Sustained High-Level Reconstitution of the Hematopoietic System by Preselected Hematopoietic Cells Expressing a Transduced Cell-Surface Antigen

Abstract

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 ∼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.

Overview summary

We have utilized a cDNA encoding the human CD24 cell-surface antigen as a dominant selectable marker in several retroviral vectors to enable the regeneration of the hematopoietic systems of myeloablated recipient mice to very high levels with provirally marked cells. Southern blot analysis of primary and secondary recipients transplanted with retrovirally transduced, CD24⁺-selected day 4 5-fluorouracil (5-FU) murine bone marrow (BM) cells at 6 months post transplant demonstrated that hematopoiesis was regenerated almost exclusively with provirally marked cells in the vast majority of animals. The transferred CD24 gene was expressed on a significant proportion of regenerated Sca-1⁺Lin¯ bone marrow, peripheral blood leukocytes, red blood cells, spleen, thymus, and whole bone marrow cells for a minimum of 6 months post transplant. Striking vector related differences in the proportion of CD24⁺ cells was observed between recipients of murine stem cell virus (MSCV) versus myeloproliferative sarcoma virus (MPSV) vector-transduced bone marrow.