Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells

Helmut Hanenberg, Kimikazu Hashino, Haruko Konishi, Randy A. Hock, Ikunoshin Kato, David A. Williams

Research output: Contribution to journalArticle

168 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2193-2206
Number of pages14
JournalHuman Gene Therapy
Volume8
Issue number18
StatePublished - Dec 10 1997

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Fibronectins
Retroviridae
Blood Cells
Granulocyte Colony-Stimulating Factor
Genes
Cytokines
Hematopoietic Stem Cells
Bone Marrow Cells
Hexadimethrine Bromide
Thrombopoietin
Stem Cell Factor
Bone Marrow
Technology
Infection

ASJC Scopus subject areas

  • Genetics

Cite this

Hanenberg, H., Hashino, K., Konishi, H., Hock, R. A., Kato, I., & Williams, D. A. (1997). Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells. Human Gene Therapy, 8(18), 2193-2206.

Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells. / Hanenberg, Helmut; Hashino, Kimikazu; Konishi, Haruko; Hock, Randy A.; Kato, Ikunoshin; Williams, David A.

In: Human Gene Therapy, Vol. 8, No. 18, 10.12.1997, p. 2193-2206.

Research output: Contribution to journalArticle

Hanenberg, H, Hashino, K, Konishi, H, Hock, RA, Kato, I & Williams, DA 1997, 'Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells', Human Gene Therapy, vol. 8, no. 18, pp. 2193-2206.
Hanenberg H, Hashino K, Konishi H, Hock RA, Kato I, Williams DA. Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells. Human Gene Therapy. 1997 Dec 10;8(18):2193-2206.
Hanenberg, Helmut ; Hashino, Kimikazu ; Konishi, Haruko ; Hock, Randy A. ; Kato, Ikunoshin ; Williams, David A. / Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells. In: Human Gene Therapy. 1997 ; Vol. 8, No. 18. pp. 2193-2206.
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