Retroviral gene transfer of human adenosine deaminase in murine hematopoietic cells: effect of selectable marker sequences on long-term expression
| Autor: | Barry D. Luskey, JF Apperley, David A. Williams |
|---|---|
| Rok vydání: | 1991 |
| Předmět: | |
| Zdroj: | Blood. 78:310-317 |
| ISSN: | 1528-0020 0006-4971 |
| Popis: | Retroviral-mediated gene transfer of human adenosine deaminase (hADA) provides a model system for the development of somatic gene therapy as a therapy for diseases of bone marrow-derived cells. We have previously demonstrated that hADA can be observed in all hematopoietic lineages in a minority of mice transplanted with bone marrow cells infected with a simplified retroviral vector, ZipPGK-ADA. Here we report a majority of mice (six of eight) demonstrate expression of hADA in the peripheral blood at least 6 months after transplantation with bone marrow infected with this simplified retroviral vector, which contains no selectable marker. The failure to express hADA in two of eight mice was associated with the absence of the recombinant retroviral provirus in DNA prepared from bone marrow cells of these mice apparently due to failure to efficiently infect the reconstituting hematopoietic stem cell. In an effort to preselect bone marrow stem cells containing proviral integrations, we incorporated the selectable marker neo phosphotransferase (NEO) into a retroviral vector encoding hADA, N2/ZipPGK-ADATKNEO, and used G418 selection of infected bone marrow cells before transplantation. In contrast to the simplified retroviral vector, hADA expression in these recipients was short lived (less than 8 weeks), despite the continued presence of intact provirus in DNA prepared from bone marrow of these mice. To determine whether the preselection of bone marrow using G418 was responsible for the lack of sustained hADA expression, we repeated the infection with the N2/ZipPGK- ADATKNEO vector but omitted the G418 selection step. Again, the majority of recipient mice failed to express hADA long term, although the continued presence of provirus in DNA prepared from peripheral blood cell mononuclear cells was clearly demonstrated. Finally, we demonstrate clonal fluctuation of infected stem cells, and observe a temporal correlation between cessation of expression of hADA and the emergence of a dominant stem cell clone between 14 and 20 weeks posttransplantation in one recipient. These data suggest that inclusion of a second transcriptional unit that includes neo phosphotransferase sequences in this simplified vector is associated with decreased expression of the nonselectable ADA sequences. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|