Gene and Base Editing as a Therapeutic Option for Cystic Fibrosis-Learning from Other Diseases.

Patrick T. Harrison,Lúcia Santos,Karen Mention

doi:10.3390/genes10050387

Abstract

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the CFTR gene. There are at least 346 disease-causing variants in the CFTR gene, but effective small-molecule therapies exist for only ~10% of them. One option to treat all mutations is CFTR cDNA-based therapy, but clinical trials to date have only been able to stabilise rather than improve lung function disease in patients. While cDNA-based therapy is already a clinical reality for a number of diseases, some animal studies have clearly established that precision genome editing can be significantly more effective than cDNA addition. These observations have led to a number of gene-editing clinical trials for a small number of such genetic disorders. To date, gene-editing strategies to correct CFTR mutations have been conducted exclusively in cell models, with no in vivo gene-editing studies yet described. Here, we highlight some of the key breakthroughs in in vivo and ex vivo gene and base editing in animal models for other diseases and discuss what might be learned from these studies in the development of editing strategies that may be applied to cystic fibrosis as a potential therapeutic approach. There are many hurdles that need to be overcome, including the in vivo delivery of editing machinery or successful engraftment of ex vivo-edited cells, as well as minimising potential off-target effects. However, a successful proof-of-concept study for gene or base editing in one or more of the available CF animal models could pave the way towards a long-term therapeutic strategy for this disease.

Highlights

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the CysticFibrosis Transmembrane Conductance Regulator (CFTR) gene which encodes a chloride/bicarbonate channel expressed on the apical surface of secretory cells
After more than 20 clinical trials, the only clinical benefit reported so far has been a stabilisation of lung function in patients who received at least nine doses of CFTR cDNA delivered by cationic liposomes over a 12-month period [4]
Faced with the challenge of developing multiple zinc finger nucleases (ZFNs)/donor combinations for each disease-causing mutation in the human F9 gene, Kathy High and colleagues [6] used ZFN-homology-directed repair (HDR) with a novel form of donor widely known as a superexon, or partial cDNA. They created a donor comprising exons 2 to 8, flanked by a splice acceptor and a polyA site, and containing homology arms of ~800 bp either side of the construct. They designed a pair of high-specificity ZFNs to introduce a targeted double-stranded break (DSB) in the first intron of the F9 gene to drive the precise and stable integration of the SA-superexon-polyA donor into the genome, which would in turn yield the production of a full-length and functional mRNA

Summary

Introduction

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the Cystic. After more than 20 clinical trials, the only clinical benefit reported so far has been a stabilisation of lung function in patients who received at least nine doses of CFTR cDNA delivered by cationic liposomes over a 12-month period [4] Both in vivo and ex vivo gene therapy approaches have been very successful in a wide range of other diseases over the same time frame [5]. It took a key proof-of-concept experiment from Maria Jasin that a targeted double-stranded break (DSB) could substantially increase editing efficiency [10], and the development of a new set of synthetic reagents called zinc finger nucleases (ZFNs) [11] to lay the foundations for therapeutic editing It would take another decade before fully programmable ZFNs were used to correct a disease-causing mutation in human cells [12]. The impact of gene editing on the study of cystic fibrosis [19], other gene-based approaches to treating CF [20,21] and the ethics of somatic cell editing [22] are discussed elsewhere

In Vivo Editing—From Mouse to Man in Six Years

Ex Vivo Approaches for Gene Editing in Animal Models

DNA Base Editing

Cytosine Base Editors

Adenine Base Editors

DNA Base Editing—Proof-of-Concept Disease Models

Adenine Base Editing of a Dmd Mouse Model

Treatment of a Metabolic Disease by Cytosine Base Editing

Adenine Base Editing of a Mouse Model of Hereditary Tyrosinemia I

Cytosine Base Editing of PCSK9 as a Permanent Therapeutic Approach

Findings

Base Editing as a Therapeutic Option for CF