RESUMO
Premature termination codon read-through drugs offer opportunities for treatment of multiple rare genetic diseases including cystic fibrosis. We here analyzed the read-through efficacy of PTC124 and G418 using human cystic fibrosis intestinal organoids (E60X/4015delATTT, E60X/F508del, G542X/F508del, R1162X/F508del, W1282X/F508del and F508del/F508del). G418-mediated read-through induced only limited CFTR function, but functional restoration of CFTR by PTC124 could not be confirmed. These studies suggest that better read-through agents are needed for robust treatment of nonsense mutations in cystic fibrosis.
Assuntos
Códon sem Sentido/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Fibrose Cística/tratamento farmacológico , Gentamicinas/uso terapêutico , Organoides/citologia , Oxidiazóis/uso terapêutico , Células Cultivadas , Coccidiostáticos/uso terapêutico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , RNA/genéticaRESUMO
Treatment efficacies of drugs depend on patient-specific pharmacokinetic and pharmacodynamic properties. Here, we developed an assay to measure functional levels of the CFTR potentiator VX-770 in human plasma and observed that VX-770 in plasma from different donors induced variable CFTR function in intestinal organoids. This assay can help to understand variability in treatment response to CFTR potentiators by functionally modeling individual pharmacokinetics.
Assuntos
Aminofenóis/farmacocinética , Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Mucosa Intestinal , Intestinos , Organoides , Quinolonas/farmacocinética , Antimutagênicos/farmacocinética , Bioensaio , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Monitoramento de Medicamentos/métodos , Humanos , Mucosa Intestinal/metabolismo , Intestinos/patologia , Mutação/efeitos dos fármacos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Resultado do TratamentoRESUMO
BACKGROUND: Over the last decade novel monoclonal CFTR-specific antibodies have been developed. We here present a paired analysis to detect wild-type and mutant CFTR using Western blot analysis, flow cytometry and confocal microscopy in several cellular expression systems. METHODS: The following CFTR-specific antibodies were used; 217, 432, 450, 570, 769, 596, 660, L12B4 and 24.1. Mutant CFTR was detected in HEK293 cells transiently expressing the mutations; G542X, R1162X, F508del, N1303K, G551D, R117H, A455E. RESULTS: The majority of these antibodies are suitable for most applications tested. Using immunofluorescence, some antibodies can better detect mutant forms of CFTR (F508del and N1303K by mAbs 596 and 769), or display lower aspecific detection by Western blot analysis (mAbs 432, 450, 769 and 596) or immunofluorescence (mAbs 432, 450, 570 and 769). CONCLUSION: Optimal detection of CFTR by monoclonal antibodies depends on CFTR mutation and the specific research application.
