RNA-Cleaving DNA Thresholder Controlled by Concentrations of miRNA Cancer Marker.

Oligonucleotide gene therapy (OGT) agents suppress specific mRNAs in cells and thus reduce the expression of targeted genes. The ability to unambiguously distinguish cancer from healthy cells can solve the low selectivity problem of OGT agents. Cancer RNA markers are expressed in both healthy and cancer cells with a higher expression level in cancer cells. We have designed a DNA-based construct, named DNA thresholder (DTh) that cleaves targeted RNA only at high concentrations of cancer marker RNA and demonstrates low cleavage activity at low marker concentrations. The RNA-cleaving activity can be adjusted within one order of magnitude of the cancer marker RNA concentration by simply redesigning DTh. Importantly, DTh recognizes cancer marker RNA, while cleaving targeted RNA; this offers a possibility to suppress vital genes exclusively in cancer cells, thus triggering their death. DTh is a prototype of computation-inspired molecular device for controlling gene expression and cancer treatment.

Genetically Encoded and Biologically Produced All-DNA Nanomedicine Based on One-Pot Assembly of DNA Dendrimers for Targeted Gene Regulation.

All-DNA nanomedicines have emerged as potential anti-tumor drugs. DNA nanotechnology provides all-DNA nanomedicines with unlimited possibilities in controlling the diversification of size, shape, and loads of the therapeutic motifs. As DNA is a biological polymer, it is possible to genetically encode and produce the all-DNA nanomedicines in living bacteria. Herein, DNA-dendrimer-based nanomedicines are designed to adapt to the biological production, which is constructed by the flexible 3-arm building blocks to enable a highly efficient one-pot DNA assembly. For the first time, a DNA nanomedicine, D4-3-As-DzSur, is successfully genetically encoded, biotechnologically produced, and directly self-assembled. The performance of the biologically produced D4-3-As-DzSur in targeted gene regulation has been confirmed by in vitro and in vivo studies. The biological production capability will fulfill the low-cost and large-scale production of all-DNA nanomedicines and promote clinical applications.

Deoxyribozyme-Based DNA Machines for Cancer Therapy.

Soon after their discovery, RNA-cleaving deoxyribozymes (RCDZ) were explored as anticancer gene therapy agents. Despite low toxicity found in clinical trials, there is no clinically significant anticancer RCDZ-based therapy. Some of the reported disadvantages of RCDZ agents include poor accessibility to folded nucleic acids, low catalytic efficiency inside cells, and problems of intracellular delivery. On the other hand, structural DNA nanotechnology provides an opportunity to build multifunctional nano-associations that can address some of these problems. Herein we discuss the possibility of building RCDZ-based multifunctional DNA nanomachines equipped with RNA unwinding, cancer marker recognition, and RCDZ-based RNA-cleavage functions. An important advantage of such "nanomachines" is the possibility to cleave a housekeeping gene mRNA in a cancer-cell-specific manner. The proposed design could become a starting point for building sophisticated DNA-based nanodevices for cancer treatment.

Allergen-induced asthmatic responses modified by a GATA3-specific DNAzyme.

BACKGROUND: The most prevalent phenotype of asthma is characterized by eosinophil-dominated inflammation that is driven by a type 2 helper T cell (Th2). Therapeutic targeting of GATA3, an important transcription factor of the Th2 pathway, may be beneficial. We evaluated the safety and efficacy of SB010, a novel DNA enzyme (DNAzyme) that is able to cleave and inactivate GATA3 messenger RNA (mRNA). METHODS: We conducted a randomized, double-blind, placebo-controlled, multicenter clinical trial of SB010 involving patients who had allergic asthma with sputum eosinophilia and who also had biphasic early and late asthmatic responses after laboratory-based allergen provocation. A total of 40 patients could be evaluated; 21 were assigned to receive 10 mg of SB010, and 19 were assigned to receive placebo, with each study drug administered by means of inhalation once daily for 28 days. An allergen challenge was performed before and after the 28-day period. The primary end point was the late asthmatic response as quantified by the change in the area under the curve (AUC) for forced expiratory volume in 1 second (FEV1). RESULTS: After 28 days, SB010 attenuated the mean late asthmatic response by 34%, as compared with the baseline response, according to the AUC for FEV1, whereas placebo was associated with a 1% increase in the AUC for FEV1 (P=0.02). The early asthmatic response with SB010 was attenuated by 11% as measured by the AUC for FEV1, whereas the early response with placebo was increased by 10% (P=0.03). Inhibition of the late asthmatic response by SB010 was associated with attenuation of allergen-induced sputum eosinophilia and with lower levels of tryptase in sputum and lower plasma levels of interleukin-5. Allergen-induced levels of fractional exhaled nitric oxide and airway hyperresponsiveness to methacholine were not affected by either SB010 or placebo. CONCLUSIONS: Treatment with SB010 significantly attenuated both late and early asthmatic responses after allergen provocation in patients with allergic asthma. Biomarker analysis showed an attenuation of Th2-regulated inflammatory responses. (Funded by Sterna Biologicals and the German Federal Ministry of Education and Research; ClinicalTrials.gov number, NCT01743768.).

Current and future treatment options for adult chronic rhinosinusitis: Focus on nasal polyposis.

Chronic rhinosinusitis (CRS) affects more than 10% of the population in the United States and Europe. Recent findings point to a considerable variation of inflammatory subtypes in patients with CRS with nasal polyps and patients with CRS without nasal polyps. According to current guidelines, glucocorticosteroids and antibiotics are the principle pharmacotherapeutic approaches; however, they fail in a group of patients who share common clinical and laboratory markers. Several clinical phenotypes often leading to uncontrolled disease, including adult nasal polyposis, aspirin-exacerbated respiratory disease, and allergic fungal rhinosinusitis, are characterized by a common endotype: a TH2 bias is associated with a higher likelihood of comorbid asthma and recurrence after surgical treatment. As a consequence, several innovative approaches targeting the TH2 bias with humanized mAbs have been subjected to proof-of-concept studies in patients with CRS with nasal polyps with or without comorbid asthma: omalizumab, reslizumab, mepolizumab, and recently dupilumab. Future concepts using upstream targets, such as GATA-3, also focus on this endotype. This current development might result in advantages in the treatment of patients with the most severe CRS.

RNA INTERFERENCE: NEW APPROACH TO THE TREATMENT OF ALLERGIC ASTHMA (A REVIEW).

Asthma is among the most common chronic disorders of airways, which affects both children and adults. Asthma being a common disease among different segments of population, it has a high mortality rate and, in the absence of appropriate care, affects the quality of life and leads to economics losses. In a view of continuing growth in the incidence of asthma, it is important to find relevant biological targets for developing new approaches to astma therapy. Recent advances in molecular immunology, genetics, and bioinformatics allowed genes involved in the pathogenesis of asthma to be identified, which provided prerequisites for the development of new types of drugs that can regulate the activity of pathogenically significant genes. To date, a number of technologies for sequence-specific gene regulation (ASO, ribozymes, DNAzymes, EGS, DNA-decoys, U 1-adapters) are available, but RNA interference is the most promising approach in both terms of efficacy and financial cost. This review focuses on the generalization and analysis of experimental data regarding the use of RNA interference technology for the treatment of astma.

Substantial Antiviral Potential of Deoxyribozymes Fixed on Anatase Nanoparticles Against Influenza A Viruses in vitro and in vivo.

Influenza A viruses (IAV) are a high threat to humanity because of a lack of proper effective antiviral drugs and resistance of viruses to existing vaccines. We describe the sufficient anti-IAV effect of Ans/PL-Dz nanocomposites that contain deoxyribozymes (Dz) immobilized on anatase TiO2 nanoparticles (Ans) through polylysine linker (PL). The Dz-containing nanocomposites appear to be more efficient than the Ans/PL-ODN nanocomposites that contain common oligodeoxyribonucleotides (ODN) targeted to the same RNA regions of the viral genome. The simultaneous use of nanocomposites that contain Dz and ODN, which are targeted to different sites of viral RNA provides a higher overall effect than the independent action of each of them (synergism). The inhibition of IAV with the proposed nanocomposites was shown to be effective, sequence-specific, and dose-dependent. The most efficient Ans/PL-Dz nanocomposite exhibited a high antiviral effect in vivo on mice models. The efficiency of IAV inhibition with this nanocomposite in vitro and in vivo is higher than that for the approved antiflu drug oseltamivir. The results open the prospect of creating a unique antiviral agent suitable for IAV suppression.

DNAzymes: Expanding the Potential of Nucleic Acid Therapeutics.

Nucleic acids drugs have been proven in the clinic as a powerful modality to treat inherited and acquired diseases. However, key challenges including drug stability, renal clearance, cellular uptake, and movement across biological barriers (foremost the blood-brain barrier) limit the translation and clinical efficacy of nucleic acid-based therapies, both systemically and in the central nervous system. In this study we provide an overview of an emerging class of nucleic acid therapeutic, called DNAzymes. In particular, we review the use of chemical modifications and carrier molecules for the stabilization and/or delivery of DNAzymes in cell and animal models. Although this review focuses on DNAzymes, the strategies described are broadly applicable to most nucleic acid technologies. This review should serve as a general guide for selecting chemical modifications to improve the therapeutic performance of DNAzymes.

DNAzyme loaded nano-niosomes attenuate myocardial ischemia/reperfusion injury by targeting apoptosis, inflammation in a NF-κB dependent mechanism.

Although DNAzymes have been found to reduce injury after myocardial ischemia/reperfusion (MI/R), their efficiency have been limited due to rapid degradation in vivo. Thus, this study was conducted to extend their half-life by encapsulation into nano­niosomes and examine their cardioprotective effects in a rat model of myocardial infarction (MI). In order to synthesize nano­niosomes, surface active agent film hydration method was used. Characterization of nano­niosomes was performed using the atomic force microscopy (AFM). In order to establish MI/R model in rats, left anterior descending coronary artery (LAD) was ligated for 30 min. A single dose (150µL) of drug formulations was injected into the infarcted region. The cardiac function was evaluated using echocardiography. The expression of pro-inflammatory cytokines, apoptotic factors, and nuclear factor-κB (NF-κB) were evaluated using Western blot and immunohistochemistry, respectively. Particle size of only nano-niosomes was in the range of 60-90 nm, while a shift to 70-110 nm was seen after DNAzyme encapsulation. MI rats treated with DNAzyme­loaded nano­niosomes could markedly reduce Bax, caspase3, TNF-α, IL-1ß, and NF-κB as well as increase Bcl-2 compared to only MI/R group. Collectively, our finding show that nano­niosomes can be considered excellent drug delivery platforms to extend half-life and stability of DNAzyme, when it is used to reduce myocardial I/R injury.

A therapeutic approach to nasopharyngeal carcinomas by DNAzymes targeting EBV LMP-1 gene.

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has been known to have oncogenic properties during latent infection in nasopharyngeal carcinoma (NPC). Genetic manipulation of LMP1 expression may provide a novel strategy for the treatment of NPC. DNAzymes are synthetic, single-stranded DNA catalysts that can be engineered to bind and cleave the target mRNA of a disease-causing gene. By targeting the LMP1 mRNA, we successfully obtained a phosphorothioate-modified ''10-23'' DNAzyme namely DZ1, through screening a series of DNAzymes. DZ1 could significantly down-regulate the expression of LMP1 in NPC cells, inhibit cell proliferation, metastasis, promote apoptosis and enhance radiosensitivity of NPC through interfering signal pathways which are abnormally activated by LMP1, including NF-κB, AP-1 and STAT3 signal pathways. Together, interfering LMP1 signaling pathway could be a promising strategy to target the malignant phenotypes of NPC.

Nucleic acid-cleaving catalytic DNA for sensing and therapeutics.

DNAzymes with nucleic acid-cleaving catalytic activity are increasing in versatility through concerted efforts to discover new sequences with unique functions, and they are generating excitement in the sensing community as cheap, stable, amplifiable detection elements. This review provides a comprehensive list and detailed descriptions of the DNAzymes identified to date, classified by their associated small molecule or ion needed for catalysis; of note, this classification clarifies conserved regions of various DNAzymes that are not obvious in the literature. Furthermore, we detail the breadth of functionality of these DNA sequences as well as the range of reaction conditions under which they are useful. In addition, the utility of the DNAzymes in a variety of sensing and therapeutic applications is presented, detailing both their advantages and disadvantages.

Core-Shell Nanosystems for Self-Activated Drug-Gene Combinations against Triple-Negative Breast Cancer.

The combination of gene therapy with chemotherapeutics provides an efficacious strategy for enhanced tumor therapy. RNA-cleaving DNAzyme has been recognized as a promising gene-silencing tool, while its combination with chemotherapeutic drugs has been limited by the lack of an effective codelivery system to allow sufficient intracellular DNAzyme activation, which requires specific metal ions as a cofactor. Here, a self-activatable DNAzyme/drug core-shell codelivery system is fabricated to combat triple-negative breast cancer (TNBC). The hydrophobic chemotherapeutic, rapamycin (RAP), is self-assembled into the pure drug nanocore, and the metal-organic framework (MOF) shell based on coordination between Mn2+ and tannic acid (TA) is coated on the surface to coload an autophagy-inhibiting DNAzyme. The nanosystem efficiently delivers the payloads into tumor cells, and upon endocytosis, the MOF shell is disintegrated to release the therapeutics in response to an acidic endo/lysosome environment and intracellular glutathione (GSH). Notably, the coreleased Mn2+ serves as the cofactor of DNAzyme for effective self-activation, which suppresses the expression of Beclin 1 protein, the key initiator of autophagy, resulting in a significantly strengthened antitumor effect of RAP. Using tumor-bearing mouse models, the nanosystem could passively accumulate into the tumor tissue, impose potent gene-silencing efficacy, and thus sensitize chemotherapy to inhibit tumor growth upon intravenous administration, providing opportunities for combined gene-drug TNBC therapy.

DNAzymes and their therapeutic possibilities.

Our increasing understanding of the regulatory mechanisms involved in the pathogenesis of disease is opening up opportunities for therapeutic intervention. To tackle the unmet disease burden, the last decade has seen the emergence of gene-targeting small-molecule nucleic acid-based strategies, such as antisense oligodeoxynucleotides, ribozymes, small interfering RNA and DNAzymes. DNAzymes represent promising candidates for drug therapy in a wide range of diseases, such as cancer and cardiovascular disorders. This brief review will discuss recent developments in DNAzymes and their therapeutic potential.

Effective prevention and therapy of experimental allergic asthma using a GATA-3-specific DNAzyme.

BACKGROUND: Allergic bronchial asthma is a chronic inflammatory disease of the airways. The transcription factor GATA-3 was shown to play an important role in TH2 cell activation, but also in the regulation of other cell types involved in bronchial asthma including mast cells, eosinophils, and epithelial cells. DNAzymes represent a new class of antisense molecules that combines the specificity of DNA base pairing with an inherent RNA-cleaving enzymatic activity. OBJECTIVE: To develop a GATA-3 mRNA-specific DNAzyme and analyze its allergy-preventing activity in murine models of experimental allergic asthma. METHODS: The most active DNAzyme (termed gd21) was selected by in vitro cleavage assays. Allergic airway inflammation was assessed by inflammatory cell and cytokine analysis within bronchoalveolar lavage. Lung histology, including goblet cell hyperplasia and lung function, was analyzed using head-out body-plethysmography. RESULTS: Intranasal administration of gd21 prevented airway inflammation and mucus production and inhibited development of airway hyperresponsiveness to methacholine in models of acute allergic airway inflammation. Similar effects were also detected in a model of chronic experimental asthma. Interestingly, gd21 was at least as effective as other antisense molecules, and off-target effects were not detected. Further experiments indicated that pulmonary surfactant may facilitate the cellular uptake of gd21 by acting as an endogenous transfectant. CONCLUSION: These results indicate that topical application of the GATA-3-specific DNAzyme is a promising novel approach for the treatment of allergic bronchial asthma.

DNAzyme technology and cancer therapy: cleave and let die.

Novel molecules are constantly being discovered and developed to find better means of managing debilitating and fatal diseases, which include cancer in its multiple forms. Among these molecules, and as a direct consequence of a better understanding of the molecular basis of diseases, are those falling within the class of gene therapeutics. Among these players, deoxyribozymes (DNAzymes) have come a long way from being just another analytic tool available to molecular biologists. Recent studies have shown the potential DNAzymes to serve as drugs both in cell-based assays and preclinical models of cancer. It is anticipated that with the development of smart delivery systems for DNAzymes, better pharmacokinetics and pharmacodynamics will be possible, expediting DNAzyme march toward the clinic. Also, the ability of DNAzymes to yield to such phenomena as light-induced activation may be exploited for targeted therapy. This review documents the rise of DNAzymes in the fight against cancer and serves as a forecast for this promising biotechnology in this context.