Gene induction for the treatment of methylmalonic aciduria.
BACKGROUND
Methylmalonic aciduria is an autosomal recessive inborn error of the propionate metabolic pathway. One form of this disorder is caused by mutations in methylmalonyl-coenzyme A mutase (MCM), resulting in reduced levels of enzyme activity. The pharmacological up-regulation of residual mutase activity is one approach to advance treatment strategies for individuals affected by this disorder. We describe the construction, characterization and use of a cellular genomic reporter assay for MCM expression that will potentially identify therapeutic pharmacological agents for methylmalonic aciduria treatment.


METHODS
Homologous recombination was used to insert an enhanced green fluorescent protein (EGFP) cassette inframe before the last codon of exon 13 of the MCM gene (MUT) in a BAC clone. The construct was used to generate stable HeLa cell lines. EGFP expression was measured by flow cytometry and the real-time reverse transcriptase-polymerase chain reaction was used to quantify changes in MUT gene mRNA levels.


RESULTS
The genomic reporter assay used to screen a selection of compounds. Cisplatin, zidovudine and adefovir were found to increase the levels of MCM mRNA and EGFP expression, providing support for the possible efficacy of these pharmacological compounds in treating methylmalonic aciduria.


CONCLUSIONS
This assay has the potential of being used in high-throughput screening of chemical libraries for the identification of novel compounds that specifically modulate the expression of MCM.
Hu
Ruimei
R
Cell and Gene Therapy Group, Murdoch Childrens Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Victoria, Australia.
Buck
Nicole E
NE
Khaniani
Mahmoud S
MS
Wood
Leonie
L
Wardan
Hady
H
Benoist
Jean-Francois
JF
Li
Lingli
L
Vadolas
Jim
J
Sarsero
Joseph P
JP
Ioannou
Panos A
PA
Peters
Heidi L
HL
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D002945:Cisplatin / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008661:Metabolism, Inborn Errors / Q000235:genetics / Q000628:therapy*; D008765:Methylmalonyl-CoA Mutase / Q000172:deficiency* / Q000235:genetics*; D063065:Organophosphonates / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D015215:Zidovudine / Q000494:pharmacology
D063065:Organophosphonates; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D015215:Zidovudine; C053001:adefovir; D008765:Methylmalonyl-CoA Mutase; D000225:Adenine; D002945:Cisplatin