Evaluation of Aminoglycoside and Non-Aminoglycoside Compounds for Stop-Codon Readthrough Therapy in Four Lysosomal Storage Diseases.
Nonsense mutations are quite prevalent in inherited diseases. Readthrough drugs could provide a therapeutic option for any disease caused by this type of mutation. Geneticin (G418) and gentamicin were among the first to be described. Novel compounds have been generated, but only a few have shown improved results. PTC124 is the only compound to have reached clinical trials. Here we first investigated the readthrough effects of gentamicin on fibroblasts from one patient with Sanfilippo B, one with Sanfilippo C, and one with Maroteaux-Lamy. We found that ARSB activity (Maroteaux-Lamy case) resulted in an increase of 2-3 folds and that the amount of this enzyme within the lysosomes was also increased, after treatment. Since the other two cases (Sanfilippo B and Sanfilippo C) did not respond to gentamicin, the treatments were extended with the use of geneticin and five non-aminoglycoside (PTC124, RTC13, RTC14, BZ6 and BZ16) readthrough compounds (RTCs). No recovery was observed at the enzyme activity level. However, mRNA recovery was observed in both cases, nearly a two-fold increase for Sanfilippo B fibroblasts with G418 and around 1.5 fold increase for Sanfilippo C cells with RTC14 and PTC124. Afterwards, some of the products were assessed through in vitro analyses for seven mutations in genes responsible for those diseases and, also, for Niemann-Pick A/B. Using the coupled transcription/translation system (TNT), the best results were obtained for SMPD1 mutations with G418, reaching a 35% recovery at 0.25 μg/ml, for the p.W168X mutation. The use of COS cells transfected with mutant cDNAs gave positive results for most of the mutations with some of the drugs, although to a different extent. The higher enzyme activity recovery, of around two-fold increase, was found for gentamicin on the ARSB p.W146X mutation. Our results are promising and consistent with those of other groups. Further studies of novel compounds are necessary to find those with more consistent efficacy and fewer toxic effects.
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, IBUB, CIBERER, Barcelona, Spain.
Marta
M
Gómez-Grau
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, IBUB, CIBERER, Barcelona, Spain.
Elena
E
Garrido
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, IBUB, CIBERER, Barcelona, Spain.
Mónica
M
Cozar
CIBBIM-Nanomedicina, Vall d'Hebron Institut de Recerca (VHIR), CIBERER, Hospital Universitari Vall d'Hebron, Barcelona, Spain.
Víctor
V
Rodriguez-Sureda
CIBBIM-Nanomedicina, Vall d'Hebron Institut de Recerca (VHIR), CIBERER, Hospital Universitari Vall d'Hebron, Barcelona, Spain.
Carmen
C
Domínguez
Departament d'Estadística, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.
Concepción
C
Arenas
David Geffen/UCLA School of Medicine, Departments of Pathology & Laboratory Medicine, and Human Genetics, Los Angeles, United States of America.
Richard A
RA
Gatti
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, IBUB, CIBERER, Barcelona, Spain.
Bru
B
Cormand
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, IBUB, CIBERER, Barcelona, Spain.
Daniel
D
Grinberg
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, IBUB, CIBERER, Barcelona, Spain.
Lluïsa
L
Vilageliu
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D018389:Codon, Nonsense / Q000187:drug effects / Q000235:genetics; D018388:Codon, Terminator / Q000187:drug effects / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology; D005839:Gentamicins / Q000627:therapeutic use*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D009084:Mucopolysaccharidosis III / Q000188:drug therapy / Q000235:genetics*; D009087:Mucopolysaccharidosis VI / Q000188:drug therapy / Q000235:genetics*; D052536:Niemann-Pick Disease, Type A / Q000188:drug therapy / Q000235:genetics; D052537:Niemann-Pick Disease, Type B / Q000188:drug therapy / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics
D018389:Codon, Nonsense; D018388:Codon, Terminator; D005839:Gentamicins; D012333:RNA, Messenger; C010680:antibiotic G 418