Our study provides robust evidence that rAAV-mediated gene transfer of a truncated CFTR functionally rescues the CF phenotype across the nasal mucosa of CF mice and in patient-derived organoids. These results underscore the clinical potential of rAAV-CFTRΔR in offering a cure for all patients with CF in the future.

Gene therapy provides a mutation-independent approach to treat or even cure CF airway disease. To develop a clinical candidate for CF gene therapy, a thorough examination of preclinical efficacy in relevant cell and animal models is a prerequisite. For a long time, the CF field was struggling with a lack of appropriate animal models for CF airway pathology. Since 2008, many different and complementary animal models have been generated that develop hallmarks of CF airway disease, including the CF pig, ferret, and rat. With this, a new era has arisen that allows investigating the efficacy of gene therapy beyond molecular and electrophysiological end-points. Successful gene therapy most likely requires an appropriate time window. CF lung pathology progresses with age and therefore an early treatment would be beneficial to prevent irreversible damage. In that regard, newborn screening programs and prenatal diagnosis already provide a basis to facilitate future preventive gene-based treatment. If successful, gene therapy for CF airway disease would markedly reduce the treatment burden and improve life quality and life expectancy of CF patients.

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Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome 1,2 or hyperoxic injuries of the neonatal lung Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies 7 .In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep 8, and even in a clinical setting 9 , but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection [10][11][12] , (ultrasound-guided) intrapulmonary injection 13,14 and intravenous administration into the yolk sac vessels 15,16 or umbilical vein 17 . Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques 18 . Video LinkThe video component of this article can be found at

Gene therapy holds promise to cure a wide range of genetic and acquired diseases. Recent successes in recombinant adeno-associated viral vector (rAAV)-based gene therapy in the clinic for hereditary disorders such as Leber's congenital amaurosis and hemophilia B encouraged us to reexplore an rAAV approach for pulmonary gene transfer. Only limited clinical successes have been achieved for airway gene transfer so far, underscoring the need for further preclinical development of rAAV-based gene therapy for pulmonary disorders. We sought to determine the preclinical potential of an airway-tropic serotype, rAAV2/5, encoding reporter genes when delivered to mouse airways. Although several groups have assessed the stability of gene transfer using a nonintegrating rAAV in mouse airways, long-term stability for more than a year has not been reported. Additionally, an extensive quantitative analysis of the specific cell types targeted by rAAV2/5 using cell-specific markers is lacking. We obtained sustained gene expression in upper and lower airways up to 15 months after vector administration, a substantial proportion of the lifespan of a laboratory mouse. In addition, we demonstrated that readministration of rAAV2/5 to the airways is feasible and increases gene expression 14 months after primary vector administration, despite the presence of circulating neutralizing antibodies. Finally, identification of transduced cell types revealed different subpopulations being targeted by rAAV2/5, with 64% of β-galactosidase-positive cells being ciliated cells, 34% club cells in the conducting airways, and 75% alveolar type II cells in the alveoli at 1 month postinjection. This underscores the therapeutic potential of a nonintegrating rAAV vector to develop a gene therapeutic drug for a variety of pulmonary disorders, such as cystic fibrosis, primary ciliary dyskinesia, and surfactant deficiencies.

Progressive familial intrahepatic cholestasis type 1 (PFIC1) is caused by mutations in the gene encoding the phospholipid flippase ATP8B1. Apart from severe cholestatic liver disease, many PFIC1 patients develop extrahepatic symptoms characteristic of cystic fibrosis (CF), such as pulmonary infection, sweat gland dysfunction and failure to thrive. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel essential for epithelial fluid transport. Previously it was shown that CFTR transcript levels were strongly reduced in livers of PFIC1 patients. Here we have investigated the hypothesis that ATP8B1 is important for proper CFTR expression and function. We analyzed CFTR expression in ATP8B1-depleted intestinal and pulmonary epithelial cell lines and assessed CFTR function by measuring short-circuit currents across transwell-grown ATP8B1-depleted intestinal T84 cells and by a genetically-encoded fluorescent chloride sensor. In addition, we studied CFTR surface expression upon induction of CFTR transcription. We show that CFTR protein levels are strongly reduced in the apical membrane of human ATP8B1-depleted intestinal and pulmonary epithelial cell lines, a phenotype that coincided with reduced CFTR activity. Apical membrane insertion upon induction of ectopically-expressed CFTR was strongly impaired in ATP8B1-depleted cells. We conclude that ATP8B1 is essential for correct apical localization of CFTR in human intestinal and pulmonary epithelial cells, and that impaired CFTR localization underlies some of the extrahepatic phenotypes observed in ATP8B1 deficiency.

Intratracheal injection of nanoparticles is feasible in the pregnant pig and does not cause procedural lung damage. Using an injection volume of 3 mL, all lung lobes were efficiently targeted. This nanoparticle delivery model to fetal airways opens perspectives for therapeutic interventions. © 2016 John Wiley & Sons, Ltd.

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