Abstract
Gene therapy has always been a promising therapeutic approach for Cystic Fibrosis (CF). However, numerous trials using DNA or viral vectors encoding the correct protein resulted in a general low efficacy. In the last years, chemically modified messenger RNA (cmRNA) has been proven to be a highly potent, pulmonary drug. Consequently, we first explored the expression, function and immunogenicity of human (h)CFTR encoded by cmRNAhCFTRin vitro and ex vivo, quantified the expression by flow cytometry, determined its function using a YFP based assay and checked the immune response in human whole blood. Similarly, we examined the function of cmRNAhCFTRin vivo after intratracheal (i.t.) or intravenous (i.v.) injection of the assembled cmRNAhCFTR together with Chitosan-coated PLGA (poly-D, L-lactide-co-glycolide 75:25 (Resomer RG 752 H)) nanoparticles (NPs) by FlexiVent. The amount of expression of human hCFTR encoded by cmRNAhCFTR was quantified by hCFTR ELISA, and cmRNAhCFTR values were assessed by RT-qPCR. Thereby, we observed a significant improvement of lung function, especially in regards to FEV0.1, suggesting NP-cmRNAhCFTR as promising therapeutic option for CF patients independent of their CFTR genotype.
Highlights
Gene therapy has always been a promising therapeutic approach for Cystic Fibrosis (CF)
We provide a proof of concept of NP- cmRNAhCFTR mediated, Enzyme-linked immunosorbent assays (ELISAs) quantified, hCFTR expression in the lungs of Cftr−/− mice, leading to significantly reduced chloride secretion and, more importantly, restored criticial lung function parameters, including the most important parameter to evaluate mortality and morbidity of CF patients, the forced expiratory volume (FEV) in 1s or 0.1s in small animals, respectively[22,23,24]
Much progress has been achieved since the discovery of the CFTR gene 25 years ago, there is still a substantial need to restore robust CFTR function in patients suffering from cystic fibrosis[8]
Summary
(c)mRNAhCFTR and hCFTR protein quantification in vitro. To evaluate the influence of chemical nucleoside modification, we first conducted a set of in vitro analyses to characterize the expression and functionality of hCFTR protein. Transfected CFBE41o- cells showed an average of 22.8% of the protein expression compared to hCFTR observed in 16HBE14o- cells, which increased 4.1-fold to 94.0% at 72 h (P ≤ 0.05; Fig. 1C). All tested samples show a higher amount of hCFTR positive cells compared to the negative control (CFBE41o- cells; Fig. 2A). All cmRNAhCFTR showed a very similar result in cytokine expression as observed for negative controls: the IFN-α levels did not reach the detection limit of the ELISA; IL-8 and TNF-α responses were not statistically significant at 6 h and 24 h, respectively (Fig. 2C). In vivo lung function measurements in cmRNAhCFTR and pDNAhCFTR treated Cftr−/− mice by i.t. route. No immune response had been observed apart from positive control in groups treated intratracheally (i.t) (Fig. 7B)
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