A Novel G542X CFTR Rat Model of Cystic Fibrosis Is Sensitive to Nonsense Mediated Decay

Abstract

Nonsense mutations that lead to the insertion of a premature termination codon (PTC) in the cystic fibrosis transmembrane conductance regulator (CFTR) transcript affect 11% of patients with cystic fibrosis (CF) worldwide and are associated with severe disease phenotype. While CF rat models have contributed significantly to our understanding of CF disease pathogenesis, there are currently no rat models available for studying CF nonsense mutations. Here we created and characterized the first homozygous CF rat model that bears the CFTR G542X nonsense mutation in the endogenous locus using CRISPR/Cas9 gene editing. In addition to displaying severe CF manifestations and developmental defects such as reduced growth, abnormal tooth enamel, and intestinal obstruction, CFTR G542X knockin rats demonstrated an absence of CFTR function in tracheal and intestinal sections as assessed by nasal potential difference and transepithelial short-circuit current measurements. Reduced CFTR mRNA levels in the model further suggested sensitivity to nonsense-mediated decay, a pathway elicited by the presence of PTCs that degrades the PTC-bearing transcripts and thus further diminishes the level of CFTR protein. Although functional restoration of CFTR was observed in G542X rat tracheal epithelial cells in response to single readthrough agent therapy, therapeutic efficacy was not observed in G542X knockin rats in vivo. The G542X rat model provides an invaluable tool for the identification and in vivo validation of potential therapies for CFTR nonsense mutations.

Sheep models of F508del and G542X cystic fibrosis mutations show cellular responses to human therapeutics

“…Also, the CFTR-null models do not represent the same molecular mechanisms seen in patients with the most common mutations, F508del and G542X, as the random disruption of the gene may lead to unpredictable effects on protein synthesis. Currently, a few F508del and G542X CF animal models have been established, including pig 35 , rat 36,37 , and mouse 38 for the F508del mutation, and mouse 39 and rat 40 for the G542X mutation. The F508del mutants have similar abnormalities as seen in their respective CFTR-null models of the same species, except with less severe damage in some organs due to residual CFTR function 35,36 (Reviewed in 41 ).…”

“…CF pig models, on the other hand, require a minimum of four offspring to maintain the pregnancy 43 , and normally have 6-10 offspring per pregnancy. G542X CF mice and rats were generated recently 39,40 and exhibit a similar phenotype to their CFTR-null models with no residual CFTR functionality. However, organoids isolated from the intestine of G542X CF mice and epithelial cells isolated from the trachea of G542X CF rats indicated that CFTR protein synthesis could be rescued by G418 antibiotic treatment, thus, partially restoring the CFTR channel.…”

“…CF pig models, on the other hand, require a minimum of four offsprings to maintain the pregnancy, 43 and normally have 6-10 offspring per pregnancy. G542X CF mice and rats were generated recently 39,40 and exhibit a similar phenotype to their CFTR-null Effect of G418 and SMG1-i on G542 cells. Cells were pretreated with SMG1-i (1 μM) plus G418 (50 μM) for 24 h with or without VX-661 (3.3 μM), followed by acute treatment as described above models with no residual CFTR functionality.…”

Recoding of Nonsense Mutation as a Pharmacological Strategy

“…Additionally, gene therapies may be a useful treatment option for CF patients carrying nonsense mutations, and the G542X rat model could serve as an important tool for future treatment testing. Multi-agent therapy to augment readthrough could be an alternative approach [ 89 ].…”

Gene therapy for cystic fibrosis: new tools for precision medicine

“…Nevertheless, rats possess a 76% amino acid sequence identity to h CFTR [ 99 ] and have submucosal glands in the large airways [ 97 , 100 ]. Rat models have also provided the groundwork for exploring new genetic advancements in CF modelling, like the generation of the first G542X CF nonsense mutation rat model with CRISPR-Cas9 [ 101 ], and a new F508del rat models that may be invaluable in the development of therapeutics [ 97 ].…”