Optimization of Fibronectin-Assisted Retroviral Gene Transfer into Human CD34⁺Hematopoietic Cells

Abstract

Efficient retroviral gene transfer into hematopoietic stem and progenitor cells can be achieved by co-localizing retrovirus and target cells on specific adhesion domains of recombinant fibronectin (FN) fragments. In this paper, we further optimize this technology for human CD34+ cells. Investigating the role of cytokine prestimulation in retrovirus-mediated gene transfer on plates coated with the recombinant FN CH-296 revealed that prestimulation of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) CD34+ cells was essential to achieve efficient gene transfer into clonogenic cells. The highest gene transfer occurred by prestimulating PB CD34+ cells for 40 hr with a combination of stem cell factor (SCF), G-CSF, and megakaryocyte growth and development factor (MGDF) prior to retroviral infection on CH-296. Surprisingly, a prolonged simultaneous exposure of primary CD34+ PB cells to retrovirus and cytokines in the presence of CH-296 lowered the gene transfer efficiency. Gene transfer into cytokine prestimulated CD34+ bone marrow (BM) cells was not influenced by increasing the coating concentrations of a recombinant FN fragment, CH-296, nor was it adversely influenced by increasing the number of CD34+ target cells, suggesting that the amount of retroviral particles present in the supernatant was not a limiting factor for transduction of CD34+ BM cells on CH-296-coated plates. The polycation Polybrene was not required for efficient transduction of hematopoietic cells in the presence of CH-296. Furthermore, we demonstrated that repeated exposure of CH-296 to retrovirus containing supernatant, called preloading, can be employed to concentrate the amount of retroviral particles bound to CH-296. These findings establish a simple and short clinically applicable transduction protocol that targets up to 68% of BM or G-CSF-mobilized PB CD34+ cells and is capable of genetically modifying up to 17% of CD34+CD38-/dim PB cells.

Oncogenic Vav1 induces Rac-dependent apoptosis via inhibition of Bcl-2 family proteins and collaborates with p53 deficiency to promote hematopoietic progenitor cell proliferation

“…LDBM cells were prestimulated for 2 days in IMDM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum Vav1 signaling in hematopoietic progenitor growth Y Gu et al (FCS, HyClone, Logan, UT, USA), 2% penicillin and streptomycin (P/S), and 100 ng/ml of recombinant rat SCF, megakaryocyte growth and development factor (MGDF) and granulocyte colony-stimulating factor (G-CSF) (all from Amgen, Thousand Oaks, CA, USA). Then, retrovirusmediated transduction of LDBM cells was performed by published methods (Hanenberg et al, 1997). At 2 days after transduction, cells were stained with a phycoerythrin (PE)-conjugated anti-mouse CD117 antibody (c-Kit, PharMingen, San Diego, CA, USA).…”

A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase

“…Recent preclinical work using specific combinations of cytokines 31 and the recombinant fibronectin CH-296 fragment 24 have improved transduction and engraftment to meaningful levels. Our prior experience with transfer of the MDR1 gene by retroviral gene transfer resulted in similar levels of gene transfer between infused and engrafted cell population, with 5-15% of the bone marrow progenitor cells containing vector 1 year after transplantation.…”

“…Second, the transduction protocol was modified to include preloading of the CH-296-coated plates as previous preclinical studies had suggested such vector 'preloading' improved transduction of CD34 þ cells with lower titer virus supernatant. 24 Interestingly, transduction efficiencies for the remaining study participants were not significantly increased but the modifications did appear to significantly increase the transduction of engrafting cells (discussed below).…”

“…After 48 h of prestimulation, up to 3 Â 10 7 cells were plated on non-tissue culture dishes treated with the fibronectin fragment CH-296 (Takara Shuzo, Japan). 23,24 Cells were exposed to the retroviral vector for 4 h, then collected and resuspended in fresh media with cytokines and incubated overnight. The transduction was repeated the next day and the cells were incubated overnight prior to cryopreservation.…”

“…Preloading was accomplished by adding retroviral supernatant to each CH-296-coated plate for 15 min four times prior to the transduction procedure. 24,25 Hematopoietic progenitor assays Isolated CD34 þ cells (5 Â 10 2 ) or MNC from peripheral blood samples or bone marrow samples (5 Â 10 4 ) were plated in 35 mm tissue culture dishes containing 1.1% methylcellulose (Stem Cell Technologies, Vancouver, Canada), 30% fetal bovine serum, 50 mM beta-mercaptoethanol, 50 ng/ml stem cell factor, 10 ng/ml GM-CSF, 10 ng/ml IL-3, and 3 U/ml erythropoietin. Cultures were incubated at 371C in 5% CO 2 in 100% humidity.…”