title: 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.
authors.affiliation: Herman B Wells Center for Pediatric Research, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis 46202-5225, USA.
authors.forename: H
authors.initials: H
authors.lastname: Hanenberg
authors.forename: K
authors.initials: K
authors.lastname: Hashino
authors.forename: H
authors.initials: H
authors.lastname: Konishi
authors.forename: R A
authors.initials: RA
authors.lastname: Hock
authors.forename: I
authors.initials: I
authors.lastname: Kato
authors.forename: D A
authors.initials: DA
authors.lastname: Williams
mesh_terms: D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD; D000943:Antigens, Differentiation; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects; D002478:Cells, Cultured; D005353:Fibronectins / Q000235:genetics* / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006583:Hexadimethrine Bromide; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects; D008562:Membrane Glycoproteins; D009244:NAD+ Nucleosidase; D017463:Receptors, Complement 3b; D012190:Retroviridae; D019089:Stem Cell Factor / Q000494:pharmacology; D013997:Time Factors; D014170:Transformation, Genetic; D014771:Virion
chemical_list: D015703:Antigens, CD; D000943:Antigens, Differentiation; D005353:Fibronectins; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D017463:Receptors, Complement 3b; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor; D006583:Hexadimethrine Bromide; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1