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Transplantation and gene transfer of the human glucocerebrosidase gene into
immunoselected primate CD34+Thy-1+ cells
RE Donahue, ER Byrne, TE Thomas, MR Kirby, BA Agricola, SE Sellers, G Gaudernack, S Karisson and PM Lansdorp
Hematology Branch, National Heart, Lung, and Blood Institute, Rockville, MD
20850, USA.
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.
Volume 88,
Issue 11,
pp. 4166-4172,
12/01/1996
Copyright © 1996 by The American Society of Hematology
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