Highly Efficient Retroviral Gene Transfer Based on Centrifugation-Mediated Vector Preloading of Tissue Culture Vessels

Abstract

Efficient retroviral gene transfer into primary cells is a prerequisite for various gene therapeutic strategies. We have developed a transduction protocol based on the preloading of tissue culture vessels with retroviral particles by low-speed (1000g) centrifugation. We show that vector-preloaded tissue culture vessels allow highly efficient gene transfer into various target cells. We obtained transduction rates of up to 85% for primary T lymphocytes after just a single round of transduction. Under clinically relevant conditions using a vector developed for suicide gene therapy and produced under good manufacturing practice (GMP) conditions, the described method allowed generation of large numbers (>2x10(9)) of gene-modified T cells. The preloading concept ensures transduction of target cells in their optimal growth medium regardless of the medium used for vector production. This facilitated highly efficient gene transfer into quite different target cells such as CD34(+) and AC133(+) bone marrow progenitor as well as mesenchymal stem cells. The presented method combines high gene-transfer rates with a great potential for standardization in accordance with GMP guidelines and is consequently well suited for both research and clinical applications. (c)2002 Elsevier Science (USA).

A novel ‘sort-suicide’ fusion gene vector for T cell manipulation

“…Currently the most suitable of these clones is processed under GMP conditions to generate a master cell bank and subsequent retroviral supernatant for clinical use. High-titer retroviral supernatant in conjunction with efficient gene transfer protocols 12,30 will allow high transduction rates after just one infection cycle (at day 3 after initial stimulation). In combination with a selection method based on magnetic cell sorting, the whole procedure may, therefore, be shortened to 5 days.…”

T lymphocytes isolated from patients with advanced colorectal cancer are suitable for gene immunotherapy approaches

“…It is clear that higher levels of transduction are preferable particularly with respect to use in a clinical context. Protocols have been developed that have reported high-level gene transfer using longer antibody-mediated preactivation periods (Movassagh et al, 2000), through the use of retronectin to enhance infection (Hanenberg et al, 1996) or by preloading plates with retrovirus (Kuhlcke et al, 2002). The levels of retroviral transduction generated here represent those achieved using a short period of centrifugation (spin-fection) and is adaptable to transduction on a large scale.…”

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.…”

“…Although an encouraging human study using a retrovirus demonstrated the success of T-cell-directed gene therapy against severely combined immunodeficiency disease (SCID) owing to adenosine deaminase deficiency (ADA), 1,3 it has been difficult to use this strategy in cases of other diseases, because the gene transfer efficiency is usually a critical factor limiting the outcome. Therefore, current efforts are now focused on the development of pseudotyped vectors 4 and transduction techniques [5][6][7][8] for the use of retroviral vectors for clinical applications.…”