Robert E. Donahue1,2,3,4, Ellen R. Byrne1,2,3,4, Terry E. Thomas1,2,3,4, Martha R. Kirby1,2,3,4, Brian A. Agricola1,2,3,4, Stephanie E. Sellers1,2,3,4, Gustav Gaudernack1,2,3,4, Stefan Karlsson1,2,3,4, Peter M. Lansdorp1,2,3,4
1Hematology Branch, National Heart, Lung, and Blood Institute, Rockville, MD
2Terry Fox Laboratory, Vancouver, BC, Canada
3The Norwegian Radium Hospital, Oslo, Norway
4Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders and Stroke, Bethesda, MD
Tóm tắt
Abstract
In an attempt to improve our gene transfer efficiency into hematopoietic stem cells and to evaluate the capacity of immunoselected CD34+Thy-1+(CDw90) cells to reconstitute hematopoiesis following myeloablation, bone marrow (BM) transplantation was performed using autologous, immunoselected CD34+Thy-1+ cells in rhesus macaques. BM samples were positively selected for cells that express CD34, further subdivided using high gradient immunomagnetic selection for cells that express Thy-1, and transduced using a 7-day supernatant transduction protocol with a replication-defective retroviral vector that contained the human glucocerebrosidase (GC) gene. Circulating leukocytes were evaluated using a semiquantitative polymerase chain reaction (PCR) assay for the human GC gene, with the longest surviving animal evaluated at day 558. Provirus was detected at all time points in both CD20+ B cells and CD2+ dim T cells, but long-term gene transfer was not observed in the granulocyte population. The CD2+ dim population was phenotypically identified as being CD8+ natural killer cells. By day 302 and day 330, both the CD2+ bright and dim cell populations and sorted CD4+ and CD8+ cells had detectable provirus. Vector-derived GC mRNA was detected by reverse transcriptase (RT)-PCR analysis as far out as day 588. Thus, CD34+Thy-1+ cells isolated using high gradient magnetic separation techniques can engraft, be transduced with a replication-defective retroviral vector, and contribute to CD20+ B lymphocytes, CD8+ T lymphocytes, and CD4+ T lymphocytes; making them a suitable cell population to target for gene therapies involving lymphocytes.
