Delivering on the promise of gene editing for cystic fibrosis

Abstract

In this review, we describe a path for translation of gene editing into therapy for cystic fibrosis (CF). Cystic fibrosis results from mutations in the CFTR gene, with one allele predominant in patient populations. This simple, genetic etiology makes gene editing appealing for treatment of this disease. There already have been success in applying this approach to cystic fibrosis in cell and animal models, although these advances have been modest in comparison to advances for other disease. Less than six years after its first demonstration in animals, CRISPR/Cas gene editing is in early clinical trials for several disorders. Most clinical trials, thus far, attempt to edit genes in cells of the blood lineages. The advantage of the blood is that the stem cells are known, can be isolated, edited, selected, expanded, and returned to the body. The likely next trials will be in the liver, which is accessible to many delivery methods. For cystic fibrosis, the biggest hurdle is to deliver editors to other, less accessible organs. We outline a path by which delivery can be improved. The translation of new therapies doesn't occur in isolation, and the development of gene editors is occurring as advances in gene therapy and small molecule therapeutics are being made. The advances made in gene therapy may help develop delivery vehicles for gene editing, although major improvements are needed. Conversely, the approval of effective small molecule therapies for many patients with cystic fibrosis will raise the bar for translation of gene editing.

Keywords: CFTR gene; CRISPR/Cas9; Cystic fibrosis; Gene editing; Gene therapy.

Figure 1

The structure of the human CFTR transcription unit, coding sequence and protein. The CFTR transcription unit is slightly less than 189,000 bases long with 27 exons. The most common disease-causing mutations are shown, as are three intronic mutations which lead to aberrant splicing (arrows). The CFTR protein forms a membrane channel with twelve transmembrane domains (TM), two nucleotide-binding domains (NBD1 and NBD2) and a regulatory domain (R).

Figure 2

Cas9 sequence recognition and cleavage. a) The complex of Cas9 protein and guide RNA bind DNA at the sequence NGG (the protospacer-adjacent motif or PAM). b–c) If the DNA strand opposite NGG and immediately downstream can base-pair with the guide RNA, then hybridization with the remainder of the guide RNA is attempted. c–d) If hybridization to the guide RNA is complete, then the two nuclease domains of Cas9 are activated. The HNH nuclease domain of Cas9 cuts the DNA strand paired with the guide RNA 3 nucleotides from the PAM. The Cas9 RuvC nuclease domain cuts the unpaired strand 3 to 6 nucleotides from the PAM. e) The most frequent product is a DNA double-strand break with short 5′ overhangs.

Figure 3

The repair of DNA double-strand breaks generated by Cas9 occurs by multiple pathways that can lead to correction, insertion and deletion. The break can be bridged by hybridization with a transduced single-stranded oligonucleotide (HDR, top left), the overhangs of the double strand break are removed by exonucleolytic digestion followed by DNA synthesis and ligation leading to correction if nucleotide substitutions (in red) are included in the oligonucleotide. Small insertions (NHEJ, top right) can occur if the 5′ overhangs are filled in by cellular DNA synthesis, followed by ligation. Small deletions (NHEJ, bottom left) can occur if the overhangs are digested and the DNA ends ligated. Large deletions (MMEJ, bottom right) can occur if microhomologous sequences on either side of the break are recognized, leading to pairing and ligation.

Figure 4

The mechanism of a C to T base editor (BE3 of ⁴⁸). The base editor, consisting of a protein fusion of Cas9, cytidine deaminase and uracil glycosylase inhibitor (UGI), binds to a specific DNA target as shown in Fig. 2. The RuvC-like nuclease domain of Cas9 is inactivated and thus only the DNA strand paired with the gRNA is cut. The cytidine deaminase is only able to reach the sixth nucleotide on the unpaired strand, and if this base is a cytidine it is deaminated to uracil. The uracil glycosylase inhibitor prevents cellular enzymes from removing the uracil, and the break on the opposite strand stimulates removal of the mis-paired G and thus the templated change to T.

Figure 5

The airway epithelium and stem cell hierarchy (modified from Montoro et al⁸⁰). The airway epithelium is a stratified epithelium with apical club, ciliated, goblet, tuft, ionocyte, pulmonary neuroendocrine cells (PNEC) as well as basal cells. The basal cells are stem cells which produce all apical cell types. Club cells are progenitor cells for goblet and ciliated cells. Under conditions of severe depopulation, club cells can dedifferentiate to basal stem cells (dotted arrow).