TRANSPLANTATION AND GENE-TRANSFER OF THE HUMAN GLUCOCEREBROSIDASE GENE INTO IMMUNOSELECTED PRIMATE CD34(-1(+) CELLS()THY)

In an attempt to improve our gene transfer efficiency into hematopoiet ic stem cells and to evaluate the capacity of immunoselected CD34(+)Th y-1(+)(CDw90) cells to reconstitute hematopoiesis following myeloablat ion, bone marrow (BM) transplantation was performed using autologous, immunoselected CD34(+)Thy-1(+)cells in rhesus macaques. BM samples wer e positively selected for cells that express CD34, further subdivided using high gradient immunomagnetic selection for cells that express Th y-1, and transduced using a 7-day supernatant transduction protocol wi th a replication-defective retroviral vector that contained the human glucocerebrosidase (GC) gene. Circulating leukocytes were evaluated us ing a semiquantitative polymerase chain reaction (PCR) assay for the h uman GC gene, with the longest surviving animal evaluated at day 558. Provirus was detected at all time points in both CD20(+) B cells and C D2(+) 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 33 0, both the CD2(+) bright and dim cell populations and sorted CD4(+) a nd CD8(+) cells had detectable provirus. Vector-derived GC mRNA was de tected by reverse transcriptase (RT)-PCR analysis as far out as day 58 8. Thus, CD34(+)Thy-1(+) cells isolated using high gradient magnetic s eparation techniques can engraft, be transduced with a replication-def ective retroviral vector, and contribute to CD20(+) B lymphocytes, CD8 (+) T lymphocytes, and CD4(+) T lymphocytes; making them a suitable ce ll population to target for gene therapies involving lymphocytes. (C) 1996 by The American Society of Hematology.