RETROVIRUS-MEDIATED GENE-TRANSFER AND EXPRESSION OF HUMAN ORNITHINE DELTA-AMINOTRANSFERASE INTO EMBRYONIC FIBROBLASTS

Ornithine delta aminotransferase (OAT) is a nuclear-encoded mitochondr ial matrix enzyme that catalyzes the reversible transamination of orni thine to glutamate semialdehyde. In humans, genetic deficiency of OAT results in gyrate atrophy of the choroid and retina, a blinding chorio retinal degeneration usually beginning in late childhood. This disorde r has been shown to be autosomal recessive, and is often caused by mis sense, 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 regulat ory elements derived from the MoMLV long terminal repeat (LTR). The co nstruct was transfected into the retroviral packaging cell lines GP E - 86 and psi CRIP to produce virus particles. Supernatant from these OAT retrovirus producer cell lines were used to transduce mouse C57Bl /6 embryonal fibroblasts. We showed that the recombinant retrovirus tr ansfers the OAT gene to the recipient cells, which produce an OAT RNA transcript when analyzed by Northern blot. Western blot analysis and e nzymatic assays confirmed the presence of an OAT polypeptide that has a high enzymatic activity in the transduced cell lines, even after a l ong period of time in vitro.