Developing a clinically applicable protocol for gene transfer using retroviral infection on fibronectin

H. Hanenberg, C. Ding, K. Hashing, H. Konishi, K. Asada, I. Kalo, M. C. Dinauer, P. A. Williams

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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 fibronectin fragments (FNF) (Hanenberg et al. Nat. Med. 2, 1996: 876). We further optimized this technology to target primary human clonogenic CD34+ cells obtained from bone marrow (BM) or from peripheral blood (PB) after mobilization with G-CSF. CD34+ BM ceils were prestimulated overnight with SCF and IL6. The next day, plates were coated with increasing concentrations (2, 8, 20, 50, 100 ug/cm2) of the FNF CH296 to which cells suspended in SN with an amphotropic NEO RV were added. GT efficiency assessed 12-16 days later as the number of G418' colonies did not increase with coating concentrations above 8 ug/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 on CH-296. Next, 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 and then infected for two 4h transduction periods with NEO RV. The highest gene transfer efficiency occurred with a 40h prestimulation with SCF, G-CSF and MGDF (Artigen) achieving gene delivery of up to 69% of the clonogenic BM and PB CD34+ cells. Finally, we tested the following transduction protocols using SCF+IL6 or SCF+G-CSF+MGDF: A) no prestimulation, 40h RV+FN; B) 24h prestimulation, 24h RV+FN: C) 40h prestimulation, 8h RV+FN. Surprisingly, a prolonged incubation with RV+FN lowered the GT efficiency into clonogenic cells significantly (A vs. C). Preliminary data showed that a protocol using 40h prestimulation with 8h RV+FN can transduce NOD/SCID repopulating human PB CD34+ cells. These data demonstrate that FN-mediated genetic transduction of primary CD34+ cells may be well suited for genetic modifications of hematopoietic cells with retroviral vectors in clinically applicable protocols.

Original languageEnglish
Number of pages1
JournalExperimental Hematology
Issue number8
StatePublished - Dec 1 1997


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