Gene therapy for cystic fibrosis: Challenges and prospects

Abstract

Cystic fibrosis (CF) is a life-threatening autosomal-recessive disease caused by mutations in a single gene encoding cystic fibrosis transmembrane conductance regulator (CFTR). CF effects multiple organs, and lung disease is the primary cause of mortality. The median age at death from CF is in the early forties. CF was one of the first diseases to be considered for gene therapy, and efforts focused on treating CF lung disease began shortly after the CFTR gene was identified in 1989. However, despite the quickly established proof-of-concept for CFTR gene transfer in vitro and in clinical trials in 1990s, to date, 36 CF gene therapy clinical trials involving ∼600 patients with CF have yet to achieve their desired outcomes. The long journey to pursue gene therapy as a cure for CF encountered more difficulties than originally anticipated, but immense progress has been made in the past decade in the developments of next generation airway transduction viral vectors and CF animal models that reproduced human CF disease phenotypes. In this review, we look back at the history for the lessons learned from previous clinical trials and summarize the recent advances in the research for CF gene therapy, including the emerging CRISPR-based gene editing strategies. We also discuss the airway transduction vectors, large animal CF models, the complexity of CF pathogenesis and heterogeneity of CFTR expression in airway epithelium, which are the major challenges to the implementation of a successful CF gene therapy, and highlight the future opportunities and prospects.

Keywords: airway delivery; animal models; cystic fibrosis; gene therapy; viral vectors.

FIGURE 1

Worldwide distribution of disease-causing CFTR variants. More than 2,000 different mutations in CFTR have been identified, and ∼400 mutations are confirmed to be disease-causing. (http://www.genet.sickkids.on.ca/app). Pie chart represents the frequencies of common CF mutations (>0.3%) in population worldwide. Notably, the distribution and frequency of CFTR variants vary in different countries and ethnic groups.

FIGURE 2

Human airways and major airway epithelial cell types. (A) Anatomy. The human airways are divided into the upper airways, lower airways and the lung parenchyma. The upper airways include the nose and nasal passages, paranasal sinuses, the pharynx, and the portion of the larynx above the vocal folds (cords). The lower airways include conducting zone from generation (G) G0 to G16, and respiratory zone from G17 and beyond. The trachea and proximal bronchi (generation G0 to G6) are considered as large airways. The small airways are distal bronchioles whose diameter is smaller than 2mm, starting from G7 the terminal bronchioles in conducting zone to the respiratory bronchioles in respiratory zone. After G23, the airway epithelium merges with the alveolar epithelium with pneumocyte type I and type II cells (alveolar type I and II cells, ATI and ATII cells) in lung parenchyma. (B) Airway epithelium is a continuous cellular layer with epithelial cell populations and functions vary along the respiratory tree. It is pseudostratified in the nasal and large airways, becoming columnar and cuboidal in the small airways. Submucosal glands (SMG) are majorly found in large airways. Major surface epithelial cell types are columnar ciliated cells, goblet cells, club cells and basal cells. Their composition is similar in large and small airways, but with gradually less ciliated cells, and the secretory cells shift from goblet cells to the dome-shaped club cells. Airway basal cells have the capacity to differentiate into the major cell types of the airways, and a subset of club cells can differentiate to ciliated cells and goblet cells. Pulmonary ionocytes express high level CFTR, but the mechanism by which they contributed to cystic fibrosis are still largely unknown. Ionocytes may be absent in the small airways. Pulmonary neuroendocrine cells (PNEC) serve as key communicators between the immune and nervous system.

FIGURE 3

CFTR mutation types and CREIPSR-based Gene Editing. CRIPSR/Cas editing approaches rely on the generation of double stranded DNA break (DSB) and the following repair via non-homologous end joining (NHEJ) or homologous recombination (HR). NHEJ can be used for the allele specific repair of splicing mutation in the intronic sequence, such as the 3272-26A>G (c.3140–26A>G) and 3,849 + 10kbC>T (c.3718–2477C>T) CFTR mutations. Homology-independent targeted insertion (HITI) is an approach based on NHEJ. Theoretically, HITI can universally address all CFTR variants independent of genotypes, the donor DNA can be a splicing acceptor associated CFTR cDNA sequence to be inserted into the intronic sequence of CFTR to reconstitute a mutation free mini gene, or a CFTR expression cassette to be inserted into the safe harbor of the chromosome, such as AAVS1. Homology-directed repair (HDR) requires a template for HR to correct point mutation or small deletion/deletion. Base editing and prime editing do not require the generation of DSB and DNA template for correction. Base editing is only used to correct single base point mutation, and primer editing can be used to correct point mutation as well as short deletion/insertion. Same as HDR, the correction options of base editing and prime editing are mutation dependent.