Correction of F508del CFTR in airway epithelium using nanoparticles delivering triplex-forming PNAs

Cystic fibrosis (CF) is a lethal genetic disorder most commonly caused by the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is not readily amenable to gene therapy because of its systemic nature and challenges including in vivo gene delivery and transient gene expression. Here, we use triplex-forming PNA molecules and donor DNA in biodegradable polymer nanoparticles to correct F508del. We confirm modification with sequencing and a functional chloride efflux assay. In vitro correction of chloride efflux occurs in up to 25% of human cells. Deep sequencing reveals negligible off-target effects in partially homologous sites. Intranasal application of nanoparticles in CF mice produces changes in nasal epithelium potential differences consistent with corrected CFTR, with gene correction also detected in lung tissue. This work represents facile genome engineering in vivo with oligonucleotides using a nanoparticle system to achieve clinically relevant levels of gene editing without off-target effects. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Correspondence to: Marie Egan, [email protected]. †The first two authors contributed equally to this work Authors contributions Experiments were designed by N.A.M., K.A., C.C., P.M.G., W.M.S., and M.E.E. Experiments were performed and data analyzed by N.A.M., K.A., R.J.F., C.C., S.K., A.G., E.Q, L.P., Y.K., and M.E.E. Important reagents were prepared and provided by E.Q., R.B., and J.G. Written manuscript was prepared by N.A.M., K.A., E.Q., P.M.G., W.M.S., and M.E.E. Conflict of financial interests: The authors have no conflicts of interest to disclose. Accession codes: The sequences generated by deep sequencing have been deposited in SRA database under the accession codes (awaiting upload). Sequences have been uploaded to http://www.ncbi.nlm.nih.gov/bioproject/276283. HHS Public Access Author manuscript Nat Commun. Author manuscript; available in PMC 2015 October 27. Published in final edited form as: Nat Commun. ; 6: 6952. doi:10.1038/ncomms7952. A uhor M anscript