Genetic Engineering Publications - GEG Tech top picks

EMBL scientists have developed a new variant of the Sleeping Beauty transposase which could be used for genome engineering of stem cells and therapeutic T cells. As such it is extremely valuable for use in regenerative medicine and cancer immunotherapy. The underlying genome engineering procedures will in the future also reduce costs and improve the safety of genome modifications.

https://www.nature.com/articles/s41587-019-0291-z

Transposons are unique DNA elements that can be integrated into and removed from chromosomes. Here, the scientists attempted to utilize the piggyBac transposon system to generate iHeps by integrating a transgene consisting of Hnf4a and Foxa3, and successfully obtained functional iHeps. They then demonstrated removal of the transgene to obtain transgene-free iHeps, which still maintained hepatocyte functions. This non-viral, transgene-free reprogramming method using the piggyBac vector may facilitate clinical applications of iHeps in upcoming cell therapy.

Here, the scientists examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Through hydrodynamic tail vein injections, they delivered a PB-CRISPR library into mouse liver. Rapid tumor formation could be observed in less than 2 mo. By sequencing analysis of PB-mediated gRNA insertions, they identified corresponding genes mediating tumorigenesis. Their results demonstrate that PB is a simple and nonviral choice for efficient in vivo delivery of CRISPR libraries for phenotype-driven screens.

Sleeping Beauty (SB) is the first synthetic DNA transposon that was shown to be active in a wide variety of species. Here, the authors review studies from the last two decades addressing both basic biology and applications of this transposon. They discuss how host–transposon interaction modulates transposition at different steps of the transposition reaction. They also discuss how the transposon was translated for gene delivery and gene discovery purposes. They critically review the system in clinical, pre-clinical and non-clinical settings as a non-viral gene delivery tool in comparison with viral technologies.

In this study, the scientists used a transposon mutagenesis strategy based on a two-step sleeping beauty (SB) forward genetic screen to identify and validate new tumor suppressors (TS) in this disease. They generated 120 siRNAs targeting 40 SB-identified candidate breast cancer TS genes and used them to downregulate expression of these genes in four human TNBC cell lines. Their validation of several new TS genes in TNBC demonstrate the utility of two-step forward genetic screens in mice and offer an invaluable tool to identify novel candidate therapeutic pathways and 

Juan Cadinanos and colleagues perform an insertional mutagenesis screen with a single-copy inactivating Sleeping Beauty transposon to look for Pten-cooperating tumor suppressor genes in mice. They find novel candidate cancer genes, verify their clinical relevance in patient cohorts and functionally demonstrate their synergistic relationship to Pten in tumor suppression.

In this work, the scientists describe the generation of a set of plasmid vector tools that allow the rapid generation of complex-interacting stable transgenes in immortalized and primary cells. This system incorporates MultiSite-Gateway cloning for the rapid generation of vectors allowing flexible choice of promoters and transgenes, and Sleeping Beauty transposase technology for efficient incorporation of multiple transgenes in into host cell DNA. The vectors and a library of compatible Gateway Entry clones are available from the non-profit plasmid repository Addgene.

In this study, the scientists evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR. They found that SB-mediated genetic transposition and stimulation resulted in 2,200- to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients. These results support further clinical development of this nonviral gene therapy approach.