Production of high-titer helper-free retroviruses by transient transfection.

TLDR

The method aims to enable and broaden helper‑free retroviral gene transfer for gene therapy applications. High‑titer, helper‑free retroviruses were generated by transiently transfecting 293T cells stably expressing retroviral packaging functions. The BOSC 23 cell line produced >10⁶ infectious units/ml within 72 h, showed no replication‑competent virus, and enabled high‑titer production of difficult‑to‑propagate retroviral vectors.

Abstract

The generation of high-titer, helper-free retroviruses by transient transfection has been achieved by using the highly transfectable 293T cell line into which are stably introduced constructs that express retroviral packaging functions. The resulting ecotropic virus packaging cell line BOSC 23 produces infectious retrovirus at > 10(6) infectious units/ml of supernatant within 72 hr after CaPO4-mediated transfection. A stringent assay for replication-competent virus showed that no helper virus was present. The system can produce high titers of retroviral vectors expressing genes that are extremely difficult to propagate at high titer in stable producer lines. This method should facilitate and extend the use of helper-free retroviral gene transfer, as well as be useful for gene therapy.