Retrovirus-Mediated Gene Transfer and Expression of Human Ornithine δ-Aminotransferase into Embryonic Fibroblasts

Abstract

Ornithine δ aminotransferase (OAT) is a nuclear-encoded mitochondrial matrix enzyme that catalyzes the reversible transamination of ornithine to glutamate semialdehyde. In humans, genetic deficiency of OAT results in gyrate atrophy of the choroid and retina, a blinding chorioretinal degeneration usually beginning in late childhood. This disorder has been shown to be autosomal recessive, and is often caused by missense, nonsense, and/or frameshift mutations in the OAT gene.

With the view of applying gene therapy, a Moloney murine leukemia virus (MoMLV)-based recombinant retrovirus vector has been constructed. The human OAT cDNA was placed under the control of the enhancer–promoter regulatory elements derived from the MoMLV long terminal repeat (LTR). The construct was transfected into the retroviral packaging cell lines GP + E – 86 and ψCRIP to produce virus particles. Supernatant from these OAT retrovirus producer cell lines were used to transduce mouse C57B1/6 embryonal fibroblasts. We showed that the recombinant retrovirus transfers the OAT gene to the recipient cells, which produce an OAT RNA transcript when analyzed by Northern blot. Western blot analysis and enzymatic assays confirmed the presence of an OAT polypeptide that has a high enzymatic activity in the transduced cell lines, even after a long period of time in vitro.

Overview summary

In humans, a genetic deficiency of OAT results in gyrate atrophy of the coroid and retina. As a prelude to human gene therapy, we made a recombinant retrovirus vector bearing a human OAT cDNA. Supernatant from retrovirus producer cell lines were used to transduce cells in vitro. We showed that the retrovirus transfers the OAT to the recipient cells, which produce an OAT transcript and an immunoreactive OAT that was shown to be enzymatically active.