(622) DEVELOPMENT OF IN VITRO TRANSCRIBED mRNA THERAPEUTICS FOR CYSTIC FIBROSIS

Background: Cystic fibrosis (CF) is a relatively common recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Absent/non-functional CFTR leads to thick, sticky mucus in the lung which results in chronic bacterial infection and inflammation. In vitro transcribed (IVT) mRNA has emerged in the last few years as a new approach for CFTR protein replacement therapy but problems of delivery must be overcome.

Methods: We have developed novel, receptor-targeted nanocomplex (RTN) formulations consisting of liposomes and receptor-targeting peptide for delivery of CFTR IVT mRNA. The RTNs were first optimised in transfections of primary cystic fibrosis bronchial epithelial cells (CFBE) using reporter IVT mRNAs. We then assessed delivery efficiency of CFTR mRNA in CFBE cells at submerged culture and ALI culture and assessed uptake by qRT-PCR and demonstrated protein expression by Western blot.

Results: We first optimized the RTN formulations, comparing combinations of three different cationic liposomes and five peptides for their biophysical properties and transfection efficiency. We identified a novel formulation (size 130 nm, PDI 0.28, charge 38 mV) for mRNA delivery that achieved almost 100% cellular uptake efficiency and 90% transfection efficiency. There were no differences in transfection efficiency between primary normal human bronchial epithelial (NHBE) cells and CFBE cells. The same RTN formulation was able to deliver the mRNAs in ALI cultured cells and mouse lungs where luciferase expression in mouse lungs was ~200-fold higher than plasmid DNA encoded luciferase. CFTR mRNA was successfully delivered to NHBE and CFBE cells. In addition, we co-packaged the CFTR corrector VX-809 with CFTR mRNA into RTNs and found that this improved the expression of CFTR protein in CFBE cells in submerged culture by approximately 2 to 2.5-fold. CFTR protein translated from the IVT mRNA was detected by immunoblotting from 4-hour to at least 48-hour after the CFBE mRNA transfections. CFTR protein expression was also shown in transfections of ALI culture of CFBE cells by functional analysis of ion transport in Ussing chamber. The ∆Is was 10-fold larger in cells transfected with CFTR mRNA than the untransfected cells before/after Forskolin added and it was approximately 59.0% of Non-CF.

Conclusions: In conclusion, CFTR IVT mRNA delivery is a promising novel therapeutic for cystic fibrosis. In addition, the flexibility of the RTN formulation allows co-delivery of CFTR mRNA with VX-809, which significantly improved CFTR expression.

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