On the Corner of Models and Cure: Gene Editing in Cystic Fibrosis

Abstract

Cystic fibrosis (CF) is a severe genetic disease for which curative treatment is still lacking. Next generation biotechnologies and more efficient cell-based and in vivo disease models are accelerating the development of novel therapies for CF. Gene editing tools, like CRISPR-based systems, can be used to make targeted modifications in the genome, allowing to correct mutations directly in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Alternatively, with these tools more relevant disease models can be generated, which in turn will be invaluable to evaluate novel gene editing-based therapies for CF. This critical review offers a comprehensive description of currently available tools for genome editing, and the cell and animal models which are available to evaluate them. Next, we will give an extensive overview of proof-of-concept applications of gene editing in the field of CF. Finally, we will touch upon the challenges that need to be addressed before these proof-of-concept studies can be translated towards a therapy for people with CF.

Keywords: CF animal models; CF cell models; CFTR; cystic fibrosis; gene editing; gene therapy; humanized CF animals.

FIGURE 1

Cystic fibrosis cell models for the evaluation of gene editing strategies. Different cell-based models are available to evaluate gene editing for CF. While heterologous, CFTR overexpressing models allow the study of any CFTR variant, and are easiest to work with, their read-outs are limited to molecular and functional analysis of CFTR and genetic correction at the cDNA or minigene level. Immortalized cells, expressing the endogenous CFTR, allow correction of the CFTR gene, but are only available for a limited number of the more common CFTR genotypes. Gene editing however, has allowed to generate immortalized lines for additional genotypes (*) (Valley et al., 2019). Primary cell models are the most relevant, since they can be used for CF specific read-outs that are important in the CF phenotype, including mucus and immune defects. Traditional molecular CFTR endpoints remain challenging for primary cells. Depending on the origin of the primary cells, genotypes might be exceedingly hard to acquire i.e., human bronchial epithelial (HBE) cells from explant lungs. On the other hand, human intestinal organoids (HIO) and human nasal epithelial (HNE) cells are more readily obtained via minimally invasive procedures. Alternatively, iPSCs were edited (†) to generate isogenic lines with different genotypes (Ruan et al., 2019). A combination of different models allows the most complete cell-based evaluation of novel gene editing strategies. Abbreviations: FIS, forskolin induced swelling; HBE, human bronchial epithelial; HIO, human intestinal organoids; HNE, human nasal epithelial; ICC, immunocytochemistry; iPSCs, induced pluripotent stem cells; Isc, short circuit currents; HS-YFP, halide sensitive yellow fluorescent protein.

FIGURE 2

Gene editing strategies for CF from proof-of-concept to their translation into a therapy. Gene editing for CF consists of correcting mutations in CFTR either by an ex vivo stem cell therapy approach or by directly editing target cells in vivo. To transfer gene editors as nucleic acids or proteins into cells, it is likely necessary, particularly for the in vivo approach, to encapsulate them with a vector in order to protect them and facilitate their entry into target cells. For their clinical translation, in vitro/ex vivo and in vivo models are essential to determine the best formulation that allows efficient and safe gene editing. In vitro and ex vivo models allow evaluating gene editing efficacy at three levels: genomic, protein and physiological. Summarized, this starts by demonstrating a genetic correction with low off-targets, followed by a normalization of CFTR expression, folding and function, to end with a rescue of pathophysiological defects induced by mutant CFTR. Patient derived cell models furthermore allow developing personalized formulations for each patient. Animal models mimicking CF pathophysiology are a major asset to study the efficacy of the gene editing delivery vehicle in a clinically relevant environment. This, as extracellular barriers must be overcome such as thick and viscous mucus, pathogens or endonucleases to allow efficient gene transfer. Knock-out, knock-in and humanized models each have their specific advantages and limitations that should be considered for an in vivo evaluation. Abbreviations: ASL, airway surface liquid; BAC, bacterial artificial chromosome; Cas, CRISPR associated protein; KI, Knock-in models; KO, Knock-out models; PCL, periciliary liquid; sgRNA, single guide RNA; YAC, yeast artificial chromosome.