Abstract
Genetically engineered retroviral vectors can be used to transfer genes into eucaryotic cells. The application of these vectors for human gene therapy is being evaluated in vitro and in animal models. The time course of vector mediated gene transfer into the human erythroleukemia cell line, K562, was followed by in situhybridization (ISH) to determine if this technique could be used to follow retroviral mediated gene transfer. The initial binding of vector to K562 cells was readily detected. Little vector RNA was detected during particle uptake, conversion of the vector RNA to dsDNA, and integration of the dsDNA into the host genome. However, by 18h after vector binding cytoplasmic RNA from the transferred gene was readily detected with steady state levels reached by 48h, demonstrating the ability of ISH to follow vector mediated gene transfer. In an autologous monkey bone marrow transplant model attempts to transfer a human adenosine deaminase (hADA) gene has resulted in hADA activity 0.5% that of endogenous monkey ADA. To determine if this represents a low level of activity in a large number of cells or the converse, ISH was used to show that 6 of 673 peripheral blood mononuclear cells contained retroviral RNA, implying an appropriate level of hADA gene expression in those cells infected by the vector. Similar percentages of gene transfer are seen in in vitro studies with primate and human bone marrow colony forming assays. The ability of ISH to detect expression of transferred genes in single cells provides a powerful technique for the refinement of methods for human gene therapy.
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Moen, R., Blaese, R. & Anderson, W. EVALUATION OF RETROVIRAL MEDIATED GENE TRANSFER BY IN SITU HYBRIDIZATION. Pediatr Res 21 (Suppl 4), 292 (1987). https://doi.org/10.1203/00006450-198704010-00752
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DOI: https://doi.org/10.1203/00006450-198704010-00752