[PubMed] [Google Scholar] [5] Leong KW, Mao HQ, Truong-Le VL, Roy K, Walsh SM, August JT, DNA-polycation nanospheres as non-viral gene delivery vehicles, J Control Release, 53 (1998) 183C193. intracellular kinases (e.g. PLK1) can significantly enhance non-viral transgene expression for applications in biotechnology and medicine. gene delivery vehicles have already shown promise as potential therapeutic strategies [1, 2]. However, limitations associated with immunogenicity [3], cost [4], and limited cargo load [5] associated with viral vectors motivate the development of non-viral gene delivery methods, including novel polymers as delivery vehicles[6C9]. Unfortunately, current non-viral gene delivery methods demonstrate lower transgene expression levels than those obtained using viral techniques. Most approaches for improving non-viral gene BRL-15572 delivery therefore focus on creative approaches towards optimizing the delivery vector to overcome physical barriers within the cell [10, 11]. In particular, cellular uptake and endosomal escape have been significantly improved with the development of novel cationic polymers and lipids [12C14]. At the cellular level, we and others have previously shown that modulating intracellular trafficking by inhibiting cytoplasmic histone deacetylases (HDAC6) leads to enhanced transgene expression [15]. Despite these advances, key intracellular targets (biomarkers) that act as significant barriers to non-viral transgene expression remain poorly comprehended [16, 17]. In addition to physical barriers (e.g. cell / endosomal / nuclear membranes), transgene delivery may also be limited by specialized biochemical defenses that specifically safeguard cells against foreign DNA. For example, Toll-Like Receptor 9 (TLR9) binds unmethylated cytosine-guanine (CpG) base pairs within bacterial and viral DNA that are taken up in the endosomes of macrophages and dendritic cells (host CpGs are methylated and do not activate TLR9) [18, 19]. Once TLR9 binds the unmethylated DNA, it initiates a signaling cascade involving several different kinases (IRAK-1, IRAK-4, TAK1, IKK, and MAPK) that activate a set of transcription factors (NF-B, AP-1, and IRF7). This, in turn, leads to expression of interleukins and interferons leading to the induction of an immune response[20]. TLR9 activation also reduces the magnitude and duration of transgene expression[21], but removing CpGs from plasmid DNA decreases inflammation and enhances transgene expression in lung tissue for up to 56 days[22]. The kinases involved in the TLR9 pathway are just one example of the 500 kinases in the human kinome [23] that play key roles in intracellular processes, including endocytosis (PI3K [24] and EGFR[25]), cell cycle progression (CDK, PLK, Aurora)[26], and gene transcription (JAK/STAT [27] and JNK[28]). Therefore, we hypothesized that kinases are likely to be involved in cellular uptake and trafficking, nuclear import, and / or transgene expression following plasmid DNA delivery. Some studies in the literature have investigated the role of kinases in delivery. For example, ur Rehman et al. showed that inhibition of Protein Kinase A (PKA) enhances gene delivery 2C3 fold by promoting lipoplex and polyplex (polymer-plasmid DNA complex) uptake by caveolae instead of clathrin-coated pits[29]. Inhibition of Rho kinase by Y-27632 also increases lentiviral transduction by 20% in keratinocytes[30]. In contrast, inhibition of PI3K has been shown to reduce adenoviral transduction, since PI3K plays a key role in integrin-associated endocytosis of viruses.(11) The tyrosine kinase inhibitor genistein also decreases polyplex uptake by up to 50% by inhibiting cavaeolar uptake[31]. Therefore, there exists some evidence to indicate that kinase inhibition has the potential to either increase or decrease the efficacy of transgene expression. In this work, we carried out a kinome-level screen of small molecule inhibitors in order to identify kinases that influence the efficacy of transgene expression following non-viral (polymer-mediated) delivery of plasmid DNA. While the screen resulted in the identification of several kinases that enhanced polymer-mediated transgene expression, treatment with small-molecule inhibitors of the cell cycle regulator Polo-Like Kinase 1 (PLK1) resulted in the highest enhancement of transgene expression in cancer cell lines, indicating a pivotal role for this kinase in transgene delivery. Simultaneous inhibition of PLK1, histone deacetylase 1 (HDAC1), and Janus Kinase (JAK, another kinase identified in our screen) resulted in further enhancement of transgene expression relative to inhibition of each individual target. These results demonstrate that inhibition of key intracellular kinase targets using small-molecule inhibitors can enhance transgene expression and potentially improve gene therapy efficacy. MATERIALS AND METHODS Cell Culture PC3-PSMA prostate cancer cells[32],.Most importantly, combinations of small-molecule inhibitors resulted in significant enhancement of transgene expression compared to individual inhibitors acting alone. findings indicate that inhibition of specific intracellular kinases (e.g. PLK1) can significantly enhance non-viral transgene expression for applications in biotechnology and medicine. gene delivery vehicles have already shown promise as potential therapeutic strategies [1, 2]. However, limitations associated with immunogenicity [3], cost [4], and limited cargo load [5] associated with viral vectors motivate the development of non-viral gene delivery methods, including novel polymers as delivery vehicles[6C9]. Unfortunately, current non-viral gene delivery methods demonstrate lower transgene expression levels than those obtained using viral techniques. Most approaches for BRL-15572 improving non-viral gene delivery therefore focus on creative approaches towards optimizing the delivery vector to overcome physical barriers within the cell [10, 11]. In particular, cellular uptake and endosomal escape have been significantly improved with the development of novel cationic polymers and lipids [12C14]. At the cellular level, we and others have previously shown that modulating intracellular trafficking by inhibiting cytoplasmic histone deacetylases (HDAC6) leads to enhanced transgene expression [15]. Despite these advances, key intracellular targets (biomarkers) that act as significant barriers to non-viral transgene expression remain poorly comprehended [16, 17]. In addition to physical barriers (e.g. cell / endosomal / nuclear membranes), transgene delivery may also be BRL-15572 limited by specialized biochemical defenses that specifically safeguard cells against foreign DNA. For example, Toll-Like Receptor 9 (TLR9) binds unmethylated cytosine-guanine (CpG) base pairs within bacterial and viral DNA that are taken up in the endosomes BRL-15572 of macrophages and dendritic cells (host CpGs are methylated and do not activate TLR9) [18, 19]. Once TLR9 binds the unmethylated DNA, it initiates a signaling cascade involving several different kinases (IRAK-1, IRAK-4, TAK1, IKK, and MAPK) that activate a set of transcription factors (NF-B, AP-1, and IRF7). This, in turn, leads to expression of interleukins and interferons leading to the induction of an immune response[20]. TLR9 activation also reduces the magnitude and duration of transgene expression[21], but removing CpGs from plasmid DNA decreases inflammation and enhances transgene expression in lung tissue for up to 56 days[22]. The kinases involved in the TLR9 pathway are just one example of the 500 kinases in the human kinome [23] that play key roles in intracellular processes, including endocytosis (PI3K [24] and EGFR[25]), cell cycle progression (CDK, PLK, SLC5A5 Aurora)[26], and gene transcription (JAK/STAT [27] and JNK[28]). Therefore, we hypothesized that kinases are likely to be involved in cellular uptake and trafficking, nuclear import, and / or transgene expression following plasmid DNA delivery. Some studies in the literature have investigated the role of kinases in delivery. For example, ur Rehman et al. showed that inhibition of Protein Kinase A (PKA) enhances gene delivery 2C3 fold by promoting lipoplex and polyplex (polymer-plasmid DNA complex) uptake by caveolae instead of clathrin-coated pits[29]. Inhibition of Rho kinase by Y-27632 also increases lentiviral transduction by 20% in keratinocytes[30]. In contrast, inhibition of PI3K has been shown to reduce adenoviral transduction, since PI3K plays a key role in integrin-associated endocytosis of viruses.(11) The tyrosine kinase inhibitor genistein also decreases polyplex uptake by up to 50% by inhibiting cavaeolar uptake[31]. Therefore, there exists some evidence to indicate that kinase inhibition has the potential to either increase or decrease the efficacy of transgene expression. In this work, we carried out a kinome-level screen of small molecule inhibitors in order to identify kinases that influence the efficacy of transgene expression following non-viral (polymer-mediated) delivery of plasmid DNA. While the screen resulted in the identification of several kinases that enhanced polymer-mediated transgene expression, treatment with small-molecule inhibitors of the cell cycle regulator Polo-Like Kinase 1 (PLK1) resulted in the highest enhancement of transgene expression in cancer cell lines, indicating a pivotal role for this kinase in transgene delivery. Simultaneous inhibition of PLK1, histone deacetylase 1 (HDAC1), and Janus Kinase (JAK, another kinase identified in our screen) resulted in further enhancement of transgene expression.