An Efficient Large-Scale Retroviral Transduction Method Involving Preloading the Vector into a RetroNectin-Coated Bag with Low-Temperature Shaking

Abstract

In retroviral vector-mediated gene transfer, transduction efficiency can be hampered by inhibitory molecules derived from the culture fluid of virus producer cell lines. To remove these inhibitory molecules to enable better gene transduction, we had previously developed a transduction method using a fibronectin fragment-coated vessel (i.e., the RetroNectin-bound virus transduction method). In the present study, we developed a method that combined RetroNectin-bound virus transduction with low-temperature shaking and applied this method in manufacturing autologous retroviral-engineered T cells for adoptive transfer gene therapy in a large-scale closed system. Retroviral vector was preloaded into a RetroNectin-coated bag and incubated at 4°C for 16 h on a reciprocating shaker at 50 rounds per minute. After the supernatant was removed, activated T cells were added to the bag. The bag transduction method has the advantage of increasing transduction efficiency, as simply flipping over the bag during gene transduction facilitates more efficient utilization of the retroviral vector adsorbed on the top and bottom surfaces of the bag. Finally, we performed validation runs of endoribonuclease MazF-modified CD4+ T cell manufacturing for HIV-1 gene therapy and T cell receptor-modified T cell manufacturing for MAGE-A4 antigen-expressing cancer gene therapy and achieved over 200-fold (≥1010) and 100-fold (≥5×109) expansion, respectively. In conclusion, we demonstrated that the large-scale closed transduction system is highly efficient for retroviral vector-based T cell manufacturing for adoptive transfer gene therapy, and this technology is expected to be amenable to automation and improve current clinical gene therapy protocols.

Materials promoting viral gene delivery

“…[359][360][361] A typical workflow for transduction involves the coating of Retronectin on a solid surface, the addition of viral particles and cells followed by spin centrifugation. 362 To overcome the limitations of the laborious preparation on surfaces and the necessity of centrifugation, various alternative approaches have been devel-oped. The coating of epoxy-modified paramagnetic beads with Retronectin enables the effective capture of retroviral particles from solution and provides access to remote-controllable transduction.…”

Microfluidic Transduction Harnesses Mass Transport Principles to Enhance Gene Transfer Efficiency

“…Although previous reports indicate that RN, one of the few clinically approved transduction-enhancing compounds, may be optimized for adsorption to plastics such as polystyrene, cyclo-olefin, polyethylene, and Teflon, 26 no characterization for other materials such as polydimethylsiloxane (PDMS), the most commonly utilized polymer for soft lithography and microfluidics, has yet been assessed. To that end, we aimed to determine microfluidic compatibility with RN, which has the benefit of binding cells and virus for co-localization, although varying efficacy with different viral vectors and envelope proteins has been reported.…”

Simplified prototyping of perfusable polystyrene microfluidics

“…Retronectin works best when coated onto PS, cyclo-olefin, polyethylene, or Teflon, which is the reason a hybrid PS-PDMS device was essential for this application. 23 Silanization had no observable effect on the adsorption of Retronectin on the PS surface. The ability to emboss patterns into the PS also assists with the adhesion of cells by providing more contact surface area.…”