Date of this Version
Published in The Journal of Gene Medicine 17 (2015), pp. 33–53.
Background Design of efficient nonviral gene delivery systems is limited by the rudimentary understanding of specific molecules that facilitate transfection.
Methods Polyplexes using 25-kDa polyethylenimine (PEI) and plasmid encoding green fluorescent protein (GFP) were delivered to HEK 293T cells. After treating cells with polyplexes, microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2 h of exposure) or between flow-separated transfected cells (GFP+) and treated, untransfected cells (GFP–) at 8, 16 and 24 h after lipoplex treatment. Cell priming studies were conducted using pharmacologic agents to alter endogenous levels of the identified differentially expressed genes to determine effect on transfection levels. Differentially expressed genes in polyplex-mediated transfection were compared with those differentially expressed in lipoplex transfection to identify DNA carrier-dependent molecular factors.
Results Differentially expressed genes were RGS1, ARHGAP24, PDZD2, SNX24, GSN and IGF2BP1 after 2 h; RAP1A and ACTA1 after 8 h; RAP1A, WDR78 and ACTA1 after 16 h; and RAP1A, SCG5, ATF3, IREB2 and ACTA1 after 24 h. Pharmacologic studies altering endogenous levels for ARHGAP24, GSN, IGF2BP1, PDZD2 and RGS1 were able to increase or decrease transgene production. Comparing differentially expressed genes for polyplexes and lipoplexes, no common genes were identified at the 2-h time point, whereas, after the 8-h time point, RAP1A, ATF3 and HSPA6 were similarly expressed. SCG5 and PGAP1 were only upregulated in polyplex-transfected cells.
Conclusions The identified genes and pharmacologic agents provide targets for improving transfection systems, although polyplex or lipoplex dependencies must be considered.
Includes supplementary materials.