Microbead-assisted retroviral transduction for clinical application.

Abstract

Retroviral transduction is the most commonly used strategy to obtain long-term expression of therapeutic genes. To efficiently transduce mammalian cells, a recombinant fibronectin molecule, RetroNectin, is generally used to juxtapose viral particles and cells, and thereby enhance viral uptake. Although this strategy has become widely adopted, in particular for the genetic modification of hematopoietic cells, several limitations apply. For example, it requires the use of culture systems that allow protein coating, something that is not possible for many of the closed cell culture systems that are used in clinical trials. Furthermore, efficient transduction is obtained only when culture systems can be exposed to centrifugation, an approach termed spin transduction. Here, we describe a novel and more potent strategy for the transduction of T cells that can be applied on a clinical scale. We show that RetroNectin can efficiently be coated onto epoxy-modified paramagnetic beads. After a blocking step, these beads can subsequently bind retroviral particles from viral supernatants, rendering such supernatants largely devoid of functional viral particles. Addition of these virus-loaded beads to activated T cells results in efficient retroviral infection. Importantly, transduction does not require the use of culture systems that are compatible with protein coating, nor is it dependent on centrifugation of either the viral supernatant or the cells. Finally, cell growth, phenotype, and function of spin-transduced versus bead-transduced cells are comparable. Viral coating of microbeads should facilitate the production of genetically modified cells, in particular for use in clinical trials.