(584at) Cellular and Molecular Factors Contribute to a Cell Shutdown After DNA Complex Delivery

The design of efficient nonviral gene delivery systems offers potential uses in therapeutic applications such as tissue engineering, biomaterials, and gene therapy. Nonviral gene delivery involves the delivery of exogenous gene(s) to cells, facilitated by the electrostatic complexation of the gene (as plasmid DNA) with a nonviral vector such as a cationic polymer or lipid. Unlike viral based delivery systems, nonviral gene delivery systems can carry a larger genetic payload, overcome several safety concerns, and are easily modified to allow for cellular targeting. However, current nonviral gene delivery systems are inefficient; overcoming the function of cellular barriers to transfection by design of the nonviral gene delivery system is an important strategy for enhancing transfection. While many proposed approaches have resulted in incremental successes to transfection, our rudimentary understanding of the specific molecules that facilitate the DNA transfer process limits the design of better nonviral gene delivery systems.

We have taken a transcriptomics approach to identify up- and down-regulated endogenous genes in transfected HEK293T cells at 8 h, 16 h, and 24 h after delivery of a plasmid encoding the green fluorescent protein (GFP) transgene, complexed by polyethylenimine (PEI) or Lipofectamine® 2000. Bioinformatics analysis using a nonlinear model was used to identify the up (+) and down (-) regulated genes characteristic of transfected cells (GFP+) in comparison to untransfected cells (GFP-). For polyplex transfection, genes differentially expressed between GFP+ and GFP- populations include: 8h (+RAP1A, +NM_022155, +WDR78, +ACRC); 16h (+RAP1A, +CHORDC1, +NM_022155); and 24h (+RAP1A, +CHORDC1, +WDR78, -SNHG6, -EIF4A2, -TRA2B, -CKB, -ACTB, -ND2, -AF253979). The ENRICHR bioinformatics tool showed these genes to be implicated in cell cycle/mitogenesis effectors (E2F4, EGR1, NFKB, MEF2A), oncogenic/immunogenic action (E2F4, FLI1, RFX5, NFKB, MEF2A), apoptosis (MEF2A), gene regulation involved in development/differentiation (HNF4A, GATA1, NKX2-5, ZFHX3, EGR1, NFKB, FOXC1, SOX2), xenobiotic metabolism (ARNT), the integrin pathway (RAP1A), and nucleosome modifications (PHF8, H3K23ACH1, H3K23ME2H9, H2BK120ACH9). For lipoplex transfection, genes differentially expressed between GFP+ and GFP- populations include: 8h (+RAP1A, +PACSIN3, +IFT27, +NDUFA10, -DNAJB11); 16h (+RAP1A, +HSPA6, +ZNF548, -AI819238, -BC017896); and 24h (+RAP1A, +HSPA6). The ENRICHR bioinformatics tool showed these genes to be implicated in cell cycle/mitogenesis effectors (SMARCA4, BRAF, NTRK1, MORF, IL13, NDUFA10), integrin/cytoskeletal processes (RAP1A, PAK2), protein stabilization (HSPA1A, PACSIN3), oncogenic/immunogenic action (SMARCA4, BRAF, NTRK1, IL13), apoptosis (NTRK1, IL13), gene regulation involved in development/differentiation ( SMARCA4, BRAF, NTRK1, IL13), DNA repair (MSH3, MORF), chromatin/nucleosome modifications (TRIM28, SMARCA4), and micro RNA/chromosome overlap (TTTTGAG MIR-373, chr1p13).

These data further support the hypothesis that transfected cells utilize different genes and processes to facilitate DNA transfer, compared to untransfected cells. The results also demonstrate that while some genes and cellular processes are common to both vectors and overlapping time points, there are also time- and DNA-carrier dependent genes identified.  Our study suggests a role of these genes and processes as molecular mechanisms that facilitate transfection and offer new targets for improved gene delivery systems.