Abstract
Cystic fibrosis (CF) is caused by mutations in the gene encoding the transmembrane conductance regulator (CFTR) protein. Some CF patients are compound heterozygous or homozygous for nonsense mutations in the CFTR gene. This implies the presence in the transcript of premature termination codons (PTCs) responsible for a truncated CFTR protein and a more severe form of the disease. Aminoglycoside and PTC124 derivatives have been used for the read-through of PTCs to restore the full-length CFTR protein. However, in a precision medicine framework, the CRISPR/dCas13b-based molecular tool “REPAIRv2” (RNA Editing for Programmable A to I Replacement, version 2) could be a good alternative to restore the full-length CFTR protein. This RNA editing approach is based on the targeting of the deaminase domain of the hADAR2 enzyme fused to the dCas13b protein to a specific adenosine to be edited to inosine in the mutant mRNA. Targeting specificity is allowed by a guide RNA (gRNA) complementarily to the target region and recognized by the dCas13b protein. Here, we used the REPAIRv2 platform to edit the UGA PTC to UGG in different cell types, namely IB3-1 cells, HeLa, and FRT cells engineered to express H2BGFPopal and CFTRW1282X, respectively.
Highlights
Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP-gated anion channel, which plays a major role in regulating both secretion and absorption in many epithelial tissues [1]
Some small molecules, PTC124 derivatives that do not show the toxicity of aminoglycosides, have been suggested as a potential treatment for genetic disorders caused by nonsense mutations [8,9,10,11,12,13]
Encouraged by the results obtained with Fisher Rat Thyroid (FRT)-CFTRW1282X cells, we evaluated if the 2R.4E.PEAvIaRluva2tisoynstoefmCFcToRulRdebsceuaebFloelltooweidnigt EthdeiteinngdCogFeTnRoWu1s28C2XFTinRWIB12382-X1mHuutmatainonCeplrlsesent in IB3-1 airway epithelial cells that do not express any detectable amouEnntcsooufraegneddobgyenthoeurseCsuFlTtsRopbrtaoitneeind [w30it]h
Summary
Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP-gated anion channel, which plays a major role in regulating both secretion and absorption in many epithelial tissues [1]. 8% of CF patients worldwide and 13% in Europe were reported to be compound heterozygous or homozygous for a nonsense mutation and the percentage of these patients was remarkably high in Israel (Ashkenazi 45.5%), Italy (32.4%), and Slovenia (27.3%) [2] These patients could take advantage of a drug treatment promoting the translation read-through of the PTC to restore the CFTR full-length protein, at least to some extent. Novel approaches based on site-directed RNA editing (SDRE) to restore the full-length functional protein are worth to be investigated. The Feng Zhang lab pointed out the possibility of editing mRNA at the level of specific stop mutations before it undergoes translation This has been made possible by exploiting the deaminase domain (DD) of the human ADAR2 [25]. We edited the naturally occurring nonsense mutation in human IB3-1 airway epithelial cells (compound heterozygous F508del/W1282X) that do not express any detectable amounts of endogenous CFTR protein [30]
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