All- trans Retinoic Acid Facilitates Oncoretrovirus-Mediated Transduction of Hematopoietic Repopulating Stem Cells

Abstract

A major limiting factor in achieving high levels of gene transfer into hematopoietic stem cells is the ability to retain significant repopulating activity of the stem cells during the ex vivo exposure to oncoretroviral vectors. Recently, we reported that pharmacological levels (1 μM) of all-trans retinoic acid (ATRA) enhanced the maintenance of in vivo repopulating hematopoietic stem cells during liquid suspension culture. Therefore, we investigated the use of ATRA to improve transduction of hematopoietic repopulating cells. Hematopoietic precursors cultured and transduced with a GFP-containing oncoretroviral vector with or without ATRA were transplanted immediately post-transduction (day 3 post-culture initiation) or following extended culture without further transduction (day 7 post-culture initiation). Mice transplanted with 3-day ATRA-treated cells had four-fold more donor cells than the untreated cells. In contrast, there were more GFP-expressing donor cells in recipients of cells cultured without ATRA (31.31 ± 8.47% no ATRA vs. 16.52 ± 9.35% ATRA). After 7 days of culture, however, the repopulating ability of the hematopoietic precursors was the same for both treatment groups, but the ATRA-treated cells had significantly more green fluorescence protein (GFP)-expressing donor cells (5.57 ± O.53% no ATRA vs. 13.67 ± 2.14% ATRA). Secondary recipients of marrow from recipients of the 3 day cultured cells had similar donor cell levels, but the percentage of GFP-expressing cells within the donor cell population was higher in the recipients of ATRA-treated cells (3.25± 0.70% no ATRA vs. 7.97 ± 2.71% ATRA). Our data show that the addition of ATRA to cultures of hematopoietic precursors resulted in increased gene transfer into murine hematopoietic repopulating cells. These data suggest that ATRA may be useful in clinical gene therapy protocols using oncoretroviral vectors.