Viral Vectors, Animal Models, and Cellular Targets for Gene Therapy of Cystic Fibrosis Lung Disease

Abstract

After more than two decades since clinical trials tested the first use of recombinant adeno-associated virus (rAAV) to treat cystic fibrosis (CF) lung disease, gene therapy for this disorder has undergone a tremendous resurgence. Fueling this enthusiasm has been an enhanced understanding of rAAV transduction biology and cellular processes that limit transduction of airway epithelia, the development of new rAAV serotypes and other vector systems with high-level tropism for airway epithelial cells, an improved understanding of CF lung pathogenesis and the cellular targets for gene therapy, and the development of new animal models that reproduce the human CF disease phenotype. These advances have created a preclinical path for both assessing the efficacy of gene therapies in the CF lung and interrogating the target cell types in the lung required for complementation of the CF disease state. Lessons learned from early gene therapy attempts with rAAV in the CF lung have guided thinking for the testing of next-generation vector systems. Although unknown questions still remain regarding the cellular targets in the lung that are required or sufficient to complement CF lung disease, the field is now well positioned to tackle these challenges. This review will highlight the role that next-generation CF animal models are playing in the preclinical development of gene therapies for CF lung disease and the knowledge gaps in disease pathophysiology that these models are attempting to fill.

Keywords: animal models; cellular targets; cystic fibrosis; gene therapy; pathophysiology; viral vectors.

Figure 1.

Cellular expression patterns of CFTR in the proximal human airway. (A) Major cell types in the proximal human cartilaginous airways. Brush cells and PNEC are not shown. Goblet cells are classified at the proximal airway secretory cell. (B) CFTR mRNA expression levels in various proximal human airway cell types as determined by scRNAseq. Data were extracted and visualized from the Broad Institute Single Cell Portal. A subset of cells with lower levels of CFTR mRNA expression is visualized with an enlarged y-axis in the lower panel. (C) Schematic summary of CFTR mRNA expression levels for the various cell types marked in (A). (D) CFTR expression (yellow) within a subpopulation of basal intermediate cells, fated to become goblet secretory cells, suggests potential roles for CFTR in differentiation toward other CFTR-expressing cell types. B→S: Basal→Secretory; B→C: Basal→Ciliated. CFTR, cystic fibrosis transmembrane conductance regulator; PNEC, pulmonary neuroendocrine cells; scRNAseq, single-cell RNA sequencing.

Figure 2.

Models for the multicellular integration of CFTR function in ion and fluid movement across the proximal airway epithelium. (A) Model-1 in which CFTR-expressing ionocytes perform the majority of Cl^– and HCO₃^– transport required for regulating fluid movement. As proposed, ionocytes are electrically coupled with ciliated cells through gap junctions (gray circles). (B) Model-2 in which ciliated cells (or alternatively goblet secretory cells, not shown) participate in CFTR-dependent Cl^– movement across the epithelium, whereas CFTR in ionocytes functions to transport HCO₃^–. In both models, pH regulation of the ASL controls ENaC activation, through either ionocyte CFTR-dependent HCO₃^– secretion to raise the ASL pH or ATP12A-dependent H⁺ secretion to lower the ASL pH. Lower ASL pH leads to ENaC activation and fluid absorption (left panels of A, B) and higher ASL pH leads to ENaC inactivation and fluid secretion (right panels of A, B). (C) Potential model for CFTR involvement in mucin exocytosis and unfolding in goblet secretory cells. The model proposes that CFTR (yellow) expression in mucin secretory vesicles may control Cl^– and/or HCO₃^– movement required for exocytosis and/or mucin unfolding. ASL, airway surface liquid; ATP12A, nongastric H⁺/K⁺ ATPase; BK, voltage-dependent K⁺ channel; CaCC, calcium-activated chloride channel; ENaC, epithelial sodium channel.