Stable correction of maple syrup urine disease in cells from a Mennonite patient by retroviral-mediated gene transfer
| Autor: | David T. Chuang, Rody P. Cox, H. Koyata |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Enzyme complex
Blotting Western Genetic Vectors Molecular Sequence Data Mutant Biology Biochemistry 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) law.invention Viral vector Maple Syrup Urine Disease Multienzyme Complexes law medicine Humans Lymphocytes RNA Messenger Molecular Biology G alpha subunit Base Sequence Maple syrup urine disease Genetic transfer Gene Transfer Techniques Ketone Oxidoreductases Genetic Therapy Cell Biology Blotting Northern medicine.disease Molecular biology Recombinant Proteins Blotting Southern Retroviridae Cell culture Recombinant DNA Research Article |
| Zdroj: | Biochemical Journal. 295:635-639 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj2950635 |
| Popis: | We have successfully used retroviral gene transfer to correct the deficiency of the branched-chain alpha-oxo acid dehydrogenase complex in lymphoblasts from a homozygous Mennonite maple syrup urine disease (MSUD) patient. The mutation in Mennonites is a Tyr-393 to Asn substitution in the branched-chain alpha-oxo acid decarboxylase (E1)alpha subunit of the enzyme complex. This promotes improper assembly of mutant E1 alpha with E1 beta subunits, leading to degradation of both polypeptides. For transduction studies, a full-length human E1 alpha CDNA was inserted into the retroviral vector LXSN to produce the recombinant LSN-E1 alpha. High-titre [6 x 10(5) colony-forming units/ml] amphotropic retroviral preparations free of helper viruses were obtained by co-cultivation of infected GP+E86 with PA317 cells. Transduction of MSUD lymphoblasts from the Mennonite patient with LSN-E1 alpha viruses restored the decarboxylation of alpha-oxo[1-14C]isovalerate to the normal level. The normal decarboxylation activity in transduced MSUD cells remained stable without G418 selection during the 14 weeks studied. Southern-blot analysis indicated that the recombinant E1 alpha cDNA was integrated into the host genome. Northern and Western blotting showed that both the normal E1 alpha mRNA and the subunit were properly expressed in transduced MSUD cells. However, the level of E1 beta subunits is lower than that of normal cells, suggesting competition of the recombinant E1 alpha with the mutant form for assembly with E1 beta. The results provide a paradigm for the development of somatic gene therapy for disorders involving mitochondrial multienzyme complexes. |
| Databáze: | OpenAIRE |
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