SUSTAINED HIGH-LEVEL RECONSTITUTION OF THE HEMATOPOIETIC SYSTEM BY PRESELECTED HEMATOPOIETIC-CELLS EXPRESSING A TRANSDUCED CELL-SURFACE ANTIGEN

Despite improvements in retrovirus-mediated gene transfer to primitive murine hematopoietic cells, high-level reconstitution with provirally marked cells with continued expression of the transferred gene(s) rem ains a challenge in many situations. We evaluated a physical preselect ion strategy for isolating transduced cells after their infection with different vectors. The small (240-bp) cDNA coding region for the huma n CD24 cell-surface antigen was inserted into myeloproliferative sarco ma virus (MPSV) and murine stem cell virus (MSCV)-based retroviral vec tors such that expression of CD24 was under the control of the viral l ong terminal repeat (LTR). After infection of (Ly-5.1) mouse bone marr ow (BM), those expressing CD24 were isolated by fluorescence-activated cell sorting (FACS) and a transplant dose estimated to contain simila r to 12 +/- 4 longterm competitive repopulating cells (CRU) injected i nto lethally irradiated congenic Ly-5.2 recipients. Six months later, virtually all recipients showed high-level (>80%) reconstitution of th eir BM and thymus with Ly-5.1 (transplant-derived) cells, the majority of which were also transduced (mean of 2.5 or 2.6 proviral copies for the two vectors). All spleen colonies generated in secondary recipien ts of cells obtained from the BM of the 6-month-old primary mice conta ined the provirus. However, only in recipients of MSCVCD24-infected ma rrow was a correspondingly high level of CD24 expression seen: a maxim um of 88% for whole BM (all mice positive), 58% for peripheral blood l eukocytes (all mice positive), and 21% for thymocytes (11 of 13 mice p ositive). CD24 was also readily detected on the regenerated Sca-1(+)Li n(-) cells present in the primary and secondary recipients when these were studied 6 months post-transplant, but again on more of the Sca-1( +)Lin(-) cells in recipients of MSCVCD24-infected cells as compared to recipients of MPSVCD24-infected cells. These results point to the uti lity of preselection strategies and suggest an approach for the develo pment of better vectors for achieving regulated, lineage-specific or s tage-specific gene expression patterns in particular subsets of hemato poietic cells.