Inducible,High-Level Production of Infectious Murine Leukemia Retroviral Vector Particles Pseudotyped with Vesicular Stomatitis Virus G Envelope Protein

Abstract

Murine leukemia viruses (MuLV) have been adapted for use as gene transfer vectors for experimental and human gene therapy applications. Their utility for these purposes has been circumscribed by the limited host range and relatively low titer of available producer clones. Pseudotyping of MuLV particles with the vesicular stomatitis virus envelope protein (VSV-G), expressed transiently in cells producing MuLV Gag and Pol proteins, has yielded vector preparations with a broader host range that can be concentrated by ultracentrifugation. We have explored the use of steroid-inducible and tetracycline-modulated promoter systems (necessary because the VSV-G protein is toxic to cells when constitutively expressed) to derive stable producer cell lines capable of substantial production of VSV-G pseudotyped MuLV particles. A packaging cell line and producer clones capable of expressing a chimeric transcription factor, composed of the tetracycline repressor (tet^R) and the VP16 trans-activating sequences of herpes simplex virus VP16 gene and containing the VSV-G coding sequences linked to a minimal promoter having seven tandem copies of the tetracycline responsive operator (tet°), exhibited high levels of VSV-G protein expression when cultured in the absence of tetracycline. Vector particles, produced at titers of 10⁵–10⁶ infectious colony forming units per ml (cfu/ml), could be concentrated effectively by ultracentrifugation yielding vector preparations having a titer of 10⁹ cfu/ml. These cell lines grew normally when VSV-G protein expression was repressed by tetracycline. Such producer clones hold promise for future human gene therapy applications.

Overview summary

Murine leukemia viruses, when pseudotyped with the envelope protein of vesicular stomatitis virus (VSV-G), can be concentrated to high titer and have a broad host range. VSV-G protein is cytotoxic, preventing derivation of producer clones in which it is constitutively expressed. This paper describes the use of an inducible promoter system, based on utilization of components of the tetracycline resistance operon in combination with a strong eukaryotic transcriptional trans-activator, to achieve inducible expression of VSV-G protein. A packaging cell line and producer clones have been derived that generate large numbers of pseudotyped vector particles that can be concentrated to greater than 10⁹ infectious units/ml. Such pseudotyped vector particles are likely to be useful for gene therapy applications.