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

Abstract

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.

Maple Syrup Urine Disease in Cypriot Families: Identification of Three Novel Mutations and Biochemical Characterization of the p.Thr211Met Mutation in the E1α Subunit

“…We have shown that in the chaperonin-assisted E1 assembly pathway, the E1b subunit maintains its solubility through assembly with the E1a subunit as a heterodimeric intermediate, which subsequently dimerizes into an active heterotetramer (Chuang et al, 1999;Wynn et al, 2000). This thesis is corroborated by our previous studies that showed that transfection of the wild-type E1a subunit into the E1a-affected MSUD cells restores the assembly and presence of the normal E1b subunit in cultured cells from Mennonite MSUD patients (Fisher et al, 1991;Koyata et al, 1993). A total of five prenatal diagnoses were performed for this family (family I), and all five fetuses were found to be affected (four of the five prenatal diagnoses were performed both by DNA analysis and by measurement of the activity of the BCKD complex activity in chorionic villi).…”

Neonatal gene therapy achieves sustained disease rescue of maple syrup urine disease in mice

“…The lack of sufficient expression in other tissues, or the production of a non-functional and/or misfolded BCKD enzyme in hepatocytes due to overexpression may also play a role. The recombinantly expressed E1 protein has been shown to depend on molecular chaperones for proper folding and heterotetrameric assembly in cells 26 , 43 , 44 .…”

Production and characterization of murine models of classic and intermediate maple syrup urine disease

“…The most significant opportunity presented by the MSUD mouse models is to test novel treatments such as gene [ 38 - 41 ] and cell based therapies (e.g., hepatocytes [ 42 , 43 ] or embryonic stem cells [ 44 ]). Relevant to gene therapy, recent problems with gene therapy in humans highlight the importance and critical need of animal models of genetic diseases for preclinical studies.…”