Deoxyribozymes As Catalytic Nanotherapeutic Agents Levon M

Published OnlineFirst February 13, 2019; DOI: 10.1158/0008-5472.CAN-18-2474

Cancer Review Research

Deoxyribozymes as Catalytic Nanotherapeutic Agents Levon M. Khachigian

Abstract

RNA-cleaving deoxyribozymes (DNAzymes) are synthet- and nanosponges, and the emerging role of adaptive ic single-stranded DNA-based catalytic molecules that can immunity underlying DNAzyme inhibition of cancer be engineered to bind to and cleave target mRNA at growth. DNAzymes represent a promising new class of predetermined sites. These have been used as therapeutic nucleic acid–based therapeutics in cancer. This article dis- agents in a range of preclinical cancer models and have cusses mechanistic and therapeutic insights brought about entered clinical trials in Europe, China, and Australia. This by DNAzyme use as nanotools and reagents in a range of review surveys regulatory insights into mechanisms of basic science, experimental therapeutic and clinical appli- disease brought about by use of catalytic DNA in vitro and cations. Current limitations and future perspectives are also in vivo, including recent uses as nanosensors, nanoﬂowers, discussed.

DNAzyme Catalysts: Mechanistic and transfected with commercial delivery agents or electroporated Design Considerations into cells. DNAzyme use in experimental animal models can be hampered by delivery issues, especially in regard to systemic DNAzymes are synthetic single-stranded enzymatic DNA mole- administration (10). This has motivated local delivery meth- cules that bind to their target mRNA via Watson–Crick base odologies in animals, such as intracardiac, intratumoral, and pairing and cleave a specific interbase junction in the mRNA by intraarticular injection, or tissue immersion. Novel biodegrad- a deesterification reaction (1–3). This involves metal-assisted able template-based DNAzymes have recently been developed 0 deprotonation of 2 -hydroxyl in the RNA, producing RNA frag- that facilitate cancer cell recognition and internalization (11). 0 0 0 ments terminating in a 2 ,3 -cyclic phosphate and a 5 -hydroxyl That said, oligonucleotides may not necessarily require a spe- (Fig. 1; ref. 4). DNAzymes were originally developed as catalysts cific delivery vehicle for endosomal or lysosomal seques- in vitro and then used as gene-silencing agents within cells where tration (12), and indeed there are numerous examples of endogenous mRNA is targeted. Breaker and Joyce reported the first DNAzyme efficacy in experimental models without use of any DNAzyme in late 1994, cleaving a ribonucleotide linkage by way carrier. In experimental tumors, this may involve an enhanced 2þ of a transesterification reaction requiring Pb (5). Studies have permeability and retention (EPR) effect (13). DNAzymes have since demonstrated that DNAzymes also use other divalent ions been used in the clinic with or without a specifictransfection 2þ 2þ 2þ as cofactors, including Mg ,Zn , and Ca (6). DNAzyme agent as discussed below. Second, the half-life of the target RNA action is not confined to RNA cleavage; these agents can also would ideally be short and background mRNA expression of 0 catalyze bond formation, such as ligation between the 3 -hydroxyl the target low. Cleavage destabilizes RNA and accelerates its 0 and 5 -triphosphate terminus in RNA (7, 8). The first high- degradation, meaning less protein is produced. In our own resolution (2.8 Å) crystal structure of a DNAzyme in its post- work, we have generated DNAzymes targeting immediate-early catalytic state complexed with RNA was recently reported with genes that are poorly expressed in normal tissues and have one such DNAzyme. Ponce-Salvatierra and colleagues found that relatively short half-lives. For example, c-jun mRNA (targeted the 44 nucleotide RNA-ligating DNAzyme 9DB1 forms a double by the DNAzyme Dz13; ref. 14) rapidly decays with a half-life pseudoknot in complex with a 15-nucleotide RNA strand (9). of 20 minutes (15). The half-life of early growth response-1 This review focuses mainly on RNA-cleaving DNAzymes. (Egr-1) mRNA (targeted by ED5; ref. 16) is even shorter (17). There are several considerations when designing DNAzymes These genes are expressed at low or undetectable levels in as endogenous RNA-cleavage–based inhibitors. First, there uninjured, growth quiescent arteries (14, 16, 18). Third, should be sufficient intracellular accumulation of the oligonu- although target site specificity is prescribed by the choice of cleotide and access to mRNA. Typically in vitro, DNAzymes are bases in the binding arms, DNAzymes, like other antisense- based targeting strategies, can have "off-target" effects, arising from partial complementarity of DNAzyme arm sequences Vascular Biology and Translational Research, School of Medical Sciences, with an unintended target or Toll-like receptors that recognize Faculty of Medicine, University of New South Wales, Sydney, Australia. CpG-motif–containing DNA (TLR-9; ref. 19). This can be Corresponding Author: Levon M. Khachigian, University of New South Wales, addressed experimentally using DNAzymes as controls bearing High Street, Gate 9, Sydney, NSW 2052, Australia. Phone: 61-2-9385-2537; single-nucleotide mutations (e.g., G6>C6) in the 15 nucleotide Fax: 61-2-9385-1797; E-mail: [email protected] 10–23 catalytic domain (i.e., 50-GGC TAG6 CTA CAA CGA-30; doi: 10.1158/0008-5472.CAN-18-2474 refs.1,4),renderingtheDNAzymeunabletocleavebut 2019 American Association for Cancer Research. remaining identical to the test DNAzyme in all other

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R Y O 2’ O O P O

3’ OOH O O - 5’ Figure 1. O HO Mechanism of action by the Y RNA-cleaving 10–23 DNAzyme. The 10–23 DNAzyme (depicted here with

R O þ OOH

O 9 9 nucleotide arms) hybridizes O N P OOH N with its target RNA through O N N

O N – O Watson Crick base pairing and N – N N N N N catalyzes cleavage between an N N 3’ N R N N unpaired purine (R) and a paired N N N N N N N pyrimidine (Y) through an internal N N G N 5’ N N A phosphoester transfer reaction N G 0 N G whereby the 2 -hydroxyl attacks the 5’ N N C N adjacent phosphodiester bond. This 3’ N C T results in one fragment ending with a 10-23 20,30-cyclic phosphate and the other A DNAzyme A ending with a 50-hydroxyl. Other A G sugar phosphates in the DNAzyme or C C RNA are not shown for simplicity. A T R, A or G (purine); Y,CorU (pyrimidine); N, G or A or T or C. © 2018 American Association for Cancer Research

respects (20). Another way is to test for TLR9/NF-kBactivation rat carotid arteries, a finding later confirmed by Liu and col- alongside reference oligonucleotides (21). Such "off-target" leagues who used ED5 to inhibit Egr-1 and TGFb expression, effects should not be confused with DNAzymes targeting resulting in increased levels of nitric oxide synthase and NO master regulators, such as transcription factors, where and preservation of endothelial cell function (31). Wang and that DNAzyme would also be expected to affect other genes colleagues further found ED5 reduced levels of Egr-1 and reliant on the targeted factor. Fourth, DNAzyme stability platelet-derived growth factor (PDGF)-BB, cdk4, cyclin D1, against nucleolytic attack is typically achieved by structural monocyte chemotactic factor (MCP)-1, and intercellular adhe- modifications such as inverted bases, phosphorothioate or sion molecule (ICAM)-1 (32). In a rat autologous vein graft phosphoramidate linkages, locked nucleic acids, and 20-meth- model, Liu and colleagues used essentially the same DNAzyme oxy substitutions. These modifications can be incorporated (EDRz) to suppress Egr-1 expression, SMC proliferation, and during oligonucleotide manufacture. Fifth, although there are intimal thickening (33). EDRz also reduced formation of potentially hundreds of target sites in a typical mRNA, con- abdominal aortic aneurysm in rats, suppressing Egr-1, matrix ventional sequence selection for a DNAzyme is time-consum- metalloproteinase (MMP)-2, and MMP-9 levels (34). Egr-1 ing, labor-intensive, and cumbersome (22). Investigators have DNAzymes also inhibited intimal thickening in a rat model used predictions on RNA folding, low free energy, and second- of carotid ligation following adventitial delivery (35), reduced ary structure (23, 24), but this does not ensure DNAzyme myocardial infarct size in the area at risk in a rat model of efficacy (14). Finally, it should be noted that DNAzyme cata- myocardial ischemia–reperfusion injury (36), in-stent resteno- lytic efficacy also depends on the length of binding arms even sis in a porcine model of stenting (37), and improved for a single target site (6, 21, 25). left ventricular systolic function in porcine models of myocardial ischemia–reperfusion injury and intracoronary deliv- DNAzymes as Versatile Experimental ery (38, 39). Dickinson and colleagues further demonstrated the effects of Egr-1 suppression on pulmonary vascular remo- Drugs in Noncancerous Settings deling in an experimental flow-associated pulmonary arterial Since the discovery of the 10–23 DNAzyme by Santoro and hypertension rat model. Intravenous administration of ED5 (in Joyce reported in 1997, so named from the 23rd clone from the DOTAP) reduced pulmonary vascular Egr-1 expression, vascu- 10th round of an in vitro selection procedure (1, 4), DNAzymes lar remodeling and neointima formation, and reduced expres- have been used as experimental drugs in a diverse range of models. sion of PDGF-B, TGFb,IL6,andp53(40).ED5alsoinhibited New monovalent cation-dependent RNA-cleaving DNAzymes, pulmonary vascular resistance, right ventricular (RV) systolic such as Ag10c (26, 27), Ce13d (28), EtNa (29), and NaA43 (30), pressure, and RV hypertrophy. Nakamura and colleagues deliv- with catalytic domains that differ from 10–23 have also emerged, ered ED5 into interstitial fibroblasts in vivo by electroporation and it will be interesting to see how these progress within via the ureter that reduced interstitial fibrosis in obstructed preclinical systems. kidneys. The DNAzyme inhibited Egr-1, TGFb,andtypeI The first in vivo demonstration of DNAzyme efficacy in an collagen expression (41). animal model of any kind was ED5, a 10–23 DNAzyme DNAzymes have been investigated in a broad range of other targeting Egr-1 (16). ED5 inhibited vascular smooth muscle noncancerous applications in vitro and in vivo. Examples include cell (SMC) growth and intimal thickening in balloon-injured DNAzymes targeting tumor necrosis factor-a (TNFa), GATA3,

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2019 American Association for Cancer Research. Downloaded from www.aacrjournals.org Table 1. Examples of DNAzymes evaluated in preclinical models and clinical trials Preclinical models Allograft or Carrier or Administration Target DNAzyme Tumor type Xenograft vehicle route and dose Ref c-jun Dz13 Basal cell carcinoma Xenograft DOTAP/DOPE Intratumoral injection (10–100 (20) mg) Squamous cell carcinoma Allograft DOTAP/DOPE or Intratumoral injection (10–100 (20, 79) cancerres.aacrjournals.org FuGENE6 mg) or coadministration (50 Published OnlineFirstFebruary13,2019;DOI:10.1158/0008-5472.CAN-18-2474 mg) Melanoma Allograft FuGENE6 Coadministration (750 mg) (74) Allograft DOTAP/DOPE Intratumoral injection (100 mg) (59) Breast Xenograft Saline Coadministration (0.4 mmol/L) (75) Prostate Xenograft Saline Coadministration (0.4 mmol/L) (75) Osteosarcoma Xenograft Chitosan or saline Intraperitoneal or intratibial (0.4 (75, 77, 78) mmol/L or 250 ng) with doxorubicin Xenograft Doxorubicin/ Intratumoral injection (150 ng) (54) chitosan Liposarcoma Xenograft FuGENE6 Coadministration (250 ng) (76) on September 26, 2021. © 2019American Association for Cancer Research. Bcl-xL DT882 Prostate cancer Xenograft Saline Osmotic minipump (12.5 mg/kg/ (58) d) with intraperitoneal Taxol (25 mg/kg) Bcl-2 DT912 Prostate cancer Xenograft Saline Osmotic minipump (12.5 mg/kg/ (102) d) with intraperitoneal Taxol (25 mg/kg) Egr-1 ED5 Breast carcinoma Xenograft FuGENE6 Intratumoral injection (20 mg) or (68) coadministration (750 mg) DzF Breast carcinoma Xenograft Saline Intratumoral injection (10 mg) (69) ssDNA encoding Breast carcinoma Xenograft Saline Intratumoral (0.5 mmol/L in 50 mL (11) complementary per 50 mm3 tumor volume) sequence of hEgr-1 DNAzyme (hED5, ref. 16), Agents Nanotherapeutic Catalytic as Deoxyribozymes survivin DNAzyme and AS1411 aptamer VEGFR1 DT18 Melanoma Allograft FuGENE6 Coadministration (100 mg) (103) Nasopharyngeal Xenograft FuGENE6 Intratumoral injection (100 mg) (103) carcinoma VEGFR2 DNAzyme Breast carcinoma Xenograft Histidine-lysine Intratumoral injection (2.9 mg) (67)

acrRs 95 ac ,2019 1, March 79(5) Res; Cancer polymer or saline b1 integrin b1DE Colon adenocarcinoma Xenograft Saline or Intratumoral injection (1.25 mg) (72, 73, 104) unspeciﬁed b1DE Prostate Xenograft Saline or Intratumoral injection (1.25 mg) (72, 73) unspeciﬁed Aurora DZ2 Prostate Xenograft Saline Intratumoral injection (8 mg) (87) kinase A (Continued on the following page) 881 882 Downloaded from acrRs 95 ac ,2019 1, March 79(5) Res; Cancer Khachigian

Table 1. Examples of DNAzymes evaluated in preclinical models and clinical trials (Cont'd ) cancerres.aacrjournals.org Preclinical models Allograft or Carrier or Administration Published OnlineFirstFebruary13,2019;DOI:10.1158/0008-5472.CAN-18-2474 Target DNAzyme Tumor type Xenograft vehicle route and dose Ref EBV-LMP1 DZ1 Nasopharyngeal Xenograft FuGENE6 Intratumoral injection (100 mg) (85) carcinoma DZ1 Nasopharyngeal Xenograft FuGENE HD Intratumoral injection (100 mg) (61) carcinoma plus radiotherapy DZ1 Nasopharyngeal Xenograft Not speciﬁed Intratumoral injection (dose not (83) carcinoma speciﬁed) plus radiotherapy DZ509 Nasopharyngeal Xenograft Phosphate- Intratumoral injection (33 mg) (86) carcinoma buffered saline Akt1 DZ2 Nasopharyngeal Xenograft FuGENE6 Intratumoral (10 mg) (105) on September 26, 2021. © 2019American Association for Cancer Research. carcinoma HSP70 ssDNA with HSP70 Breast carcinoma Xenograft Saline Intravenous (100 mL of 0.1 mg/L (71) DNAzyme repeats stock), irradiation (2 W/cm2, 10 minutes) MMP9 AM9D Breast carcinoma Transgenic Phosphate- Intratumoral (10, 25 mg) (57) buffered saline Clinical trials Target DNAzyme Patient type Phase Trial size (n) Carrier/vehicle Administration route and dose Ref c-jun Dz13 Basal cell carcinoma 1 9 DOTAP/DOPE Single intratumoral injection (60) (10–100 mg) EBV-LMP1 DZ1 Nasopharyngeal 1 40 Saline Multiple intratumoral injections (61) carcinoma (6 mg) plus radiotherapy GATA3 SB010 (hgd40) Allergic asthma 2A 40 Saline Multiple inhalations (10 mg) (62, 90) Healthy and asthma 1 108 Saline Single (5–40 mg) and multiple (63) inhalations (5-20 mg) Eosinophilic chronic 2A 23 Saline Multiple inhalations (10 mg) (92) obstructive pulmonary disease SB011 (hgd40) Atopic eczema 2 25 Water/Oil/Water emulsion Multiple topical applications (10 Completed, mg) NCT02079688 SB012 (hgd40) Ulcerative colitis 1/2 18 Saline Multiple intrarectal applications Completed, (SECURE) (7.5 mg/mL in 30 mL, maximum NCT02129439 daily dose 225 mg) acrResearch Cancer Published OnlineFirst February 13, 2019; DOI: 10.1158/0008-5472.CAN-18-2474

Deoxyribozymes as Catalytic Nanotherapeutic Agents

influenza A virus, thioredoxin-interacting protein (TXNIP), xylo- (Table 1). Carriers facilitating delivery have included Super- syltransferase-1 (XYLT1), inducible nitric oxide synthase (iNOS), fect (16, 51), Fugene6 (32, 35–39), DOTAP (40), Lipofecta- plasminogen activator inhibitor 1 (PAI-1), and c-jun. Local injec- mine (33, 46), chitosan (54, 55), nanoparticles (42), electro- tion of DNAzyme functionalized gold nanoparticles targeting poration (41), aptamer targeting (11), transcatheter arterial TNFa in a rat model of acute myocardial infarction reduced chemoembolization with Fugene6 and Lipiodol (assists drug inflammation and improved acute cardiac function (42). Intrar- retention within the tumor; ref. 56), or simply no carrier (47, 49, ectal delivery of GATA3 DNAzymes (hgd40) prevented colitis in 50, 57, 58). We used Dz13 complexed with DOTAP-DOPE both mice and reduced IL6, IL9, and IL13 expression (43). Sel and preclinically (20, 59) and clinically (60). This complex has colleagues delivered 10–23 DNAzymes (gd21) targeting GATA3 high polydispersity and primary particle size 100–200 nm (60). in a murine model of experimental allergic asthma via intranasal DNAzymes have also been used with signs of efficacy in human administration. The DNAzyme prevented airway inflammation subjects without a carrier (61–63). and mucus production. gd21 also inhibited airway hyperrespon- siveness to methacholine. Similar effects were observed using Targeting angiogenesis gd21 in a mouse model of chronic experimental asthma, and One of Judah Folkman's many legacies is the notion that off-target effects were not detected (44). The authors suggested tumors cannot grow beyond a few millimeters without an active that pulmonary surfactant may serve as an endogenous transfec- blood supply (64, 65). Indeed, in metastatic melanoma, sur- tant supporting cellular uptake. Dicke and colleagues did not vival time after primary diagnosis or appearance of first metas- detect TLR-9–mediated cell activation by GATA3 DNAzymes in tasis appears to be shorter in those with highly vascular macrophage cell lines or primary innate immune cells, suggesting tumors (66). The first study to demonstrate inhibition of tumor the absence of nonspecific innate immune cell activation (45). growth in vivo with a DNAzyme was that by Zhang and collea- DNAzymes targeting influenza A virus M2 (Dz114) inhibited viral gues, who generated a 10–23 DNAzyme targeting the transla- replication in host cells in a dose-dependent fashion whereas tional start site in VEGFR2 mRNA (67). The DNAzyme, admin- Mt-Dz114, bearing a single G14>C14 mutation in 10–23, had no istered in saline by intratumoral injection, retarded breast cancer activity (46). Huang and colleagues used DNAzymes targeting growth in a xenograft model. It reduced ki-67 expression, TXNIP to reduce extracellular matrix deposition in kidneys of increased apoptosis, and lowered blood vessel density within streptozotocin-induced diabetic rats. TXNIP DNAzymes, deliv- the tumors (67). The 10–23 DNAzymes targeting Egr-1 (DzF ered in saline using implanted osmotic minipumps, also andED5)wereusedtoinhibitthegrowthoftwohumanbreast reversed autophagosome accumulation and lowered autophagic cancers (MDA-MB-231 and MCF-7) as xenografts in mice (68, clearance in tubular cells from human diabetic compared with 69). ED5 blocked neovascularization in mice bearing Matrigel nondiabetic kidneys (47). TXNIP DNAzymes reduce inflamma- plug and angiogenesis in tumor-bearing mice and inhibited some signaling, oxidative stress, and interstitial fibrosis in the FGF-2 (68). Later work by Yao and colleagues showed ED5 tubulointerstitium of diabetic rats (48). DNAzymes targeting suppression of Egr-1 in mouse cornea, reduction of herpetic þ XYLT1 delivered by osmotic minipump without a carrier stromal keratitis and inhibition of CD31 staining, FGF-2, and increased cortical spinal tract growth in rats and provided VEGFA (70). Jin and colleagues developed a biodegradable behavioral improvement after 19 weeks (49). Intraperitoneal cancer therapeutic system, termed DNA nanoflowers (DNF), delivery of iNOS DNAzymes, also without a carrier, reduced that uses a rolling circle amplification (RCA) template to pro- LPS-induced lethal systemic inflammation and enhanced sur- duce single-stranded DNAs targeting cancer cells with multiple vival (50). Xiang and colleagues suppressed PAI-1 in per-infarct molecular targets. The aptamer (AS1411) in the self-assembly endothelium within 2 days of intracardiac delivery of PAI-1 DNF enables targeted delivery to nucleolin-expressing cancer DNAzymes (E2) and levels remained so for at least 2 weeks. cells and receptor-mediated endocytosis. Two 10–23 DNAzyme þ Intravenous administration of CD34 cells containing angio- sequences in the template caused suppression of Egr-1 and blasts with intramyocardial E2 increased neovascularization survivin in MCF-7 tumor xenografts, increased apoptosis þ over that stimulated by CD34 cells alone. Moreover, combi- and reduced tumor size (11). Recent studies with DNAzyme- þ nation of intramyocardial E2 with intravenous CD34 cells based nanosponges, in which long single-stranded DNAzyme improved functional recovery of left ventricular ejection fraction sequences targeting heat shock protein 70 (HSP70) synthesized within 2 weeks (51). DNAzyme inhibition of PAI-1 increased by RCA were assembled with cationic polymer, sensitized MCF- myocardial neovascularization and lowered cardiomyocyte apo- 7 cells to heat and, after intravenous administration, localized ptosis after myocardial infarction (52). Dz13, targeting c-jun within tumors via the EPR effect and improved photothermal mRNA, has been used to block H5N1 influenza virus replication therapeutic efficiency (71). Future studies with other key molec- in human lung epithelial cells and reduce production of TNFa, ular targets should determine the wider utility of these clinically IL6, and IFNb. In mice infected with H5N1, intranasal delivery relevant complexed approaches. of Dz13 improved survival (55.5% in the Dz13 group compared Niewiarowska and colleagues produced a 20-O-methyl-substi- with 11% in the scrambled DNAzyme group) and reduced tuted 10–23 DNAzyme targeting human b1 integrin (hb1DE) pulmonary inflammatory response and viral burden. Dz13 may or mouse b1 integrin (mb1DE) subunit (72). hb1DE inhibited be useful to combat conditions involving a cytokine storm b1 integrin expression in human colon adenocarcinoma cells including viral pneumonia (53). and prostate carcinoma cells. mb1DE also reduced angiogenesis and xenograft growth following intratumoral administration without a carrier (72). DNAzyme was more proficient than Preclinical Anticancer Effects of DNAzymes siRNA in blocking colon adenocarcinoma and prostate carci- DNAzymes have been evaluated in a wide range of experi- noma growth even in circumstances where 5-fold less DNAzyme mental allograft, xenograft, and transgenic models of cancer was delivered than siRNA (73). Recently, Zhang and colleagues

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used a TACE strategy to deliver VEGFR1 DNAzymes into tumors was delivered in saline by way of abdominal osmotic pumps, in a rabbit hepatocellular carcinoma model and observed revealed chemosensitization of prostate cancer xenografts suppression of VEGFR expression and tumor microvascular when DNAzyme treatment was combined with taxol (58). permeability (56).

Targeting proliferation and survival DNAzymes in Clinical Trials Dz13hasbeenusedinarangeofcancermodels.Dz13 At least three different 10–23 DNAzymes have been evalu- inhibited B16 melanoma growth as subcutaneous tumors in ated in clinical trials in over 250 patients with cancer and other immunocompetent mice (74). Tan and colleagues demonstrat- disease with no serious adverse events (Table 1). ed tumor growth inhibition by Dz13 in a range of tumor xenograft models, including prostate and breast cancer (75). c-jun Dz13 suppressed the expression of MMP2, MMP9, and MT1- We conducted a comprehensive preclinical toxicological MMP (75). Dz13 also inhibited growth of liposarcoma (76) analysis of a clinically ready DNAzyme formulation (20). and osteosarcoma (77) and sensitized SaOS-2 tumor–bearing Dz13 had no adverse effects on hematologic and thrombotic mice to tumor growth inhibition by doxorubicin (78). Dz13 parameters in tumor-bearing mice. In non-human primates inhibited basal cell carcinoma (BCC) and squamous cell injected daily intravenously with Dz13 for up to 28 days at carcinoma (SCC) growth as intradermal xenograft tumors (20, doses >30-fold higher than the highest clinical dose (100 mg), 79) and reduced lung nodule formation in an intravenous therewerenosignificant clinical hematologic, biochemical, or model of SCC metastasis. Dz13 also inhibited tumor angio- organ histologic treatment-related changes. Biodistribution genesis in zebrafish (20) and retinal neovascularization in studies in mice revealed transient accumulation of Dz13 in mice (80). Dz13 tumor regression was more profound in the liver as tumor-associated Dz13 decreased (20). Dass and immunocompetent mice than in immunocompromised ani- Choong (88) found that Dz13, administered intravenously in mals (20). Dz13-mediated tumor regression was reversed by a bolus repeated dose or by pump in an in utero assay, had no administration of CD4/CD8 antibodies (20). This provided effect on blood cell populations or biochemical parameters in the first demonstration that inhibition of tumor growth by a normal mice. Dz13 had no adverse effect on wound healing, DNAzyme involves induction of antitumor immunity. More bone, or placental development except for slight hepatotox- recent studies demonstrate that Dz13 inhibits neighboring icity (determined histologically), which was also observed melanoma growth by way of an abscopal (i.e., distal) effect with the scrambled DNAzyme control. These preclinical find- þ þ in a manner dependent upon CD4 /CD8 T cells. That Dz13 ings suggested that Dz13 would likely be well tolerated treatment of melanoma prevented growth of subsequent new clinically. melanomas in the same animal (59) suggests that Dz13 may The DISCOVER trial (60) was the first reported human trial prevent tumor relapse through adaptive immunity. Marquardt of a DNAzyme. Nine patients with nodular BCC were recruited, and colleagues used Dz13 as a prototype in newly developed with each patient receiving a single intratumoral injection of protective drug-delivery systems to preserve DNAzyme integ- Dz13 (10–100 mgin50mL). Tumors were surgically removed 2 rity and enhanced skin penetration efficiency for dermal appli- weeks after Dz13 injection and compared with preinjection cations (81, 82). punch biopsy by IHC and histologic means. All patients DZ1, a 10–23 DNAzyme targeting Epstein Barr Virus (EBV)- completed the trial with no drug-related serious adverse events encoded latent membrane protein (LMP)1 that has oncogenic and no detectable systemic Dz13 exposure. c-Jun levels were properties in nasopharyngeal carcinoma, inhibited LMP1 reduced in the tumors of all patients treated with Dz13. expression and nasopharyngeal carcinoma (NPC) cell prolif- Moreover, the DNAzyme increased levels of a range of apo- eration in vitro. It also increased apoptosis and enhanced ptotic markers within the tumors. Dz13 also stimulated radiosensitivity when a suboptimal dose of radiation was inflammatory and immune cell infiltration. Notably, the combined with injection of DNAzyme (83). DZ1 caused down- majority of subjects had a reduction in histologic tumor depth. regulation of EBV lytic proteins BZLF1 and BMRF1 (84) and Dz13 was deemed safe, well tolerated, and potentially effica- reduced luciferase expressing human NPC compared with cious in humans (60). tumors treated with saline. Efficacy in response to radiotherapy was improved when DZ1 was given as adjunctive therapy to EBV-LMP1 radiotherapy, demonstrating the radiosensitizing effects of The EBV-LMP1–targeted DNAzyme DZ1 was evaluated in DZ1 (61). DZ1 reduces VEGF expression and inhibits survival China in patients with NPC in combination with standard of LMP1-positive NPC cells after irradiation (85). Ke and radiotherapy in a randomized double-blind study (61). Forty colleagues produced a different DNAzyme targeting LMP1 LMP1-positive patients received 6 mg (in 100 mL) DZ1 in saline (DZ509) and found that DZ509 suppressed LMP1 expression (n ¼ 20) or saline alone (n ¼ 20) by intratumoral injection and growth of C666-1 human NPC cells and induced apoptosis (twice a week for 7 weeks in conjunction with radiotherapy in vitro. In a C666-1 xenograft model, intratumoral injection of 5 times per week) and primary efficacy (tumor regression) was DZ509 suppressed tumor growth and LMP1 expression (86). assessed by MRI. Tumor regression at 3 months was higher in Yang and colleagues (83) generated a DNAzyme targeting the DZ1 group compared with saline. Although molecular AKT1 (Dz2). Dz2 suppressed AKT1 expression, inhibited NPC analysis of the effect of DZ1 on target gene expression in NPC cell proliferation, induced apoptosis, and reduced xenograft tissue was not performed (due to ethics restrictions) EBV growth (83). Other targets of DNAzymes have included aurora DNA copy number in plasma declined faster in DZ1 patients kinase A (87) and MMP9 (57). Studies by Yu and colleagues compared with saline patients. No adverse events were attrib- using DNAzymes targeting Bcl-xL (DT888), in which DT888 uted to DZ1 injection, no significant cardiovascular, hepatic, or

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Deoxyribozymes as Catalytic Nanotherapeutic Agents

renal events were reported, and DZ1 treatment did not increase sents an invaluable and potentially complementary research radiation-induced toxicity (61). This suggests that DNAzymes tool. DNAzymes cause transient repression of gene expression may be useful as adjunctive therapy to radiotherapy. akin to pharmacologic intervention, whereas CRISPR/Cas9 effects a stable genetic change. CRISPR/Cas9 has the potential GATA3 to correct genetic mutations whereas neither RNA-cleaving DNA- – The 10 23 DNAzymes have also been tested in humans in zymes nor RNAi currently have that capacity. The latter technol- noncancerous settings. DNAzymes targeting the transcription ogies produce so-called hypomorphic phenotypes (i.e., partial but factor GATA3 were evaluated in a randomized, double-blind, not complete loss in gene function brought about by reduced placebo-controlled multicenter phase IIA trial involving 7 sites mRNA/protein). Stable gene editing through CRISPR/Cas9 has in Germany specializing in respiratory research. Interestingly, important therapeutic implications in a range of genetic disorders > GATA3 is one of three genes mutated in 10% of breast such as cystic fibrosis and sickle cell anemia, assuming current cancers (89). The DNAzyme formulation, SB010 (nebulization challenges can be overcome (93). Hence, although DNAzymes solution containing hgd40) was administered (10 mg in 2 mL and CRISPR/Cas9 both represent loss-of-function technologies, by inhalation once daily for 28 days) to patients with allergic preference as to which to use will depend on factors such as asthma (21 received SB010, 19 received placebo). SB010 intended gene knockdown or knockout, transient or stable phe- reduced the late asthmatic response by 34% and attenuated notypic changes, and potential for off-target effects, as recently the early asthmatic response by 11%, whereas placebo considered with RNAi (94). increased the late and early response by 1% and 10%, respec- Then there is the question of "druggability." Transcription fl tively. Moreover, SB010 reduced the Th2-regulated in amma- factors outside of the nuclear hormone receptor family have often tory response. No serious adverse events were observed during been difficult drug targets for small molecule pharmacologic the study period (62). Eosinophil counts may provide a rele- intervention (95). Moreover, the inherent nuclear location of vant biomarker in SB010-treated patients (90). This followed transcription factors may make it difficult to access with small an earlier phase I trial of SB010 in 108 healthy and asthmatic molecules. In contrast, nuclear receptor transcription factors that subjects that showed the formulation safe (63) and Fuhst and translocate from the cell surface to the nucleus after ligand fi colleagues' ndings that hgd40 was well tolerated after delivery binding are considered druggable. Recent work using rationally by aerosolic inhalation or slow intravenous infusion in pre- designed cell-penetrating peptides (CPP) targeting ATF5 (96) or clinical models (91). Recent studies by Greulich and colleagues STAT3 (97) as inhibitors of tumor growth demonstrates that with SB010, delivered using a controlled inhalation system in transcription factors can be druggable. Because of their capacity 23 eosinophilic chronic obstructive pulmonary disease to target mRNA, rather than protein, DNAzymes can potentially patients, showed that GATA3 DNAzyme reduced sputum eosin- avoid limitations in druggability. Indeed, c-jun and GATA3 DNA- fi ophilia counts (92). These ndings suggest that DNAzymes zymes provide such proof of principle. delivered directly into lungs by aerosolic administration may DNAzyme versatility is not confined to therapeutics. also potentially be useful in lung cancers. GATA3 DNAzymes, For example, DNAzymes have been developed as diagnostic incidently, are under evaluation in phase I/II trials for atopic agents. Early cancer detection is important to reducing mor- eczema (ClinicalTrials.gov ID: NCT02079688) and ulcerative bidity and mortality. He and colleagues have developed colitis (ClinicalTrials.gov ID: NCT02129439). AAI2-5, an RNA-cleaving fluorogenic DNAzyme (RFD) probe, that can detect human MDA-MB-231 cells in lysates equi- Further Perspectives valent to 5,000 cells/mL. AAI2-5 can distinguish MDA-MB- Identification of an ideal RNA-cleaving DNAzyme can be 231 cells from normal cells and other types of tumor cell challenging. Effective screening systems are needed. Potaczek and types, including other breast cancer subtypes. The RFD also colleagues screened 226 candidate 10–23 DNAzymes targeting recognized human breast tumors whether benign or malig- human rhinovirus RNA, which resulted in two lead candidates nant (98). This suggests the potential use of the RFD approach after optimization of binding arm length possessing significant as clinical diagnostics. Moreover, because aberrant DNA cleavage and antiviral efficacy (21). The recent development of methylation can drive malignant transformation, Li and high-throughput kinetic assays may provide a less costly and more colleagues (99) designed a simple, label-free, colorimetric efficient way of selecting candidate DNAzyme sequences and methylation-responsive DNAzyme strategy to study methyl- mRNA target sites, albeit in single turnover rather than multiple transferase activity. Such versatility in application is not sur- turnover kinetics (22). In multiple turnover kinetics, low con- prising. MNAzyme strategies, whereby oligonucleotide partial centrations of enzyme are used to follow product formation in enzymes containing a split catalytic core of a DNAzyme assem- substrate excess, whereas single turnover conditions in enzyme ble into the active enzyme by means of an assembly facilitator excess enable active site characterization without catalytic cycling. (a target oligonucleotide of specific sequence), have been Eriksson and colleagues mass-screened DNAzymes to identify used as diagnostic biosensors (100). A quadruplex assay com- most efficient candidates more rapidly and in real time (22). The bining methylation-specificPCRwithMNAzymeswas field would also benefit from rule-based algorithms or potentially used to assess epigenetic biomarkers in prostate cancer that artificial intelligence tools that reliably inform DNAzyme design. detected a single methylated DNA allele on a background of Such design algorithms are commercially available for siRNA. 103 to 104 unmethylated alleles (101). This would improve cleavage and silencing efficacy rates, mini- mize off-target effects, reduce labor costs, and generate candidate DNAzymes faster. Conclusion Although the CRISPR/Cas9 gene editing system is funda- Since the first report of synthetic DNA having the capacity to mentally different from DNAzyme technology, each repre- cleave RNA, the field of DNAzymes has evolved from these

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oligonucleotides as test tube enzymes to use as molecular Disclosure of Potential Conflicts of Interest tools to interrogate functions of genes in complex systems in No potential conflicts of interest were disclosed. vitro and in vivo and their use as diagnostic agents. DNAzymes Acknowledgments have been tested in a variety of preclinical models and are at The author was supported by the National Health and Medical Research the doorstep of wider clinical testing. These have exciting Council of Australia. potential to serve as monotherapy or as adjuncts to existing therapies in cancer and a range of other diseases with unmet Received August 10, 2018; revised October 24, 2018; accepted December 14, clinical need. 2018; published first February 13, 2019.

References 1. Santoro SW, Joyce GF. A general purpose RNA-cleaving DNA enzyme. 23. Zuker M. On finding all suboptimal foldings of an RNA molecule. Science Proc Natl Acad Sci U S A 1997;94:4262–6. 1989;244:48–52. 2. Khachigian LM. Catalytic DNAs as potential therapeutic agents 24. Zuker M. Calculating nucleic acid secondary structure. Curr Opin Struct and sequence-specific molecular tools to dissect biological function. Biol 2000;10:303–10. J Clin Invest 2000;106:1189–95. 25. Fokina AA, Meschaninova MI, Durfort T, Venyaminova AG, Francois JC. 3. Khachigian LM. DNAzymes: cutting a path to a new class of therapeutics. Targeting insulin-like growth factor I with 10-23 DNAzymes: 20-O-methyl Curr Opin Mol Therap 2002;4:119–21. modifications in the catalytic core enhance mRNA cleavage. Biochemistry 4. Santoro SW, Joyce GF. Mechanism and utility of an RNA-cleaving DNA 2012;51:2181–91. enzyme. Biochemistry 1998;37:13330–42. 26. Saran R, Kleinke K, Zhou W, Yu T, Liu J. A Silver-Specific DNAzyme with a 5. Breaker RR, Joyce GF. A DNA enzyme that cleaves RNA. Chem Biol 1994;1: new silver aptamer and salt-promoted activity. Biochemistry 2017;56: 223–9. 1955–62. 6. Silverman SK. In vitro selection, characterization, and application of 27. Saran R, Liu J. A Silver DNAzyme. Anal Chem 2016;88:4014–20. deoxyribozymes that cleave RNA. Nucleic Acids Res 2005;33:6151–63. 28. Zhou W, Zhang Y, Huang P-JJ, Ding J, Liu J. A DNAzyme requiring two 7. Silverman SK. Catalytic DNA: scope, applications, and biochemistry of different metal ions at two distinct sites. Nucleic Acids Res 2016;44: deoxyribozymes. Trends Biochem Sci 2016;41:595–609. 354–63. 8. Hollenstein M. DNA catalysis: the chemical repertoire of DNAzymes. 29. Zhou W, Saran R, Chen Q, Ding J, Liu J. A New Na(þ)-Dependent RNA- Molecules 2015;20:20777–804. Cleaving DNAzyme with over 1000-fold Rate Acceleration by Ethanol. 9. Ponce-Salvatierra A, Wawrzyniak-Turek K, Steuerwald U, Hobartner C, Chembiochem 2016;17:159–63. Pena V. Crystal structure of a DNA catalyst. Nature 2016;529: 30. Torabi SF, Wu P, McGhee CE, Chen L, Hwang K, Zheng N, et al. In vitro 231–4. selection of a sodium-specific DNAzyme and its application in intracel- 10. Tan ML, Choong PF, Dass CR. DNAzyme delivery systems: getting past lular sensing. Proc Natl Acad Sci U S A 2015;112:5903–8. first base. Expert Opin Drug Delivery 2009;6:127–38. 31. Liu GN, Teng YX, Yan W. Transfected synthetic DNA enzyme gene 11. Jin Y, Li Z, Liu H, Chen S, Wang F, Wang L, et al. Biodegradable, specifically inhibits Egr-1 gene expression and reduces neointimal multifunctional DNAzyme nanoflowers for enhanced cancer therapy. hyperplasia following balloon injury in rats. Int J Cardiol 2008;129: NPG Asia Materials 2017;9:e365. 118–24. 12. Dias N, Stein CA. Antisense oligonucleotides: basic concepts and mechan- 32. Wang TR, Yang G, Liu GN. DNA enzyme ED5 depletes egr-1 and inhibits isms. Mol Cancer Ther 2002;1:347–55. neointimal hyperplasia in rats. Cardiology 2013;125:192–200. 13. Maeda H. Macromolecular therapeutics in cancer treatment: the EPR effect 33. Liu C, Zhang X, Wang S, Cheng M, Liu C, Wang S, et al. Transfected early and beyond. J Control Release 2012;164:138–44. growth response gene-1 DNA enzyme prevents stenosis and occlusion of 14. Khachigian LM, Fahmy RG, Zhang G, Bobryshev YV, Kaniaros A. c-Jun autogenous vein graft in vivo. BioMed Res Int 2013;2013:310406. regulates vascular smooth muscle cell growth and neointima formation 34. Wang S, Dong H, Liu C, Xu G, Hu X, Fan Y, et al. Early growth response after arterial injury: inhibition by a novel DNAzyme targeting c-Jun. J Biol factor-1 DNA enzyme 1 inhibits the formation of abdominal aortic Chem 2002;277:22985–91. aneurysm in rats. Exp Thera Med 2018;16:141–8. 15. Kayahara M, Wang X, Tournier C. Selective regulation of c-jun gene 35. Lowe HC, Chesterman CN, Khachigian LM. Catalytic antisense DNA expression by mitogen-activated protein kinases via the 12-o-tetradeca- molecules targeting Egr-1 inhibit neointima formation following perma- noylphorbol-13-acetate- responsive element and myocyte enhancer fac- nent ligation of rat common carotid arteries. Thromb Haemost 2002;87: tor 2 binding sites. Mol Cell Biol 2005;25:3784–92. 134–40. 16. Santiago FS, Lowe HC, Kavurma MM, Chesterman CN, Baker A, Atkins 36. Bhindi R, Khachigian LM, Lowe HC. DNAzymes targeting the transcrip- DG, et al. New DNA enzyme targeting Egr-1 mRNA inhibits vascular tion factor Egr-1 reduce myocardial infarct size following ischemia- smooth muscle proliferation and regrowth factor injury. Nat Med 1999;5: reperfusion in rats. J Thromb Haemost 2006;4:1479–83. 1264–9. 37. Lowe HC, Fahmy RG, Kavurma MM, Baker A, Chesterman CN, Khachi- 17. Kharbanda S, Nakamura T, Stone R, Hass R, Bernstein S, Datta R, et al. gian LM. Catalytic oligodeoxynucleotides define a key regulatory role for Expression of the early growth response 1 and 2 zinc finger genes during early growth response factor-1 in the porcine model of coronary in-stent induction of monocytic differentiation. J Clin Invest 1991;88:571–7. restenosis. Circ Res 2001;89:670–7. 18. Li Y, McRobb LS, Khachigian LM. MicroRNA miR-191 targets the zinc 38. Bhindi R, Fahmy RG, McMahon AC, Khachigian LM, Lowe HC. finger transcription factor Egr-1 and suppresses intimal thickening after Intracoronary delivery of DNAzymes targeting human EGR-1 reduces carotid injury. Int J Cardiol 2016;212:229–302. infarct size following myocardial ischaemia reperfusion. J Pathol 2012; 19. Bauer S, Wagner H. Bacterial CpG-DNA licenses TLR9. Curr Top Microbiol 227:157–64. Immunol 2002;270:145–54. 39. Rayner B, Figtree G, Sabaretnam T, Shang P, Mazhar J, Weaver J, et al. 20. Cai H, Santiago FS, Prado-Lourenco L, Patrikakis M, Wang B, Chong BH, Selective inhibition of Egr-1 using catalytic oligonucleotides reduces et al. DNAzymes targeting c-jun suppress skin cancer growth. Sci Translat myocardial injury and improves LV systolic function in a preclinical Med 2012;4:139ra82. model of myocardial infarction. J Am Heart Assoc 2013;2:e000023. 21. Potaczek DP, Unger SD, Zhang N, Taka S, Michel S, Akdag N, et al. 40. Dickinson MG, Kowalski PS, Bartelds B, Borgdorff MA, van der Feen D, Development and characterization of DNAzyme candidates demons- Sietsma H, et al. A critical role for Egr-1 during vascular remodelling in trating significant efficiency against human rhinoviruses. J Allergy Clin pulmonary arterial hypertension. Cardiovasc Res 2014;103:573–84. Immunol 2018; DOI: https://doi.org/10.1016/j.jaci.2018.07.026. 41. Nakamura H, Isaka Y, Tsujie M, Rupprecht HD, Akagi Y, Ueda N, et al. 22. Eriksson J, Helmfors H, Langel U. A high-throughput kinetic assay for Introduction of DNA enzyme for Egr-1 into tubulointerstitial fibroblasts RNA-cleaving deoxyribozymes. PLoS One 2015;10:e0135984. by electroporation reduced interstitial alpha-smooth muscle actin

886 Cancer Res; 79(5) March 1, 2019 Cancer Research

Deoxyribozymes as Catalytic Nanotherapeutic Agents

expression and fibrosis in unilateral ureteral obstruction (UUO) rats. 62. Krug N, Hohlfeld JM, Kirsten AM, Kornmann O, Beeh KM, Kappeler D, Gene Ther 2002;9:495–502. et al. Allergen-induced asthmatic responses modified by a GATA3-specific 42. Somasuntharam I, Yehl K, Carroll SL, Maxwell JT, Martinez MD, Che PL, DNAzyme. N Engl J Med 2015;372:1987–95. et al. Knockdown of TNF-alpha by DNAzyme gold nanoparticles as an 63. Homburg U, Renz H, Timmer W, Hohlfeld JM, Seitz F, Luer K, et al. Safety anti-inflammatory therapy for myocardial infarction. Biomaterials 2016; and tolerability of a novel inhaled GATA3 mRNA targeting DNAzyme in 83:12–22. patients with TH2-driven asthma. J Allergy Clin Immunol 2015;136: 43. Popp V, Gerlach K, Mott S, Turowska A, Garn H, Atreya R, et al. Rectal 797–800. delivery of a DNAzyme that specifically blocks the transcription factor 64. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med GATA3 reduces colitis in mice. Gastroenterology 2017;152:176–92. 1971;285:1182–6. 44. Sel S, Wegmann M, Dicke T, Henke W, Yildirim AO, Renz H, et al. Effective 65. Gimbrone MA Jr, Leapman SB, Cotran RS, Folkman J. Tumor dor- prevention and therapy of experimental allergic asthma using a GATA-3- mancyinvivobypreventionofneovascularization. J Exp Med 1972; specific DNAzyme. J Allergy Clin Immunol 2008;121:910–6. 136:261–76. 45. Dicke T, Pali-Scholl I, Kaufmann A, Bauer S, Renz H, Garn H. Absence of 66. Vlaykova T, Muhonen T, Hahka-Kemppinen M, Pyrhonen S, Jekunen A. unspecific innate immune cell activation by GATA-3-specific DNAzymes. Vascularity and prognosis of metastatic melanoma. Int J Cancer 1997;74: Nucleic Acid Thera 2012;22:117–26. 326–9. 46. Kumar B, Rajput R, Pati DR, Khanna M. Potent intracellular knock-down 67. Zhang L, Gasper WJ, Stass SA, Ioffe OB, Davis MA, Mixson AJ. Angiogenic of influenza a virus M2 gene transcript by DNAzymes considerably inhibition mediated by a DNAzyme that targets vascular endothelial reduces viral replication in host cells. Mol Biotechnol 2015;57:836–45. growth factor receptor 2. Cancer Res 2002;62:5463–9. 47. Huang C, Zhang Y, Kelly DJ, Tan CY, Gill A, Cheng D, et al. Thioredoxin 68. Fahmy RG, Dass CR, Sun LQ, Chesterman CN, Khachigian LM. interacting protein (TXNIP) regulates tubular autophagy and mitophagy Transcription factor Egr-1 supports FGF-dependent angiogenesis dur- in diabetic nephropathy through the mTOR signaling pathway. Sci Rep ing neovascularization and tumor growth. Nature Med 2003;9: 2016;6:29196. 1026–32. 48. Tan CY, Weier Q, Zhang Y, Cox AJ, Kelly DJ, Langham RG. Thioredoxin- 69. Mitchell A, Dass CR, Sun LQ, Khachigian LM. Inhibition of human breast interacting protein: a potential therapeutic target for treatment of pro- carcinoma proliferation, migration, chemoinvasion and solid tumour gressive fibrosis in diabetic nephropathy. Nephron 2015;129:109–27. growth by DNAzymes targeting the zinc finger transcription factor EGR-1. 49. Koenig B, Pape D, Chao O, Bauer J, Grimpe B. Long term study of Nucleic Acids Res 2004;32:3065–9. deoxyribozyme administration to XT-1 mRNA promotes corticospinal 70. Yao HW, Chen SH, Li C, Tung YY, Chen SH. Suppression of transcription tract regeneration and improves behavioral outcome after spinal cord factor early growth response 1 reduces herpes simplex virus 1-induced injury. Exp Neurol 2016;276:51–8. corneal disease in mice. J Virol 2012;86:8559–67. 50. Verma N, Tripathi SK, Chaudhury I, Das HR, Das RH. iNOS-targeted 10- 71. Jin Y, Liang L, Sun X, Yu G, Chen S, Shi S, et al. Deoxyribozyme- 23 DNAzyme reduces LPS-induced systemic inflammation and mortality nanosponges for improved photothermal therapy by overcoming ther- in mice. Shock 2010;33:493–9. moresistance. NPG Asia Materials 2018;10:373–84. 51.XiangG,SchusterMD,SekiT,KocherAA,EshghiS,BoyleA,etal. 72. Niewiarowska J, Sacewicz I, Wiktorska M, Wysocki T, Stasikowska O, Down-regulation of plasminogen activator inhibitor 1 expression Wagrowska-Danilewicz M, et al. DNAzymes to mouse beta1 integrin promotes myocardial neovascularization by bone marrow progenitors. mRNA in vivo: targeting the tumor vasculature and retarding cancer J Exp Med 2004;200:1657–66. growth. Cancer Gene Ther 2009;16:713–22. 52. Xiang G, Schuster MD, Seki T, Witkowski P, Eshghi S, Itescu S. Down- 73. Wiktorska M, Sacewicz-Hofman I, Stasikowska-Kanicka O, Danilewicz regulated expression of plasminogen activator inhibitor-1 augments M, Niewiarowska J. Distinct inhibitory efficiency of siRNAs and DNA- myocardial neovascularization and reduces cardiomyocyte apoptosis zymes to beta1 integrin subunit in blocking tumor growth. after acute myocardial infarction. J Am Coll Cardiol 2005;46:536–41. Acta Biochim Polonica 2013;60:77–82. 53. Xie J, Zhang S, Hu Y, Li D, Cui J, Xue J, et al. Regulatory roles of c-jun 74. Zhang G, Dass CR, Sumithran E, Di Girolimo NR, Sun L-Q, Khachi- in H5N1 influenza virus replication and host inflammation. gian LM. Effect of deoxyribozymes targeting c-Jun on solid tumor Biochim Biophys Acta 2014;1842:2479–88. growth and angiogenesis in rodents. J Natl Cancer Inst 2004;96: 54. Tan ML, Friedhuber AM, Dass CR. Co-nanoencapsulated doxorubicin 683–96. and Dz13 control osteosarcoma progression in a murine model. 75. Tan ML, Choong PF, Dass CR. Direct anti-metastatic efficacy by the DNA J Pharm Pharmacol 2013;65:35–43. enzyme Dz13 and downregulated MMP-2, MMP-9 and MT1-MMP in 55. Dass CR, Friedhuber AM, Khachigian LM, Dunstan DE, Choong PF. tumours. Cancer Cell Int 2010;10:9. Biocompatible chitosan-DNAzyme nanoparticle exhibits enhanced bio- 76. Dass CR, Galloway SJ, Clark JC, Khachigian LM, Choong PF. Involvement logical activity. J Microencapsul 2008;25:421–5. of c-jun in human liposarcoma growth: supporting data from clinical 56. Zhang L, Zhao W, Liang C, Yi X, Pei Y, Lin Y, et al. VEGFR-1 targeted immunohistochemistry and DNAzyme efficacy. Cancer Biol Ther 2008;7: DNAzyme via transcatheter arterial delivery influences tumor vasculature 1297–301. assessed through dynamic contrast-enhanced magnetic resonance imag- 77. Tan ML, Dunstan DE, Friedhuber AM, Choong PF, Dass CR. A nanopar- ing. Oncol Rep 2016;36:1339–44. ticulate system that enhances the efficacy of the tumoricide Dz13 when 57. Hallett MA, Teng B, Hasegawa H, Schwab LP, Seagroves TN, Pourmo- administered proximal to the lesion site. J Control Release 2010;144: tabbed T. Anti-matrix metalloproteinase-9 DNAzyme decreases tumor 196–202. growth in the MMTV-PyMT mouse model of breast cancer. Breast Cancer 78. Dass CR, Khachigian LM, Choong PF. c-Jun knockdown sensitizes oste- Res 2013;15:R12. osarcoma to doxorubicin. Mol Cancer Ther 2008;7:1909–12. 58. Yu X, Yang L, Cairns MJ, Dass C, Saravolac E, Li X, et al. Chemosensitiza- 79. Zhang G, Luo X, Sumithran E, Pua VSC, Barnetson RS, Halliday GM, et al. tion of solid tumors by inhibition of Bcl-xL expression using DNAzyme. Squamous cell carcinoma growth in mice and in culture is regulated by Oncotarget 2014;5:9039–48. c-Jun and its control of matrix metalloproteinase-2 and -9 expression. 59. Cai H, Cho EA, Li Y, Sockler J, Parish CR, Chong BH, et al. Melanoma Oncogene 2006;25:7260–6. protective antitumor immunity activated by catalytic DNA. Oncogene 80. Chan CWS, Kaplan W, Parish CR, Khachigian LM. Regression of retinal 2018;37:5115–26. neovascularization, improvement in forepaw reach, comparative micro- 60. Cho EA, Moloney FJ, Cai H, Au-Yeung A, China C, Scolyer RA, et al. Safety array and gene set enrichment analysis with c-jun targeting DNA enzyme. and tolerability of an intratumorally injected DNAzyme, Dz13, in patients PLoS ONE 2012;7:e39160. with nodular basal-cell carcinoma: a Phase 1 first-in-human trial (DIS- 81. Marquardt K, Eicher AC, Dobler D, Mader U, Schmidts T, Renz H, et al. COVER). Lancet 2013;381:1835–43. Development of a protective dermal drug delivery system for therapeutic 61. Cao Y, Yang L, Jiang W, Wang X, Liao W, Tan G, et al. Therapeutic DNAzymes. Int J Pharm 2015;479:150–8. Evaluation of Epstein-Barr Virus-encoded Latent Membrane Protein-1 82. Marquardt K, Eicher AC, Dobler D, Hofer F, Schmidts T, Schafer J, et al. Targeted DNAzyme for treating of nasopharyngeal carcinomas. Mol Thera Degradation and protection of DNAzymes on human skin. Eur J 2014;22:371–7. Pharm Biopharm 2017;107:80–7.

www.aacrjournals.org Cancer Res; 79(5) March 1, 2019 887

Khachigian

83. Yang L, Lu Z, Ma X, Cao Y, Sun LQ. A therapeutic approach to nasopha- 95. Konstantinopoulos PA, Papavassiliou AG. Seeing the future of can- ryngeal carcinomas by DNAzymes targeting EBV LMP-1 gene. Molecules cer-associated transcription factor drug targets. JAMA 2011;305: 2010;15:6127–39. 2349–50. 84. Liu S, Li H, Tang M, Cao Y. (-)-Epigallocatechin-3-gallate inhibition of 96. Karpel-Massler G, Horst BA, Shu C, Chau L, Tsujiuchi T, Bruce JN, et al. A Epstein-Barr virus spontaneous lytic infection involves downregulation of synthetic cell-penetrating dominant-negative ATF5 peptide exerts anti- latent membrane protein 1. Exp Thera Med 2018;15:1105–12. cancer activity against a broad spectrum of treatment-resistant cancers. 85. Yang L, Liu L, Xu Z, Liao W, Feng D, Dong X, et al. EBV-LMP1 targeted Clin Cancer Res 2016;22:4698–711. DNAzyme enhances radiosensitivity by inhibiting tumor angiogenesis via 97. Kim D, Lee IH, Kim S, Choi M, Kim H, Ahn S, et al. A specific STAT3- the JNKs/HIF-1 pathway in nasopharyngeal carcinoma. Oncotarget 2015; binding peptide exerts antiproliferative effects and antitumor activity by 6:5804–17. inhibiting STAT3 phosphorylation and signaling. Cancer Res 2014;74: 86. Ke X, Yang YC, Hong SL. EBV-LMP1-targeted DNAzyme restrains naso- 2144–51. pharyngeal carcinoma growth in a mouse C666-1 xenograft model. 98. He S, Qu L, Shen Z, Tan Y, Zeng M, Liu F, et al. Highly specific recognition Med Oncol 2011;28:S326–32. of breast tumors by an RNA-cleaving fluorogenic DNAzyme probe. 87. Qu Y, Zhang L, Mao M, Zhao F, Huang X, Yang C, et al. Effects of Anal Chem 2015;87:569–77. DNAzymes targeting aurora kinase A on the growth of human prostate 99. Li W, Liu Z, Lin H, Nie Z, Chen J, Xu X, et al. Label-free colorimetric assay cancer. Cancer Gene Ther 2008;15:517–25. for methyltransferase activity based on a novel methylation-responsive 88. Dass CR, Choong PF. Sequence-related off-target effect of Dz13 against DNAzyme strategy. Anal Chem 2010;82:1935–41. human tumor cells and safety in adult and fetal mice following systemic 100. Mokany E, Bone SM, Young PE, Doan TB, Todd AV. MNAzymes, a administration. Oligonucleotides 2010;20:51–60. versatile new class of nucleic acid enzymes that can function as 89. Koboldt DC, Fulton RS, McLellan MD, Schmidt H, Kalicki-Veizer J, biosensors and molecular switches. J Am Chem Soc 2010;132: McMichael JF, et al. Comprehensive molecular portraits of human breast 1051–9. tumours. Nature 2012;490:61–70. 101. Mokany E, Tan YL, Bone SM, Fuery CJ, Todd AV. MNAzyme qPCR with 90. Krug N, Hohlfeld JM, Buhl R, Renz J, Garn H, Renz H. Blood eosinophils superior multiplexing capacity. Clin Chem 2013;59:419–26. predict therapeutic effects of a GATA3-specific DNAzyme in asthma 102. Yang X, Li Z, Zhang L, He J, Sun LQ. Selection and antitumor activity of patients. J Allergy Clin Immunol 2017;140:625–8. anti-Bcl-2 DNAzymes. Biochem Biophys Res Commun 2016;479: 91. Fuhst R, Runge F, Buschmann J, Ernst H, Praechter C, Hansen T, et al. 544–50. Toxicity profile of the GATA-3-specific DNAzyme hgd40 after inhalation 103. Shen L, Zhou Q, Wang Y, Liao W, Chen Y, Xu Z, et al. Antiangiogenic exposure. Pulmonary Pharmacol Thera 2013;26:281–9. and antitumoral effects mediated by a vascular endothelial growth 92. Greulich T, Hohlfeld JM, Neuser P, Lueer K, Klemmer A, Schade-Brittinger factor receptor 1 (VEGFR-1)-targeted DNAzyme. Mol Med 2013;19: C, et al. A GATA3-specific DNAzyme attenuates sputum eosinophilia in 377–86. eosinophilic COPD patients: a feasibility randomized clinical trial. 104. Witkowski P, Seki T, Xiang G, Martens T, Sondermeijer H, See F, et al. A Resp Res 2018;19:55. DNA enzyme against plasminogen activator inhibitor- type 1 (PAI-1) 93. Wang D, Mou H, Li S, Li Y, Hough S, Tran K, et al. Adenovirus-mediated limits neointima formation after angioplasty in an obese diabetic rodent somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of model. J Cardiovasc Pharmacol 2007;50:633–40. Cas9-specific immune responses. Hum Gene Ther 2015;26:432–42. 105. Yang L, Xiao L, Ma X, Tang M, Weng X, Chen X, et al. Effect of DNAzymes 94. Boettcher M, McManus MT. Choosing the Right Tool for the Job: RNAi, targeting Akt1 on cell proliferation and apoptosis in nasopharyngeal TALEN, or CRISPR. Mol Cell 2015;58:575–85. carcinoma. Cancer Biol Ther 2009;8:366–71.

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Deoxyribozymes as Catalytic Nanotherapeutic Agents

Levon M. Khachigian

Cancer Res 2019;79:879-888. Published OnlineFirst February 13, 2019.

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