Demeetra, in partnership with VBI/Variation Biosciences, Inc. is proud to have been selected and presented an oral presentation at the prestigious European Society for Animal Cell Technology (ESACT) 2024 conference that ran from June 23-26, 2024 in Edinburgh, Scotland.
The presentation, titled "Complementary Gene Editing Technologies Enhance Rapid Generation of Stable Cell Lines Producing Enveloped Viral-Like Particles," highlights Demeetra’s expertise in gene editing application in producing hard-to-manufacture enveloped viral-like particles (eVLPs). The work is funded by the Coalition for Epidemic Preparedness Innovations (CEPI) focusing on accelerating the development of vaccines and other biologic countermeasures against epidemic and pandemic threats.
Jack Crawford, CEO of Demeetra, said “At Demeetra, we are dedicated to helping our clients implement gene editing by combining cutting-edge technologies with guided expertise and detailed know-how transfer. Our selection for an oral presentation at ESACT 2024 highlights the success of our approach.”
Abstract
Complementary Gene Editing Technologies Enhance Rapid Generation of Stable Cell Lines Producing Enveloped Viral-Like Particles.
Relevance to Theme Selected
The work presented highlights the production of hard-to-produce enveloped viral-like particles (eVLPs). Enhanced cell factory platforms are tailor-made to express protein components of eVLPs.
Impact/Novelty
Combining piggyBac transposase and Cas-CLOVER targeted nuclease technologies enable rapid stable cell line development expressing therapeutic candidates for development of novel coronavirus vaccines as new variants surface.
Introduction
Viral-like particles are proven to be powerful therapeutics towards infectious diseases by harnessing the power of the human immune system. Currently, new variants of SARS-CoV-2 continue to surface causing decreased efficacies of vaccinations and the requirement for rapid production of new therapeutic biologics.
Methods/Approach
Two complementary gene editing technologies, piggyBac transposase and Cas-CLOVER targeted nuclease, were utilized to create enhanced cell bioprocessing platforms to produce eVLPs. Firstly, the piggyBac transposase/transposon system was utilized to map novel sites of interest to express an eVLP stable core protein, MLV-GAG. Subsequently, since Cas-CLOVER is a dimeric targeted nuclease system, the most efficient guide RNA pair was used to independently knock-in viral-like protein cargo (GAG, spike proteins, etc.). Knock-in constructs incorporated homologous fragments to the target sites flanking integral protein components of eVLPs. Following NEON transfection, positive cells were enriched and characterized for the presence of knock-in cargo and secreted protein expression.
Results
Via the piggyBac system, five total novel sites were identified in which one of particular interest was further utilized for the differential gene expression stage. Known genomic safe harbor sites, rogi1 and gsh31, were also targeted for comparison. Three guide RNA pairs were tested per site which yielded over 35% cutting efficiencies at all sites in a single NEON electroporation. Antibiotic selection enriched the cell pools for positive gene-edited cells over 90%. Throughout the antibiotic selection, pools were validated via PCR and western blot, which showed increased target amplification and secreted protein expression, respectively. Comparison of each knock-in site revealed differential protein expression levels that can create different ratios of eVLP components.
Conclusion
These innovative cell platforms will be used to create product candidates for manufacturing novel vaccine therapies. Unlike current pipelines, our newly developed workflow will be able to rapidly deliver those novel product candidates in response to Pandemics.