We have previously demonstrated that efficient retroviral gene transfer (GT) into hematopoietic stem and progenitor cells can be achieved with retrovirus (RV) containing supernatant (SN) by colocalizing RV and target cells on specific adhesion domains of recombinant fragments of the extracellular matrix molecule fibronectin (FN) (Hanenberg et al. Nat. Med. 2, 1996: 876). We applied this technology to target primary human hematopietic cells. First, we analyzed gene transfer into T cells which constitutively express the FN receptors VLA-4 and VLA-5. Human peripheral blood (PB)-derived mononuclear cells (MNCs) were prestimulated for 2-3 days with CD3 and IL2 and then infected for 2 days with an amphotropic RV containing the murine ADA cDNA on the FN fragment CH-296. CH-296 contains the binding sites for VLA-4 (=CS1) and for VLA-5 (=CBD). After 4 days, cell cultures contained >80% T cells, the majority of which were strongly activated as indicated by their HLA-DR expression. Analysis of GT efficiency by ADA isoenzyme assay showed the activity of the murine ADA protein equal to the endogenous human ADA protein in the unselected population. Next, we analyzed gene transfer into human clonogenic CD34+ cells obtained from bone marrow (BM) or from PB after mobilization with G-CSF. The integrin receptors VLA-4 and VLA-5 are both expressed on hematopoietic precursor cells and are thought to be involved in homing of these cells in the BM microenviroment. CD34+ BM cells were prestimulated overnight with SCF and IL6. The next day, plates were coated with increasing concentrations of CH-296 and cells resuspendend in SN containing an amphotropic NEO RV were added. GT efficiency assessed 12-16 days later as the number of G418r colonies did not increase significantly with coating concentrations above 8 μg/cm2. Increasing the cell numbers from 500 to 625,000 per cm2 revealed that the GT efficiency was not influenced by the MOI over a range of 3 logs suggesting that the amount of RV particles present is not a limiting factor for transduction of CD34+ cells. We then investigated the role of cytokine prestimulation in the transduction protocol. To this end, CD34+ PB cells were prestimulated with various cytokine combinations for 1 or 2 days or directly put in the presence or absence of cytokines on CH-296 coated plates with NEO34 RV. GT was most efficient when using a 40h prestimulation period followed by two 4 hour transduction periods. The optimal cytokine mixture was the combination of SCF, G-CSF and c-mpl ligand achieving gene delivery of up to 70 % of the clonogenic CD34+ cells. These data demonstrate that genetic transduction of primary cells on CH-296 may be well suited for genetic modifications of hematopoietic cells with retroviral vectors in clinically applicable protocols.
|Original language||English (US)|
|Number of pages||2|
|Journal||Medical and Pediatric Oncology|
|State||Published - Dec 1 1998|
ASJC Scopus subject areas
- Pediatrics, Perinatology, and Child Health
- Cancer Research