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

Abstract

AbstractThe 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.Basic Protocol 1: Generation of the SB plasmidsBasic Protocol 2: Generation of a stable HEK293T cell line constitutively secreting MLV‐based eVLPsBasic Protocol 3: Evaluation of the SB constructs by immunofluorescence assayBasic Protocol 4: Validation of eVLPs by denaturing PAGE and western blotAlternate Protocol 1: Analysis of SARS‐CoV‐2 Spike protein oligomerization using blue native gel electrophoresis and western blotAlternate Protocol 2: Evaluation of eVLP quality by electron microscopy (negative staining)Basic Protocol 5: Small‐scale production of eVLPsAlternate Protocol 3: Large‐scale production of eVLPs (up to about 1 to 3 mg VLPs)Alternate Protocol 4: Large‐scale production of eVLPs (up to about 3 to 5 mg VLPs)Support Protocol: Quantification of total protein concentration by Bradford assay