Lipid nanoparticle-delivered mRNA encoding the NBD1-stabilizing nanobody T2a synergistically enhances FDA-approved CFTR corrector efficacy in rescuing F508del-CFTR trafficking and function, while cryo-EM structural analysis reveals that T2a stabilizes a novel CFTR conformation with undocked NBD1 that enables channel opening and ion conduction without canonical NBD dimerization, establishing a new paradigm for CFTR activation and a broadly applicable nanobody-based therapeutic strategy for protein misfolding diseases.

Defects in protein trafficking underlie many genetic diseases, including cystic fibrosis (CF), where the common F508del mutation destabilizes the cystic fibrosis transmembrane conductance regulator (CFTR) channel, leading to its degradation. To enhance current CFTR modulator therapies, we used lipid nanoparticles to deliver mRNA encoding T2a, a nanobody that thermally stabilizes CFTR by binding nucleotide-binding domain 1 (NBD1). When combined with clinically-approved correctors, T2a significantly improved F508del-CFTR maturation, plasma membrane expression, and channel activity. Single-channel recording revealed that nanobody binding sustained channel activity by promoting both full and sub-conductance gating states and protecting F508del-CFTR against thermal deactivation. Cryo-EM analysis identified a novel conformation of CFTR where NBD1 adopts an alternative geometry enabling pore formation in the absence of NBD dimerization. Our findings establish a new paradigm to correct protein trafficking by stabilizing misfolded domains with targeted nanobodies and demonstrate a broadly applicable framework to treat CF and related protein misfolding diseases.