Using the Sleeping Beauty (SB) Transposon to Generate Stable Cells Producing Enveloped Virus‐Like Particles (evlps) Pseudotyped with SARS‐CoV‐2 Proteins for Vaccination

The Sleeping Beauty (SB) transposon system is an efficient non‐viral tool for gene transfer into a variety of cells, including human cells. Through a cut‐and‐paste mechanism, your favorite gene (YFG) is integrated into AT‐rich regions within the genome, providing stable long‐term expression of the transfected gene. The SB system is evolving and has become a powerful tool for gene therapy. There are no safety concerns using this system, the handling is easy, and the time required to obtain a stable cell line is significantly reduced compared to other systems currently available. Here, we present a novel application of this system to generate, within 8 days, a stable producer HEK293T cell line capable of constitutively delivering enveloped virus‐like particles (eVLPs) for vaccination. We provide step‐by‐step protocols for generation of the SB transposon constructs, transfection procedures, and validation of the produced eVLPs. We next describe a method to pseudotype the constitutively produced eVLPs using the Spike protein derived from the SARS‐CoV‐2 virus (by coating the eVLP capsid with the heterologous antigen). We also describe optimization methods to scale up the production of pseudotyped eVLPs in a laboratory setting (from 100 µg to 5 mg). © Published 2022. This article is a U.S. Government work and is in the public domain in the USA.