192. Non-Viral, Long-Term Gene Expression in Hematopoietic Cells Using the Sleeping Beauty Transposon System

Abstract

We are developing the Sleeping Beauty (SB) transposon system as a non-viral gene therapy for treatment of blood related disorders. The goal of this study is to characterize the efficacy of SB transposon vectors to elicit long-term gene expression after introduction into hematopoietic cells by electroporation. Ex vivo, non-viral gene therapy strategies have the potential to be safer than viral strategies because of (1) reduced potential to generate an immune response, (2) the ability to more stringently control the manufacturing process, and (3) the essentially random insertion into the genome. The SB transposase mediates integration of the associated transposon into chromosomes via a precise cut and paste mechanism. To evaluate the potential of SB transposon plasmids to mediate long-term expression in hematopoietic cell populations, we constructed a transposon containing a PGK promoter to regulate transcription of a bicistronic mRNA encoding eGFP and neomycin phosphotransferase. SB transposase was provided by a separate plasmid carrying the SB gene regulated by an ubiquitin promoter. These two plasmids were transfected into K562 - undifferentiated myeloid cells at 1:1 ratio via a clinically approved electroporation-based cell loading system (MaxCyte GT). Greater than 90% of the cells expressed GFP when analyzed by flow cytometry at 24 hrs post-electroporation. Propidium iodide (PI) exclusion revealed the transfected cell viability to be greater than 95%. To study subpopulations of K562 cells that exhibit long-term expression of eGFP, the transfected cells were plated in methylcellulose in the presence or absence of G418. The results showed that cotransfection of the transposon and SB gene gave rise to 40% 9.5% G418 resistant colony formation, while G418 resistant colonies were not observed after cotransfection of the transposon with an SB deleted plasmid. Several G418 resistant colonies were isolated and are currently being characterized molecularly for transposition vs. random recombination.