Special Report Special Focus Issue: Frontiers in Nucleic Acid-Based Drug R&D Future For reprint orders, please contact [email protected] Medicinal 7 Chemistry Special Report 2015/08/28 DNAzyme-based therapeutics for cancer treatment Future Med. Chem. Gene-silencing strategies based on catalytic nucleic acids have been rapidly developed Shujun Fu1 & Lun-Quan Sun*,1 in the past decades. Ribozymes, antisense oligonucleotides and RNA interference 1Center for Molecular Medicine, Xiangya have been actively pursued for years due to their potential application in gene Hospital, Central South University Changsha, China 410008 inactivation. Pioneered by Joyce et al., a new class of catalytic nucleic acid composed *Author for correspondence: of deoxyribonucleotides has emerged via an in vitro selection system. The therapeutic [email protected] potential of these RNA-cleaving DNAzymes have been shown both in vitro and in vivo. Although they rival the activity and stability of synthetic ribozymes, they are limited by inefficient delivery to the intracellular targets. Recent successes in clinical testing of the DNAzymes in cancer patients have revitalized the potential clinical utility of DNAzymes. 00 Due to the simple four-nucleotide chemistry system was based on hydrolytic cleavage of and standard Watson–Crick pairing, nucleic a phosphodiester and nested PCR [1–3] . First, acids are appealing targets for exogenous they established a pool of 1014 ssDNA mol- regulation of gene expression. Utilizing ecules. Each one contained a 5′ biotin moi- hybridization to achieve artificial gene sup- ety, followed by a 50 random deoxyribonu- pression is mostly related to the involvement cleotides domain which was flanked by fixed of single-stranded oligodeoxynucleotides and sequence. Then these molecules were com- double-stranded siRNA based approaches, bined to a streptavidin affinity matrix and both of which have been developed in labora- washed with buffer to remove the unbound. tory and clinical applications. Other nucleic Next, the same buffer containing certain acids-based technologies may also find appli- cation passed through the matrix to cause cations in this field. Gene suppression medi- cation-dependent cleavage of phosphoester ated by RNA-cleaving catalytic DNA has and release the catalytic DNAs. These DNAs 13 been one of the promising strategies for such were collected and amplified by nested PCR, applications. These molecules recognize their reintroducing the 5´ biotin and target phos- targets by hybridization and then cleave the phoester. After serveral rounds of selection, target without assistance of any host-encoded DNAzymes were obtained. 2015 proteins. The nuclease-independent cata- Further biochemical characterization lytic DNA or DNAzymes open a new field revealed that the DNAzymes varied in their that is beyond the scope of RNAi-depen- sequences while all of them had two basic dent siRNA and ribonuclease H-dependent domains, catalytic domain and substrate- oligodeoxynucleotides. binding domain. Based on the structures, In 1994, Breaker and Joyce proposed that two types of DNAzymes selected via in vitro DNA could have catalytic activity as for system, the ‘8–17’ DNAzymes and ‘10–23’ ribozymes. However, no naturally occurring DNAzymes were proposed (Figure 1). DNAzyme was found because DNA always The ‘8–17’ DNAzyme was derived from presents as a complete duplex. Thus, they the 17th clone obtained after eight rounds of came up with an in vitro selection system to selective amplification and ‘10–23 DNAzyme obtain DNAzymes that cleaved RNA. The was similarly obtained from the 23rd clone part of 10.4155/fmc.15.106 © 2015 Future Science Ltd Future Med. Chem. (Epub ahead of print) ISSN 1756-8919 Special Report Fu & Sun Key terms the cell cycle, excessive signals promoting cell survive are always activated. PI3K-AKT signaling is thought to DNAzyme: A DNA molecule that has the ability to perform be one of the most important pathways, which makes catalytic action. it an ideal target for DNAzymes. Yang et al. designed LMP1: A critical oncogenic protein encoded by EBV. five DNAzymes, based on the analysis of sequences, c-jun: A transcription factor that is upregulated in many thermodynamics and site distribution within the Akt1 types of cancers. gene. One of the DNAzymes, namely Dz2, strongly inhibited Akt1 mRNA and protein expression in the after ten-round amplification. A structure of ‘8–17’ nasopharyngeal carcinoma (NPC) cells CNE1-LMP1, DNAzymes have a 13-nt-long catalytic core, which and markedly suppressed NPC xenograft growth in consists of a short internal stem-loop followed by an nude mice. Mechanistically, Dz2 was shown to affect unpaired region of 4 nt. The stem contained 3 bp and multiple key tumorigenic processes in vitro and in vivo at least two of them were G-C while the loop had a by downregulating AKT1 expression [8]. There was fixed sequence of 5´-AGC-3´. The unpaired region had a also a recent study showing that the AKT1-specific sequence of 5´-WCGR-3´ or 5´-WCGAA-3´ (W = A/T, DNAzymes significantly inhibited cell proliferation, R = A/G). These DNAzymes preferred an rG-dT induced apoptosis and inhibited invasion in thyroid wobble pair located immediately downstream from the tumor SW597 cells [9]. These DNAzymes acted via cleavage site. However, altering the length of the stem the mechanisms of inhibiting cellular proliferation by or the sequence of the loop could lead to noncatalytic direct suppression of AKT1 expression. activity. The inhibition of DNA methyltransferases The catalytic core of ‘10–23’ DNAzymes were com- (DNMTs) may lead to demethylation and expression posed of 15 nt [4–6], the eighth of which was usually T, of the silenced tumor suppressor genes. Wang et al. C or A while a T often provided the highest activity. utilized a multiplex selection system and generated The substrate-binding domain bound RNA substrates some efficient RNA-cleaving DNAzymes targeting through standard Watson–Crick pairing and these DNMT1. Introduction of the DNAzymes caused sig- DNAzymes cleaved at unpaired purine and paired nificant downregulation of DNMT1 expression and pyrimidine. reactivation of p16 gene, resulting in reduced cell pro- DNAzymes are more stable in physiological condi- liferation of bladder cancers [10] . This study suggests tions than ribozymes, even extending the half-life from that DNAzyme targeting of epigenetic modifying minutes to over 21 h in serum after modification [7]. Also enzymes may provide a novel strategy for epigenetic their well-catalytic characteristic make them promising inactivation of the genes that caused the uncontrolled in targeted gene therapy. With known target, one could proliferation. design corresponding DNAzymes to downregulate c-jun, a basic leucine-zipper (bZIP) protein and the target disease genes. To date, DNAzymes, mainly prototypic member of AP-1 transcription factor, was ‘10–23’ type, have been extensively explored for their upregulated in a variety of cancers. Previous work ability to validate target gene and therapeutic potentials. demonstrated that Dz13 could well target c-jun and effectively inhibit tumor growth [11] . Further studies Antiproliferative DNAzymes examined the dose range, sustained effect after cessa- Uncontrolled proliferation is a typical feature of cancer tion of therapy and biodistribution of Dz13, showing cells. To extend the normal life span and escape from that Dz13 was safe and tolerated in animal models [12] . Target RNA 5´ NNNNNNNA GNNNNNNN 3´ 5´ NNNNNNNR YNNNNNNN 3´ • DNAzyme 3´ NNNNNNN TNNNNNNN 5´ 3´ NNNNNNN RNNNNNNN 5´ R G C C A G C G G G W A W = A/T C C G G G A T C C R = A/G A A Y = U/C C G A T C 8–17 10–23 Figure 1. Structures of ‘8–17’ and ‘10–23’ DNAzymes. The substrate binding domain binds RNA through Watson–Crick pairing. The catalytic domain cleaves targets at the site indicated by the arrow. 10.4155/fmc.15.106 Future Med. Chem. (Epub ahead of print) future science group DNAzyme-based therapeutics for cancer treatment Special Report In this study, the dermal tumor models of squamous expression and caused suppression of tumor growth in cell carcinoma and basal cell carcinoma were devel- xenograft mouse models [17] . Importantly, the Bcl-xL- oped and Dz13 injected twice a week intratumorally. DNAzymes could sensitize the tumor cells to chemo- The effect of Dz13 sustained during the injection therapy and overcome Taxol resistance [17] . Targeting in a dose-dependent manner. No tumor growth was other genes involved in apoptosis, such as survivin and observed even 10 days after cessation of therapy when IGF-II, by DNAzymes was also shown to be a viable injected with 100 μg, comparing to 10 and 20 μg approach to cancer growth suppression [18,19]. Dz13-treated mice. They also provided the evidence that the observed effect was dependent on the catalytic DNAzymes for antimetastasis domain of the DNAzyme and the adaptive immune Metastasis is one of the hallmarks for malignant can- system of the host. To evaluate the drug potential of cers. Destroy of the basement membrane and invasion Dz13, biodistribution and toxicology analysis were of cancer cells to extracellular matrix are key steps dur- implemented. Transient liver accumulation of Dz13 ing the process of tumor metastasis. Matrix metallo- was detected after intratumoral administration, while proteinase (MMP), especially the MMP-9, is a critical no changes of liver function were observed. Phar- regulator in extracellular matrix degradation, which macokinetics of Dz13 via intratumoral, intravenous makes it a potential DNAzyme target in metastasis and intradermal routes revealed a favorable profile inhibition. Yang et al. demonstrated that a DNAzyme- for clinical use. GLP-compliant plasma distribution, targeting MMP9 significantly suppressed the invasion repeat-dose toxicology and local tolerance studies all and migration of lung cancer cells (A549) [20]. Hallett indicated Dz13 was well tolerated, with no abnormal designed anti-MMP-9 DNAzyme (AM9D) and evalu- clinical signs, necropsy findings or adverse effects [12] .