Correction of Splicing Defects in Cystic Fibrosis with Peptide-Oligonucleotide Conjugates and Small Molecule Enhancing Compounds

ABSTRACT The pathogenesis of Cystic FibrosisCFis caused by mutations in the gene for CFTRa cAMP activated chloride channel that regulates salt and water transport in epithelial tissuesespecially those in the airwaysRecent progress has resulted in small molecule therapeutic agents that partially correct defects in the CFTR protein due to certain mutationsHoweverin 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 therapeuticsIn these patients splice switching oligonucleotidesSSOsmay provide a means to correct the underlying molecular defect and permit expression of fully functional CFTR proteinSSOs have recently enjoyed success in treating other genetic disorders in patientsincluding Duchenne muscular dystrophyDMDand spinal muscular atrophySMAhowever they have not yet been applied to CFAlthough this concept is appealingthe effective delivery of oligonucleotides remains a problem for the entire field of oligonucleotide based therapeutics and may be especially challenging in the airwaysThus we intend to use a novel dual approach to attain effective delivery of corrective SSOs to airway epithelial cellsFirst we will make use of oligonucleotides that are chemically conjugated to shortpositively charged peptides that augment cellular uptake of the oligonucleotideIn particular we will use P PMOs in which the peptidePis conjugated to an unchargednon toxic morpholinoPMOoligonucleotideSecondwe will make use of recently discovered oligonucleotide enhancing compoundsOECsi esmall molecules that release oligonucleotides from unproductive entrapment in endosomes thus potentiating their actionThe 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 systemsValidation 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!!PROJECT NARRATIVE Splice switching oligonucleotidesSSOshave been used to treat genetic disorders in patientsincluding Duchenne muscular dystrophy and spinal muscular atrophyhoweverthey have not been applied to cystic fibrosisCFOur 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 oligonucleotidesValidation 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