Highly‐efficient gene transfer with retroviral vectors into human T lymphocytes on fibronectin

Abstract

Summary. Genetically modified lymphocytes have been successfully used for correction of ADA deficiency in children and in controlling graft-versus-host disease (GvHD) after allogeneic bone marrow transplantation. Low transduction efficiencies are, however, limiting for gene therapeutic strategies based on lymphocytes. In this study we compared protocols for highly efficient gene transfer into human T cells using retroviral vector-containing supernatant. We showed that infection of both human primary T cells and CD4 þ Jurkat cells is most efficient on the matrix component fibronectin. Transduction was carried out with a retroviral vector encoding both the human intracytoplasmatically truncated low-affinity nerve growth factor receptor (DLNGFR) as a gene transfer marker and the Herpes simplex virus thymidine kinase for negative selection. Based on LNGFR expression genetically modified cells were enriched to near purity by magnetic cell sorting (MACS). Enriched cells could be shown to be highly sensitive to ganciclovir.

Lentivirus-mediated gene transfer in primary T cells is enhanced by a central DNA flap

“…[12][13][14] Transduction efficiencies with the VSV-Gpseudotyped TRIP lentiviral vector were only augmented by 10-20% in the presence of fibronectin. However, it is important to note that this increase was consistent and was observed at all tested concentrations of virion particles ( Figure 2).…”

“…9,11 Finally, co-localization of retrovirus and target cells on specific adhesion domains of a recombinant fibronectin molecule has resulted in augmented transduction of T cells. [12][13][14] Despite these advances, a significant barrier to the use of MuLV-based vectors for T cell gene therapy remains the quiescent state of the majority of circulating T cells. The integration of oncoretroviruses such as MuLV requires mitosis, due to an inability to translocate the viral genome across an intact nuclear membrane.…”

Establishment of an optimised gene transfer protocol for human primary T lymphocytes according to clinical requirements

“…However, using standard supernatant infections with amphotropic vectors only low transduction efficiencies have been achieved. 1,2,5,6 In a bid to overcome this problem, different pseudotypes 7,8 and infection protocols [5][6][7][8][9][10] for human T cells have been proposed. However, several shortcomings may hinder the clinical application of these protocols.…”

“…Cells were infected at high densities (1 × 10 6 /ml) with retroviral vectors encoding as a gene transfer marker a truncated human low-affinity nerve growth factor receptor (⌬LNGFR) lacking the intracytoplasmatic domain. 6,12 The use of surface proteins as gene transfer markers allows rapid and convenient determination of gene transfer efficiencies by flow cytometry. 13 The vectors were produced by a PG13 producer cell line, 14 which utilises the Gibbon ape leukaemia virus (GALV) env protein.…”

Recombinant Sendai virus vectors for activated T lymphocytes

“…[5][6][7][8][30][31][32][33] However, in our systems, excellent gene transfer could be achieved by the simple addition of SeV vector solution without specific reagents or centrifugation. Moreover, optimal SeV-mediated gene transfer to activated T cells could be performed during a relatively brief exposure time (less than 30 min, Figure 4d), with representative results seen in nasal mucosa, 20 in the vasculature, 21 in retinal tissue, 24 as well as in human umbilical cord bloodderived CD34-positive cells.…”