Retrovirus-Mediated Gene Transfer of Ornithine-δ-Aminotransferase into Keratinocytes from Gyrate Atrophy Patients

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Abstract
Gyrate atrophy is a progressive blindness associated with deficiency of ornithine aminotransferase (OAT). The strategy of using an autologous keratinocyte graft, modified to express high levels of OAT as an ornithine-catabolizing skin-based enzyme sink, is investigated. Two OAT-containing retroviral vectors were constructed with or without a resistance gene. When packaged in a retroviral vector particle generated with the gibbon ape leukemia (GALV) virus envelope (PG13), these vectors could readily transduce >50% of target keratinocytes. The transduced keratinocytes in culture expressed up to 75-fold more OAT than normal control keratinocytes and these gene-modified cells extracted [14C]ornithine more efficiently than controls. The vector prepared without neo transduced cells more efficiently and led to higher levels of OAT expression than the neo-containing vector. Ornithine catabolism was maintained at high levels when the transduced patient keratinocytes were differentiated in vitro as a multilayered cutaneous organoid. Jensen et al. report the construction and analysis of retroviral vectors for the overexpression of OAT in keratinocytes from gyrate atrophy patients. Immunofluorescence of target patient keratinocytes was used to identify a highly efficient OAT retrovirus lacking a selectable marker. Patient keratinocytes transduced with this vector contain large amounts of enzymatically active OAT and display significantly higher levels of ornithine catabolism than observed in normal keratinocytes. The potential of using transduced keratinocytes to treat metabolic disorders including OAT deficiency is discussed.