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Utilizing cis element DNA decoys to sequester TFs is another approach to directly modulate transcription factors (Figure 1.11e). Previous studies have shown that tandem repeats of DNA in the genome that contain transcription factor binding sites could serve as decoy binding sites, which effectively sequesters a TF and inhibits its target gene expression.102-104 This endogenous form of TF competitive inhibition can be replicated exogenously by introducing synthetic double-stranded oligodeoxynucleotides (ODN) containing TF binding sites into cells. These platforms of artificial gene modulation utilizing cis element DNA decoys can be transfected into cells, sequestering their targeted TFs, and alter gene expression by inhibiting their endogenous activity (Figure 1.19b).105 There are several types of DNA decoys, usually ranging from 10-20 base pair ODNs to plasmid DNA containing multiple decoys in the sequence.106

There are numerous advantages to this approach of modulating TFs and gene expression: the TF targets are abundant and identifiable as publications of TFs and their consensus sequences become more common, which is attributable to genome

Figure 1.19. Inhibition of gene expression by DNA decoys. (a) TFs able to bind genomic DNA and

activate gene expression. (b) DNA decoys sequester TFs thereby reducing its effective concentration and subsequent endogenous activity.

technologies like chromatin immunoprecipitation sequencing (ChIP-seq),107,108 _the

synthesis of DNA decoys are relatively simple using phosphoramidite chemistry,109,110 the DNA binding domains of TFs are unlikely to acquire mutations to achieve resistance, and DNA decoys can be rationally designed to modulate any TF because the only structural information about the TF that is needed is the sequence that it binds.

One major limitation of DNA decoys, or DNA therapeutic agents in general, is their poor cellular uptake. Laboratory techniques such as cationic liposomes, heat shock, and electroporation can be used to introduce foreign DNA into cells; however, these techniques cannot be used in vivo. However, there has been progress to rectify this problem. The Hemaggulitinating Virus of Japan (HVJ)-liposome method and ultrasound- mediated gene transfer method have demonstrated enhanced gene transfer and uptake of DNA decoys.111-118 Another major limitation of DNA decoys is their rapid degradation by serum and intracellular nucleases,119,120 consequently, there have been several different modifications of DNA decoys to improve their stability (Scheme 1.1). Modified phosphodiester bonds (1.24), such as replacing a non-bridging oxygen in the phosphate linkages with sulfur to form phosphorothioate (PS) bonds (1.25), greatly increases DNA stability.121 Other modifications to the backbone including methyl phosphate and methyl phosphonate (1.26) derivatives have also demonstrated nuclease resistance, giving rise to higher stabilities.122,123 Conformational restriction of nucleosides is another successful strategy in designing potent DNA decoys. These locked nucleic acids (LNAs, 1.27) contain a 2′-O-4′-C-methylene bridge in the sugar of the DNA and have demonstrated added thermal stability and nuclease resistance while retaining their ability to bind

TFs.124,125 _{Peptide nucleic acids (PNAs, 1.28) are a class of DNA mimics in which the}

sugar-phosphate backbone is replaced by N-(2-aminoethyl)glycine units and are able to efficiently undergo Watson-Crick hybridization.126,127 Recently, PNA-DNA decoy chimeras have been shown to inhibit the NF-κB and Sp1 TFs and are fully resistant to exonucleases.128

Tethering the two hybridized strands of a DNA decoy together with one or two closed nucleotide loops on the end form either hairpin or dumbbell structures (Figure 1.20). These structures exhibit improved nuclease stability, improved sequence

O O O 3' O O O P O O 5' B B 1.24 Phosphodiester O O O 3' O O O P O S 5' B B 1.25 Phosphorothioate O O O 3' O O O P O 5' B B 1.26 Methylphosphonate Me O O O 3' O O O P O O 5' B B 1.27 Locked nucleic acids (LNAs) O O 1.28 Peptide nucleic acids (PNAs) N O B HN 5' O NH N _O B NH O N H2NOC O B

Scheme 1.1. Modifications to DNA to improve its serum and intracellular stability. B denotes any

specificity, cellular uptake, and lowered toxicity.119,129 Studies with AP-1 and STAT decoys have demonstrated that hairpin and dumbbell DNA decoys yield significantly greater TF inhibition in vitro (78% knockdown with the dumbbell versus 39% knockdown with the phosphorothioate modified). Importantly, they are more stable and effective over phosphorothioate and unmodified DNA decoys in vivo as well.130-132 Another advantage of hairpin and dumbbell DNA decoys is an added level of specificity. Changing the sequence of the loop region of a pan STAT dumbbell DNA decoy discriminates it for either STAT1 or STAT3.132,133 These studies demonstrate that hairpin and dumbbell DNA decoys are markedly improved over other chemically modified DNA decoys, and therefore, utilization of these decoys are quite relevant for therapeutic intervention and as a tool for further research.

The efficiency and versatility of DNA decoys make it an attractive approach for use as a tool and a possible therapeutic agent. Over the past couple of decades, numerous reports have validated utilizing this approach and the increased utilization over a wide breadth of applications and range in clinical areas.134,135 Several targets and applications are summarized in Table 1.1.20,135-146 Among these are regulating several manifestations of inflammation including cystic fibrosis (NF-κB),140 _{regulating neural}

stem cell differentiation (HNF4-1 and MAZ-1),141 reducing oxidative

Figure 1.20. (a) Hairpin and (b) dumbbell oligonucleotide structures formed by tethering two

stress-induced cardiac fibroblast proliferation (AP-1),142 _{and altering cell-cycle regulatory}

genes (E2F).20,145,146 Since this approach gives an efficient and specific TF inhibition, a wide-ranging list of DNA decoys have been developed to inhibit numerous TFs implicated in cancer growth and development (Table 1.1).147-154 Among these, DNA decoys designed against NF-κB, Sp1, STAT3, and MAZ have all demonstrated anti- cancer activity over many different cancer types including breast, melanoma, colorectal, pancreatic, and several more.147-150 Additionally, DNA decoys designed against hormone receptor TFs including the androgen receptor (AR), the estrogen receptor (ER), and the cyclic AMP receptor protein (CRP) have all been shown to induce apoptosis and cell death in several cancer types.151,152,155,156

The DNA decoy approach is also validated in several in vivo mice and rat models. Local administration of an NF-κB decoy to rats induced apoptosis of osteoclasts via Fas signaling, which could be used as a strategy for treatment of conditions such as osteoporosis, peri-articular osteolysis, inflammatory arthritis, and Paget’s syndrome.157 An E2F DNA decoy was administered into rats via HVJ-liposome complexes and demonstrated reduced expression of the c-myc, cdc2, and PCNA genes, which resulted in the inhibition of vascular smooth muscle cell (VSMC) proliferation.158 More recently, a phosphorthioate STAT3 DNA decoy has entered the “first-in-human” Phase 0 clinical trials for the treatment of head and neck squamous cancer.159 STAT3 expression and cell viability was reduced in the head and neck cancers injected with the decoy compared to the saline control. Additionally, since systemic administration results in degradation of the decoys, dumbbell STAT3 decoys were synthesized and attached hexaethyleneglycol

linkers to yield a cyclic STAT3 decoy. Intravenous injection of this cyclic decoy inhibited xenograft growth and downregulated STAT3 target genes within the tumor, demonstrating successful systemic administration of DNA decoys.159 Collectively, the DNA decoy strategy for the inhibition of transcription factor activity can be considered one of the most useful approaches for both a therapeutic for disease progression and as a tool to examine molecular mechanisms.