Hematopoietic cells are important targets for genetic modification with retroviral vectors. Attempts at human gene therapy of stem cells have achieved limited success partly because of low gene transfer efficiency. Chymotryptic fragments of the extracellular matrix molecule fibronectin used during infection have been shown to increase transduction of human hematopoietic progenitor cells. Here, we demonstrate that this enhanced gene transfer into mammalian target cells is due to direct binding of retroviral particles to sequences within the fibronectin molecule. Transduction of mammalian cells, including murine long-term repopulating hematopoietic cells, is greatly enhanced when cells are adherent to chimeric fragments containing these retroviral binding sequences. In addition, colocalization of retrovirus and target cells on fibronectin peptides allows targeted transduction of specific cell types by exploiting unique ligand/receptor interactions.

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.

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The new europium(III) chelate [2,2',2'',2'''-[[4'-(aminobiphenyl-4-yl)-2,2':6',2''-terpyridine- 6,6''-diyl]bis(methylenenitrilo)]tetrakis(acetato)] europium(III) (ATBTA-Eu3+) and its 4,6-dichloro-1,3,5-triazinyl and succinimidyl derivatives (DTBTA and NHS-ATBTA, respectively) were synthesized and characterized. Both labeling complexes DTBTA-Eu3+ and NHS-ATBTA-Eu3+ are luminescent. Especially DTBTA-Eu3+ is strongly luminescent, with a luminescence quantum yield of 9.1%, molar extinction coefficient of 3.1 x 10(4) cm(-1) M(-1) (335 nm), and luminescence lifetime of 1.02 ms. The excitation and emission maximum wavelengths of DTBTA-Eu3+ are 335 and 616 nm, respectively. The complex is very stable in aqueous buffers, with a conditional formation constant log K(DTBTA-Eu) of 25.0 at pH 8, and can be conjugated to DNA and proteins. The chelates are also highly resistant to thermal decomposition, photodegradation, and ozone oxidation. These properties prove that DTBTA-Eu3+ is suitable as a luminescence label in DNA assays.