1 in 2,000-3,000 live births in the Caucasian population, corresponding to approximately 30,000 people in the US. Cystic fibrosis (CF) is an autosomal recessive disease caused by defects in the cystic fibrosis transmembrane conductance regulator protein (CFTR), a protein kinase A- activated chloride channel that mediates epithelial chloride transport. Though CF is a multisystem disease affecting sweat glands, the reproductive tract, the pancreas, gastrointestinal tract, and liver, itis lung disease that most commonly leads to fatality. The only curative therapy for lung manifestations of CF is transplantation, but 20% of CF patients may never be transplanted due to acute organ shortage. The mean lifespan of CF patients is only 37 years. Since the CFTR gene was sequenced in 1989, there have been over 20 trials in gene therapy in an attempt to cure pulmonary CF. Gene therapy, however, has remained challenging, because of difficulties with in vivo delivery of exogenous genes to the lung. Viral plasmids containing exogenous CFTR are often only transiently expressed, and integrating vectors such as lenti- and retroviruses result in non-specific integration with attendant risks. In addition, the host immune response often clears viral vectors that are used for gene delivery, particularly in the lung. As a result, gene therapy trials have all but ceased in this disease, with the exception of an ongoing trial in the UK that is using nebulized pGM169/GL67A contained in a liposomal carrier. Recent advances in gene editing - as opposed to gene therapy - may make in vivo correction of the CF mutation possible. Site-specific gene editing could correct the CFTR gene at its endogenous site, resulting in permanent gene modification that is under normal regulatory control. The goal of this application is to develop a novel therapy for the pulmonary manifestations of CF. We have developed novel peptide nucleic acids (PNA) that can form a triple helical structure specifically within the CFTR gene. The triplex formation induces natural cellular host DNA repair-recombination pathways enabling correction of the F508del mutation in the CFTR gene when a donor DNA is supplied alongside. These pathways are error- free and can thus be used to edit the CFTR gene with extremely low off-target rates. We propose to use biocompatible nanoparticles made from the FDA-approved polymer PLGA for encapsulating the PNA and donor DNA molecules for in vivo delivery and gene editing of the CFTR gene in vivo. We will develop, optimize, and test novel nanoparticles containing peptide nucleic acids and complementary DNA (PNA/DNA) to effect CFTR gene correction in the lung. This therapy has the potential to ameliorate and/or cure the pulmonary manifestations of cystic fibrosis. Because the F508del CFTR mutation is the most common cause of CF, we are targeting that defect in this application. The overall goal of this program is to evaluate feasibility, reliability, safety nd efficacy of nanoparticle-based gene editing for treatment of CF.