# Application of Pseudoinfectious Viruses in Transient Gene Expression in Mammalian Cells: Combining Efficient Expression with Regulatory Compliance

## TL;DR

This paper introduces a new method for producing proteins in mammalian cells using modified viruses that are efficient and meet regulatory standards.

## Contribution

A novel transient gene expression system using pseudoinfectious alphavirus vectors that enables scalable and compliant protein production.

## Key findings

- PIV-based expression outperforms traditional plasmid and replicon vectors in protein yield.
- The system allows scalable production without bioreactors.
- Modified IMAC enables protein purification from conditioned media.

## Abstract

Transient gene expression (TGE) is commonly employed for protein production, but its reliance on plasmid transfection makes it challenging to scale up. In this paper, an alternative TGE method is presented, utilizing pseudoinfectious alphavirus as an expression vector. Pseudoinfectious viruses (PIV) and a replicable helper construct were derived from the genome of the Venezuelan equine encephalitis virus. The PIV carries a mutant capsid protein that prevents packaging into infectious particles, while the replicable helper encodes a wild-type capsid protein but lacks other viral structural proteins. Although PIV and the helper cannot independently spread infection, their combination results in increased titers in cell cultures, enabling easier scale-up of producing cultures. The PIV-driven production of a model protein outperforms that of alphavirus replicon vectors or simple plasmid vectors. Another described feature of the expression system is the modification to immobilized metal affinity chromatography (IMAC), allowing purification of His-tagged recombinant proteins from a conditioned medium in the presence of substances that can strip metal from the IMAC columns. The PIV-based expression system allows for the production of milligram quantities of recombinant proteins in static cultures, without the need for complex equipment such as bioreactors, and complies with regulatory requirements due to its distinction from common recombinant viruses.