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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.

ABSTRACTPrimary cultures of airway epithelia were used to evaluate variables pertinent to adenovirus (Ad)-mediated gene transfer efficiency and efficacy including: (i) Ad-vectors with different promoters, (ii) the duration of vector incubation with cells, (iii) the concentration and depth of vector-containing medium at constant multiplicity of infection (moi) (103), and (iv) the relative sensitivity of reverse transcription polymerase chain reaction (RT-PCR) versusfunctional analysis for the detection of transduced cystic fibrosis transmembrane conductance regulator (CFTR). An Ad5-lacZvector with a cytomegalovirus (CMV) enhancer/promoter transduced the greatest amount of ββ-galactosidase (ββ-Gal) activity, while an Ad2-lacZvector with an E1a enhancer/promoter transduced the least. Ad5-lacZvectors with the Rous sarcoma virus (RSV), E1a/RSV, or CMV enhancer/ββ-actin (CB) promoters transduced intermediate levels of ββ-Gal. Optimal gene transfer efficiency was detected with a 4––8 hr incubation of Ad5-CMVlacZwith cells, although optimal CFTR Cl¯¯transport function was detectable after only a 30 min incubation of Ad5-CBCFTRwith cells, consistent with correction of ≥≥6––10% of cells in the epithelial sheet. Ad5-CBCFTRtransduction of CF airway epithelial cells (moi = 103) was optimal when higher concentrations, lower volumes, or smaller depths of vector-containing medium were utilized. RT-PCR was at least 100-fold more sensitive for the detection of transduced CFTR than functional analysis, and could detect as few as 0.001 % Ad5-CBCFTR-infected CF cells admixed with uninfected CF cells. In summary, the variables studied clearly affect the efficiency of Ad-mediated gene transfer in vitroand potentially in vivo. They also suggest that RT-PCR is a poor marker of gene transfer efficiency and efficacy.