University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Biological Systems Engineering: Papers and Biological Systems Engineering Publications 2015 Temporal endogenous gene expression profiles in response to polymer-mediated transfection and profile comparison to lipid-mediated transfection Timothy M. Martin University of Nebraska Medical Center, [email protected] Sarah A. Plautz University of Nebraska-Lincoln, [email protected] Angela K. Pannier University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/biosysengfacpub Part of the Bioresource and Agricultural Engineering Commons, Environmental Engineering Commons, and the Other Civil and Environmental Engineering Commons Martin, Timothy M.; Plautz, Sarah A.; and Pannier, Angela K., "Temporal endogenous gene expression profiles in response to polymer-mediated transfection and profile comparison to lipid-mediated transfection" (2015). Biological Systems Engineering: Papers and Publications. 518. https://digitalcommons.unl.edu/biosysengfacpub/518 This Article is brought to you for free and open access by the Biological Systems Engineering at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Biological Systems Engineering: Papers and Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Published in The Journal of Gene Medicine 17 (2015), pp. 33–53. doi 10.1002/jgm.2822 PMID: 25663627 Copyright © 2015 John Wiley & Sons, Ltd. Used by permission Submitted 17 November 2014; revised 1 February 2015; accepted 3 February 2015 digitalcommons.unl.edu Temporal endogenous gene expression profiles in response to polymer-mediated transfection and profile comparison to lipid-mediated transfection Timothy M. Martin,1 Sarah A. Plautz,2 and Angela K. Pannier2,3,4 1 Department of Pharmaceutical Sciences, Durham Research Center II, University of Nebraska Medical Center, Omaha, NE 2 Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, NE 3 Center for Nanohybrid Functional Materials, University of Nebraska-Lincoln, Lincoln, NE 4 Mary and Dick Holland Regenerative Medicine Program, University of Nebraska Medical Center, Omaha, NE Corresponding author — Angela K. Pannier, 231 L.W. Chase Hall, Lincoln, NE 68583-0726, USA. email [email protected] Abstract 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 com- pared 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. Pharma- cologic 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, al- though polyplex or lipoplex dependencies must be considered. Keywords GFP, HEK 293, LF2000, microarray analysis, nonviral gene delivery, PEI, temporal gene expression profile Introduction with applications in functional genomics, tissue engineer- ing, medical devices and gene therapy [1]. Because un- Nonviral DNA delivery provides a mechanism to directly aided delivery of naked plasmid DNA (pDNA) to cells has alter endogenous gene expression and cellular behavior low efficacy, nonviral carriers are a common approach to 33 34 Martin, Plautz, & Pannier in The Journal of Gene Medicine 17 (2015) enhance delivery. Nonviral carriers such as cationic poly- genes and pathways utilized by transfected cells rela- mers (polyplexes) [2,3] and lipids (lipoplexes) [4–9] are tive to untransfected cells and confirmed the ability of used to electrostatically complex DNA to protect it against RAP1A and HSP6A to enhance lipoplex transfection [42] degradation and facilitate DNA transfer [10–12]. However, or RAP1A, SCG5, ACRC, ATF3, ACTA1 and IREB2 to en- current nonviral DNA delivery systems are inefficient for hance polyplex transfection [43]. Because those studies therapeutic use [11,13–15] because our rudimentary un- were performed at the 24-h time point when transgene derstanding of the molecules and processes that facili- expression is fully established, we now extend our ap- tate nonviral gene delivery limits the design of efficient proach in the present study for polyplexes by identifying delivery systems. genes at earlier time points because: (i) gene expression Previous studies have advanced the understanding profiles have been shown to vary over time [44]; (ii) tar- of nonviral gene delivery, including particle tracking to geting a single barrier or step in DNA transfer will proba- identify extracellular [16–19] or intracellular [20–28] bar- bly not lead to a therapeutically relevant transfection sys- riers to DNA transfer, or microarray analysis to understand tem [32]; and (iii) other genes and pathways largely remain the toxicogenomic response of cells and tissues to non- unidentified that are implicated at earlier stages of trans- viral gene delivery systems [29]. Those particle tracking fection (e.g. internalization, endosomal escape, nuclear lo- studies have identified compartments and components calization, nuclear entry) [3,19,30,31,39,41,45–52]. of the cell that interact with lipoplexes or polyplexes, at As a first objective of the present study, we identified the same time as providing pharmacokinetic information genes and pathways implicated at 2, 8, 16 and 24 h af- on the movement of the complexes throughout the cell. ter delivery of GFP transgene to HEK 293T cells using a Those data have been integrated into mathematical mod- cationic polymer vector (polyethylenimine; PEI), whereas els to further our understanding of the gene transfer pro- our other work focuses on the lipid vector (Lipofectamine cess [30–33]. Similarly, previous microarray studies have 2000; LF2000) [53]. PEI is a one of the most widely used provided global gene expression profiles in cells and tis- polymer gene carriers because it transfects a broad range sues exposed to cationic lipoplexes [29] or polyplexes [14] of cells and can be easily modified [54]. Relative to un- and identified specific genes involved in cytotoxicity and treated cells, transcriptome profiling of HEK 293T cells apoptosis [34], which can be dependent on the length of at 2 h post-delivery of polyplexes was used to identify time of exposure to the DNA carrier, cell type, and DNA those endogenous genes that may act in response to carrier [14,29,34]. These studies have been instrumental treatment stress such as toxicity induced by the complex in providing information necessary for targeted physico- [13] or inherent intracellular defenses against foreign nu- chemical modification of the DNA carrier [31,35–39] and, cleic acids [55]. Relative to GFP– cells, transcriptome pro- although achieving promising in vitro and in vivo transfec- filing was used to identify those endogenous genes used tion from lipid and polymer DNA carriers [40], transfection by transfected cells (GFP+) that may aid in overcom- still remains below desired levels. Those previous micro- ing cellular barriers known to occur during DNA transfer array studies were limited in that molecules that facilitate [3,19,30,31,39,41,45–52] at 8, 16 and 24 h post-delivery of transfection were not identified (i.e. comparisons of gene polyplexes. Events that typically take place 8 h after de- profiles between treated and transfected cells to treated livery of complexes to the cells include high rate of en- but untransfected cells were not made), even though it is dosomal escape [41,45], unpacking of pDNA from vector the treated but untransfected cell population that limits [45,46], some nuclear localization of complexes or pDNA the therapeutic efficacy and use of nonviral DNA delivery [19,30,31,45,47], very small amounts of nuclear internaliza- systems. A large gap in knowledge remains about mole- tion of complexes or pDNA [30,39,47] and very little pro- cules that actively facilitate DNA transfer [41], which will duction of transgenic protein [30,48]. Events that typically have to be exploited in the design of enhanced gene de- take place 16 h after delivery of complexes to the cells livery systems. include a high rate of nuclear localization