Stable correction of maple syrup urine disease in cells from a Mennonite patient by retroviral-mediated gene transfer

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

We have successfully used retroviral gene transfer to correct the deficiency of the branched-chain α-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 α-coxo acid decarboxylase (E1)α subunit of the enzyme complex. This promotes improper assembly of mutant E1α with E1β subunits, leading to degradation of both polypeptides. For transduction studies, a full-length human E1α cDNA was inserted into the retroviral vector LXSN to produce the recombinant LSN-E1α. High-titre [6 x 105 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-EIα viruses restored the decarboxylation of α-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 E1α cDNA was integrated into the host genome. Northern and Western blotting showed that both the normal E1α mRNA and the subunit were properly expressed in transduced MSUD cells. However, the level of E1β subunits is lower than that of normal cells, suggesting competition of the recombinant E1α with the mutant form for assembly with E1β. The results provide a paradigm for the development of somatic gene therapy for disorders involving mitochondrial multienzyme complexes.

Original languageEnglish (US)
Pages (from-to)635-639
Number of pages5
JournalBiochemical Journal
Volume295
Issue number3
DOIs
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Stable correction of maple syrup urine disease in cells from a Mennonite patient by retroviral-mediated gene transfer'. Together they form a unique fingerprint.

  • Cite this