The pathogenesis of Cystic Fibrosis (CF) is caused by mutations in the gene for CFTR, a cAMP activated chloride channel that regulates salt and water transport in epithelial tissues, especially those in the airways. Recent progress has resulted in small molecule therapeutic agents that partially correct defects in the CFTR protein due to certain mutations. However, in a substantial fraction of CF patients the disease is due to splicing defects in the CFTR gene that cannot be directly addressed using small molecule therapeutics. In these patients splice switching oligonucleotides (SSOs) may provide a means to correct the underlying molecular defect and permit expression of fully functional CFTR protein. SSOs have recently enjoyed success in treating other genetic disorders in patients, including Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA); however they have not yet been applied to CF. Although this concept is appealing, the effective delivery of oligonucleotides remains a problem for the entire field of oligonucleotide based therapeutics and may be especially challenging in the airways. Thus we intend to use a novel dual approach to attain effective delivery of corrective SSOs to airway epithelial cells. First we will make use of oligonucleotides that are chemically conjugated to short, positively charged peptides that augment cellular uptake of the oligonucleotide. In particular we will use P-PMOs in which the peptide (P) is conjugated to an uncharged, non-toxic morpholino (PMO) oligonucleotide. Second, we will make use of recently discovered oligonucleotide enhancing compounds (OECs) i.e. small molecules that release oligonucleotides from unproductive entrapment in endosomes thus potentiating their action. The current proposal is designed to be a proof of concept for this dual delivery approach via testing of P-PMOs and OECs in CF-relevant cell culture and animal model systems. Validation of this concept would be a key step in developing a novel therapy for a significant cohort of CF patients and may have broader ramifications for use of oligonucleotides in other airway diseases. ! 1!
Splice switching oligonucleotides (SSOs) have been used to treat genetic disorders in patients, including Duchenne muscular dystrophy and spinal muscular atrophy; however, they have not been applied to cystic fibrosis (CF). Our goal is to enhance the delivery of SSOs to human CF patient airway cell cultures and in vivo in mice by dual use of peptide-morpholino oligonucleotide conjugates and of small molecules that affect intracellular trafficking of oligonucleotides. Validation of this concept has impact in human health because it is a key step in developing a novel therapy for a significant cohort of CF patients and may have broader ramifications for use of oligonucleotides in other airway diseases.