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In the early 1980s, RNAs were shown to have catalytic functions that cleave the phosphodiester backbones of RNAs (1, 2), and the concept of using nucleic acid–based sequence-specif c agents to regulate gene expression by cleaving and functionally de-stroying mRNAs was born. T e excitement in the f eld centered around the ability of these small catalytic RNAs to cleave mRNAs in a site- or sequence-specif c manner me-diated by the base pairing of RNA catalysts with their target RNAs. T e smallest RNA molecules with site-specif c cleavage capabil-ities are the hammerhead ribozymes, which are derived from the self-cleaving domain of plant RNA viroids and virusoids. T is self-cleaving motif was adapted for application in trans, wherein the ribozyme could be engineered to base pair with mRNAs and catalyze strand cleavage of the target, fol-lowed by recycling of the ribozyme, mak-ing these agents true catalytic enzymes (3). For more than a decade, hammerhead ri-bozymes were the favored RNA-cleaving platform for targeted therapeutic appli-cations that alter biological processes—and disease states—by interfering with the synthesis of selected proteins. In this issue of Science Translational Medicine, Cai et al. (4) demonstrate that catalyticDNA—a DNAzyme (Fig. 1)—inhibits syn-thesis of the cancer-related transcription factor c-Jun and tumor growth in a mouse model of skin cancer.

On the basis of the chemistry behind ribozyme-based cleavage, scientists be-lieved that DNA could not carry out such a catalytic function because it lacks the 2ʹ hydroxyl group present in RNA. T en, in the mid-1990s, Gerald Joyce and col-leagues used an in vitro selection method-ology that employed short single-stranded

DNAs to identify DNA motifs that could ef ect site-specif c RNA strand scission (5). T ese scientists used an ingenious se-lection scheme and 10 rounds of selection to enrich for RNA-cleaving DNA motifs that displayed high catalytic turnover of mRNA. Two catalytic motifs emerged from this selection scheme: One cleaves RNA between nucleotides G and A, and the oth-er, which is more generally useful, cleaves between any pyrimidine-purine base-pair combination.

T is pioneering research established DNA as a true catalytic enzyme, and, in fact, the kinetic parameters of the DNA-zymes on RNA targets were superior to those of their RNA counterparts. Clear advantages of the DNAzyme versus other RNA-targeting agents are its ease of chemi-cal synthesis, broad target recognition properties, and high catalytic turnover. Since the initial description of DNAzymes in 1997, numerous applications of these catalytic molecules have been tested, in particular as anticancer therapeutics (6), but no clinical applications have emerged.

Despite the potential broad-based util-ity of DNAzymes, they have not caught on as therapeutic agents. T is def cit results, in part, from the fact that, like all other nucle-ic acid drugs, delivery has been a challenge for DNAzyme therapeutic applications (7). T rough careful choices of a selective ther-apeutic target—mRNA that encodes the cell proliferation–related transcription fac-tor c-Jun—and a disease setting in which the drug can be applied directly—injection into tumors in mouse models of two skin cancers—Cai et al. (4) have resurrected the potential clinical utility of DNAzymes.

In the new work, intratumoral injection of a c-jun mRNA–targeted DNAzyme that had been complexed with a DOTAP/DOPE {N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-tri-methylammonium methyl-sulfate}–based lipid carrier had profound inhibitory ef-

fects on skin cancer cell proliferation and metastases in murine models of both basal and squamous cell carcinomas. T e authors also showed that injection of the c-jun–targeting DNAzyme in immunocompetent animals triggered additional antitumor im-mune responses, resulting in even more profound tumor inhibition. Doses as high as 100 micrograms per mouse were well tol-erated, and at this dose, cessation of tumor growth was observed even when DNAzyme treatment was suspended.

In providing proof of principle for nucleic acid therapeutics, it is necessary to demonstrate not only ef cacy but also specif city. Indeed, Cai et al. demonstrated by both RNA and protein analyses that the DNAzyme reduced concentrations of c-jun mRNA and, consequently, its correspond-ing protein in Western (immune) blot and immunohistochemical assays. A mutant version of the ribozyme with an intact cata-lytic core and scrambled binding arms had no tumor inhibitory ef ects nor did it af ect the concentrations of c-Jun protein or en-coding mRNA. A point mutation in the cat-alytic core that abolished cleavage activity but still allowed the DNAzyme to base pair with its complementary mRNA did not re-sult in c-Jun down-regulation or inhibition of tumor progression. Taken together, these essential controls validated the ef cacy and specif city of the DNAzyme.

Inhibition of c-Jun synthesis had mul-tiple downstream ef ects. T ese included reduction in the expression of proliferating cell nuclear antigen, a DNA polymerase accessory factor; cyclin-dependent kinase 4, which is required for cell cycle progres-sion; f broblast growth factor–2; and tissue remodeling and metastasis-related proteins matrix metalloproteinase–2 (MMP-2), MMP-9, and vascular endothelial growth factor–A, as well as elevation in expres-sion of the tumor suppressor p53 and p53-inducible proteins p21 (CIP1/WAF1) and apoptosis-related caspases 3, 8, and 9. T e mutated DNAzyme with scrambled bind-ing arms and the DOTAP/DOPE vehicle alone had no ef ects on the production of any of these proteins.

In the world of nucleic acid therapeu-tics, ef cacy is of en tempered by unpre-dicted and undesirable toxicities and side ef ects. Cai et al. gave careful attention to many possible nonspecif c ef ects that might account for of -target modulation of amounts of c-Jun mRNA and protein (5). T e authors f rst demonstrated that

C A N C E R

Resurrecting DNAzymes as Sequence-Specif c TherapeuticsJohn J. Rossi

E-mail: jrossi@coh.org

Department of Molecular and Cellular Biology, Beck-man Research Institute of the City of Hope, Duarte, CA 91010, USA.

In a mouse model of skin cancer, intratumoral injection of a sequence-specif c mRNA-cleaving DNA enzyme caused potent inhibition of tumor growth and unusually benign pharmacodynamic prof les.

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the DNAzyme was not activating the DNA CpG receptor Toll-like recep-tor 9 (TLR9), which functions in ac-tivation of innate immunity. Cai et al. then went on to perform large-scale systemic pharmacodistribution stud-ies in cynomologous monkeys, mini-pigs, and rodents and observed rela-tively rapid clearance of the DOTAP/DOPE–complexed DNAzymes from blood and little or no accumulation in any of the major organs of these ani-mals. Moreover, the authors did not detect inhibition by the anti–c-junDNAzyme in 70 separate assays that assessed a variety of biological reac-tions associated with important phar-macological indicators, such as blood cell counts, clotting factor levels, and other hematological enzymatic ac-tivities. T us, these investigators have provided a strong set of analyses that validate the safety of their lead DNA-zyme for human clinical trials.

With respect to nucleic acid thera-peutics, the inert nature of the c-junDNAzyme with respect to toxicities is an obvious advantage over other nucleic acid–based drugs, which have been shown to trigger thrombocyto-penia, complement activation (phos-phorothioate antisense DNA oligos), and TLR activation with subsequent type I interferon responses (small in-terfering RNAs) (8–10). One possible explanation for the benign pharmaco-dynamics is that the c-jun DNAzyme has no base or backbone modif ca-tions and lacks CpG stimulatory mo-tifs. T e use of intratumoral injection in the context of a lipid-based carrier that itself is benign may be the keys to resurrecting DNAzymes as safe and ef ective nucleic acid therapeutic agents. It will be of great interest to follow the next steps in the develop-ment of the c-jun targeted DNAzyme for the treatment of skin cancers in human clinical trials.

REFERENCES AND NOTES1. C. Guerrier-Takada, K. Gardiner, T. Marsh, N. Pace,

S. Altman, The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35, 849–857 (1983).

2. A. J. Zaug, T. R. Cech, The intervening sequence RNA of Tetrahymena is an enzyme. Science 231, 470–475 (1986).

3. J. Haseloff , W. L. Gerlach, Simple RNA enzymes with new and highly specifi c endoribonuclease activities. Nature 334, 585–591 (1988).

Endocytosis

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Fig. 1. All in the delivery? Shown is the mech-anism of DNAzyme-mediated cleavage of c-jun mRNA. The cationic lipid–catalytic DNA com-plex was introduced directly into basal or squa-mous cell tumors in mice. A 10-23 version of the Santoro and Joyce DNAzyme is depicted in-teracting with the c-jun mRNA via Watson-Crick base pairing. A purine (R = G or A) at the begin-ning of the DNAzyme catalytic core pairs with a pyrimidine (Y = C or U) in the target mRNA, and cleavage takes place between the pyrimidine and purine in the target sequence. The cleav-age results in a 2′-3′ cyclic phosphate and 5′ OH on the target. Once cleaved, the mRNA is degraded by cellular ribonucleases. Inhibition of c-jun expression triggers a cascade of events leading to apoptosis of the cancer cell.

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4. H. Cai, F. S. Santiago, L. Prado-Lourenco, B. Wang, M. Patrikakis, M. P. Davenport, G. J. Maghzal, R. Stocker, C. R. Parish, B. H. Chong, G. J. Lieschke, T.-W. Wong, C. N. Chesterman, D. J. Francis, F. J. Moloney, R. St.C. Bar-netson, G. M. Halliday, L. M. Khachigian, DNAzyme tar-geting c-jun suppresses skin cancer growth. Sci. Transl. Med. 139, 139ra82 (2012).

5. S. W. Santoro, G. F. Joyce, A general purpose RNA-cleaving DNA enzyme. Proc. Natl. Acad. Sci. U.S.A. 94, 4262–4266 (1997).

6. C. R. Dass, P. F. Choong, L. M. Khachigian, DNAzyme technology and cancer therapy: Cleave and let die. Mol. Cancer Ther. 7, 243–251 (2008).

7. M. L. Tan, P. F. M. Choong, C. R. Dass, DNAzyme delivery

systems: Getting past fi rst base. Expert Opin. Drug Deliv. 6, 127–138 (2009).

8. T. L. Jason, J. Koropatnick, R. W. Berg, Toxicology of antisense therapeutics. Toxicol. Appl. Pharmacol. 201, 66–83 (2004).

9. A. Judge, I. MacLachlan, Overcoming the innate im-mune response to small interfering RNA. Hum. Gene Ther. 19, 111–124 (2008).

10. M. E. Kleinman, K. Yamada, A. Takeda, V. Chandrasek-aran, M. Nozaki, J. Z. Baffi , R. J. Albuquerque, S. Yama-saki, M. Itaya, Y. Pan, B. Appukuttan, D. Gibbs, Z. Yang, K. Karikó, B. K. Ambati, T. A. Wilgus, L. A. DiPietro, E. Sakurai, K. Zhang, J. R. Smith, E. W. Taylor, J. Ambati, Sequence- and target-independent angio-

genesis suppression by siRNA via TLR3. Nature 452, 591–597 (2008).

Funding: NIH grants AI29329 and AI42552. Competing interests: The author is the chair of the scientifi c advisory board of Dicerna Pharmaceuticals, an RNAi company target-ing cancer, and a consultant for Calando Pharmaceuticals, an RNAi delivery company.

Citation: J. J. Rossi, Resurrecting DNAzymes as sequence-specifi c therapeutics. Sci. Transl. Med. 4, 139fs20 (2012).

10.1126/scitranslmed.3004080

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Resurrecting DNAzymes as Sequence-Specific TherapeuticsJohn J. Rossi

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