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) remains a challenge in many situations. We evaluated a physical preselection strategy for isolating transduced cells after their infection with different vectors. The small (240-bp) cDNA coding region for the human CD24 cell-surface antigen was inserted into myeloproliferative sarcoma virus (MPSV) and murine stem cell virus (MSCV)-based retroviral vectors such that expression of CD24 was under the control of the viral long terminal repeat (LTR). After infection of (Ly-5.1) mouse bone marrow (BM), those expressing CD24 were isolated by fluorescence-activated cell sorting (FACS) and a transplant dose estimated to contain approximately 12 +/- 4 long-term competitive repopulating cells (CRU) injected into lethally irradiated congenic Ly-5.2 recipients. Six months later, virtually all recipients showed high-level (> 80%) reconstitution of their 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 recipients of cells obtained from the BM of the 6-month-old primary mice contained the provirus. However, only in recipients of MSCVCD24-infected marrow was a correspondingly high level of CD24 expression seen: a maximum of 88% for whole BM (all mice positive), 58% for peripheral blood leukocytes (all mice positive), and 21% for thymocytes (11 of 13 mice positive). CD24 was also readily detected on the regenerated Sca-1+Lin- 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 utility of preselection strategies and suggest an approach for the development of better vectors for achieving regulated, lineage-specific or stage-specific gene expression patterns in particular subsets of hematopoietic cells.
Pawliuk
R
R
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada.
Eaves
C J
CJ
Humphries
R K
RK
D000818:Animals; D015703:Antigens, CD / Q000235:genetics*; D001854:Bone Marrow Cells / Q000276:immunology*; D051927:CD24 Antigen; D019070:Cell Lineage / Q000235:genetics; D005260:Female; D005822:Genetic Vectors; D006410:Hematopoiesis / Q000235:genetics* / Q000276:immunology; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic
D015703:Antigens, CD; D051927:CD24 Antigen; C497889:CD24 protein, human; C497890:Cd24a protein, mouse; D008562:Membrane Glycoproteins