DNAzyme Cleavage of CAG Repeat RNA in Polyglutamine Diseases.

CAG repeat expansion is the genetic cause of nine incurable polyglutamine (polyQ) diseases with neurodegenerative features. Silencing repeat RNA holds great therapeutic value. Here, we developed a repeat-based RNA-cleaving DNAzyme that catalyzes the destruction of expanded CAG repeat RNA of six polyQ diseases with high potency. DNAzyme preferentially cleaved the expanded allele in spinocerebellar ataxia type 1 (SCA1) cells. While cleavage was non-allele-specific for spinocerebellar ataxia type 3 (SCA3) cells, treatment of DNAzyme leads to improved cell viability without affecting mitochondrial metabolism or p62-dependent aggresome formation. DNAzyme appears to be stable in mouse brain for at least 1 month, and an intermediate dosage of DNAzyme in a SCA3 mouse model leads to a significant reduction of high molecular weight ATXN3 proteins. Our data suggest that DNAzyme is an effective RNA silencing molecule for potential treatment of multiple polyQ diseases.

Metal organic framework coated MnO2 nanosheets delivering doxorubicin and self-activated DNAzyme for chemo-gene combinatorial treatment of cancer.

The RNA-cleaving DNAzyme (DZ) holds promising potential for RNA interference (RNAi) applications and is favored over siRNA owing to its high chemical stability, biocompatibility, predictable activity, and substrate versatility. However, its pharmaceutical applications for disease treatment are limited by the requirement of metal cofactor for activation, as well as the lack of effective co-delivery systems to combine with other therapeutic modalities. Herein, we designed and constructed metal organic framework (MOF) coated MnO2 nanosheets to realize the co-delivery of a survivin inhibiting DZ and doxorubicin (DOX) for chemo-gene combinatorial treatment of cancer. In our design, the DOX was adsorbed on MnO2 planar surface, and the DZ was loaded into the MOF shell layer through the coordination between Mn2+ and tannic acid. The nano-system could stably encapsulate the payloads under physiological condition, but rapidly degraded after endocytose into tumor cells in response to intracellular stimuli, resulting in triggered drugs release. Notably, the coreleased Mn2+ could act as metal cofactor for effective DZ activation. Both in vitro and in vivo studies have demonstrated the enhanced anti-tumor efficacy of the nanosystem, with co-contributions from anti-neoplastic DOX, survivin silencing effect of DZ, and to some extent, ROS generation by Mn2+. This work provides an ingenious strategy to address the key limitation of DZ for RNAi applications and realize the combination of DZ with other therapeutic modalities, in which the DZ can be in-situ activated for target gene silencing.

Inhibition of proliferation and migration of tumor cells through phenylboronic acid-functionalized polyamidoamine-mediated delivery of a therapeutic DNAzyme Dz13.

BACKGROUND: The phenylboronic acid-functionalized polyamidoamine (PP) was employed as a gene carrier for Dz13 delivery, inducing an obvious anticancer response. MATERIALS AND METHODS: The Dz13 condensation ability of PP was evaluated through gel retardation assay. The cellular uptake mechanism of PP/Dz13 nanoparticles was studied using confocal laser scanning microscope and flow cytometer. The inhibition ability of cell proliferation, migration and invasion was investigated through MTT assay, flow cytometry, wound healing and Transwell migration assays, using hepatocarcinoma cell line HepG2 as a model. Finally, Western blotting analysis was used to detect the signaling pathway associated with the inhibition of cell apoptosis and migration induced by Dz13 delivery. RESULTS: The carrier PP could efficiently condense Dz13 into stable nanoparticles at mass ratios of >1.5. The hydrodynamic diameter and zeta potential of PP/Dz13 nanoparticles were measured to be 204.77 nm and +22.00 mV at a mass ratio of 10.0, respectively. The nanoparticles could realize an efficient cellular uptake in sialic acid-dependent endocytosis manner. Moreover, the nanoparticles exhibited an obvious antiproliferation effect through the induction of cell apoptosis and cell cycle arrest due to the cleavage of c-Jun mRNA. Besides, the suppression of cell migration and invasion could be achieved after the PP/Dz13 transfection, attributing to the decreased expression level of MMP-2 and MMP-9. CONCLUSION: The PP provided a potential delivery system to achieve the tumor-targeting gene therapy.

Dermal delivery of therapeutic DNAzymes via chitosan hydrogels.

Biopharmaceutical development is progressing rapidly. It is imperative that novel drug delivery systems are designed to protect the integrity of the biopharmaceutical, and, at the same time, transport and distribute the drug efficaciously to the target site. Administration of highly specific and sensitive molecules, like therapeutic proteins or nucleic acid-based drugs, present distinct challenges. In this study, we investigate the topical drug delivery of 10-23 DNAzymes; short single-stranded oligonucleotides with RNA-cleaving properties. We developed different hydrogel formulations based on chitosan. These natural-based polymers are particularly suitable for biopharmaceuticals due to their high biocompatibility and biodegradability. We tested these hydrogels for penetration enhancement and for protective efficacy against DNAzymes degradation. Additionally, we examined the physicochemical characteristics and the storage stability of several hydrogel preparations. The formulations developed in this study demonstrate adequate antimicrobial activity, even without the addition of preservatives. A DNAse II degradation assay confirmed their ability to prevent enzymatic degradation of the oligonucleotide. The recovery of intact oligonucleotides in full thickness porcine skin samples indicated that hydrogel formulations composed of DNA/chitosan polyplexes provided satisfactory skin penetration.

A GATA3-specific DNAzyme attenuates sputum eosinophilia in eosinophilic COPD patients: a feasibility randomized clinical trial.

BACKGROUND: A subset of COPD-patients presents with eosinophilic airway inflammation. While treatment of asthmatic patients with the GATA3-specific DNAzyme SB010 attenuated sputum eosinophilia after allergen challenge, this specific treatment has not been evaluated in patients with COPD. Our objective was to evaluate the feasibility and safety of inhaled SB010 in COPD patients with sputum eosinophilia. METHODS: We conducted a randomized, double-blind, placebo-controlled, multicentre clinical trial in COPD-patients with sputum eosinophilia (≥2.5% non-squamous cells). Patients inhaled 10 mg SB010 bid or matching placebo via the controlled inhalation system AKITA2 APIXNEB for 28 days. Endpoints included the feasibility of the study (primary), patient's safety, sputum eosinophils, FENO, lung function, symptoms, and biomarkers. The study was registered in the German Clinical Trials Register: DRKS00006087. RESULTS: One hundred thirty patients were screened, 23 patients were randomized (FEV1 49.4 ± 11.5%; sputum eosinophils 8.0 ± 8.4%) and 19 patients completed the study (10 placebo, 9 SB010. After 28 days, SB010 decreased the relative sputum eosinophil count (p = 0.004) as compared to no changes in placebo-treated patients. FENO, lung function, and symptoms were not affected significantly. We found an increase in blood IFN-γ (p = 0.02) and a trend to lower IL-5 levels in patients treated with SB010. SB010 was safe and well tolerated. Thirty five AEs (22 SB010, 13 placebo including 1 SAE) were observed with 3 AEs in each group judged to be possibly treatment-related. CONCLUSION: In patients with eosinophilic COPD, sputum eosinophils could be reduced by inhalation of SB010. Long-term studies are needed to demonstrate clinical efficacy.

Multiplexed gene silencing in living cells and in vivo using a DNAzymes-CoOOH nanocomposite.

We demonstrate a novel DNAzymes-based nanocomposite that can simultaneously silence three types of genes in living cells and in vivo. The synergetic strategy for silencing three different genes can significantly enhance the knockdown efficacy and effectively inhibit the cancer cells' progression.

Delivery of therapeutic RNA-cleaving oligodeoxyribonucleotides (deoxyribozymes): from cell culture studies to clinical trials.

INTRODUCTION: Development of efficient in vivo delivery systems remains a major challenge en route to clinical application of antisense technology, including RNA-cleaving molecules such as deoxyribozymes (DNAzymes). The mechanisms of oligonucleotide uptake and trafficking are clearly dependent on cell type and the type of oligonucleotide analogue. It appears likely that each particular disease target would pose its own specific requirements for a delivery method. Areas covered. In this review we will discuss the available options for DNAzyme delivery in vitro and in vivo, outline various exogenous and endogenous strategies that have been, or are still being, developed and ascertain their applicability with emphasis on those methods that are currently being used in clinical trials. Expert opinion. The available information suggests that a practical system for in vivo delivery has to be biodegradable, as to minimize concerns over long-term toxicity, it should not accumulate in the organism. Extracellular vesicles may offer the most organic way for drug delivery especially as they can be fused with artificial liposomes to produce hybrid nanoparticles. Chemical modification of DNAzymes holds great potential to apply oligonucleotide analogs that would not only be resistant to nuclease digestion, but also able to penetrate cells without external delivery agents.

Rectal Delivery of a DNAzyme That Specifically Blocks the Transcription Factor GATA3 and Reduces Colitis in Mice.

BACKGROUND & AIMS: GATA3 is a transcription factor that regulates T-cell production of cytokines. We investigated the role of GATA3 in development of colitis in mice. METHODS: We performed quantitative polymerase chain reaction and immunofluorescence analyses of colon tissues from patients with Crohn's disease (n = 61) or ulcerative colitis (UC, n = 74) or from patients without inflammatory bowel diseases (n = 22), to measure levels of GATA3. Colitis was induced by administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid to control mice, mice with T-cell-specific deletion of GATA3, and mice with deletion of tumor necrosis factor receptor (TNFR) 1 and TNFR2 (TNFR double knockouts); some mice were given a GATA3-specific DNAzyme (hgd40) or a control DNAzyme via intrarectal administration, or systemic injections of an antibody to TNF before or during sensitization and challenge phase of colitis induction. Colon tissues were collected and immunofluorescence and histochemical analyses were performed. Lamina propria mononuclear cells and T cells were isolated and analyzed by flow cytometry or cytokine assays. Colonic distribution of labeled DNAzyme and inflammation were monitored by in vivo imaging (endoscopy) of mice. RESULTS: Levels of GATA3 messenger RNA were higher in colon tissues from patients with UC, but not ileal Crohn's disease, than control tissues; levels of GATA3 correlated with levels of inflammatory cytokines (interleukin [IL] 9, IL17A, IL6, IL5, IL4, IL13, and TNF). We observed increased expression of GATA3 by lamina propria T cells from mice with colitis compared with controls. Mice with T-cell-specific deletion of GATA3 did not develop colitis and their colonic tissues did not produce inflammatory cytokines (IL6, IL9, or IL13). The DNAzyme hgd40 inhibited expression of GATA3 messenger RNA by unstimulated and stimulated T cells, and distributed throughout the inflamed colons of mice with colitis. Colon tissues from mice given hgd40 had reduced expression of GATA3 messenger RNA, compared with mice given a control DNAzyme. Mice given hgd40 did not develop colitis after administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid; lamina propria cells from these mice expressed lower levels of IL6, IL9, and IL13 than cells from mice given the control DNAzyme. Mini-endoscopic images revealed that hgd40 and anti-TNF reduced colon inflammation over 3 days; hgd40 reduced colitis in TNFR double-knockout mice. CONCLUSIONS: Levels of GATA3 are increased in patients with UC and correlate with production of inflammatory cytokines in mice and humans. A DNAzyme that prevents expression of GATA3 reduces colitis in mice, independently of TNF, and reduces levels of cytokines in the colon. This DNAzyme might be developed for treatment of patients with UC.

VEGFR-1 targeted DNAzyme via transcatheter arterial delivery influences tumor vasculature assessed through dynamic contrast-enhanced magnetic resonance imaging.

DNAzymes are synthetic single-stranded DNA oligonucleotides that bind and cleave target mRNA in a sequence-specific manner. Although the therapeutic potential has been demonstrated in both preclinical and clinical settings, the efficient delivery and in vivo assessment of the DNAzyme efficacy remain the vital unsolved issue. In the present study, we examined the feasibility of using transcatheter arterial chemoembolization (TACE) strategy to deliver a DNAzyme targeting VEGFR-1 and monitoring its effect on tumor angiogenesis in vivo via dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). In a rabbit liver cancer model (VX2), we showed that the DNAzyme was efficiently delivered into the tumor by TACE. DCE-MRI revealed that the VEGFR-1-targeted DNAzyme affected the tumor vasculature through inhibiting VEGFR-1 expression in vivo, which was reflected by a reduction of Ktrans and Kep, the parameters of tumor microvascular permeability. Our findings offer an efficient strategy of delivery and assessment of the VEGFR-1 DNAzyme, and further demonstrate the feasibility of DNAzyme for cancer therapy.

Long term study of deoxyribozyme administration to XT-1 mRNA promotes corticospinal tract regeneration and improves behavioral outcome after spinal cord injury.

Spinal cord injury (SCI) affects approximately 3 million people around the world, who are desperately awaiting treatment. The pressing need for the development of therapeutics has spurred medical research for decades. To respond to this pressing need, our group developed a potential therapeutic to reduce the presence of proteoglycans at the injury site after acutely traumatizing the spinal cord of rats. With the aid of a DNA enzyme against the mRNA of xylosyltransferase-1 (DNAXT-1as) we adjourn the glycosylation and prevent the assembly of the proteoglycan core protein into the extracellular matrix. Hence, endogenous repair is strengthened due to the allocation of a more growth permissive environment around the lesion site. Here, we present data on a long term study of animals with a dorsal hemisection treated with DNAXT-1as, DNAXT-1mb (control DNA enzyme) or PBS via osmotic minipumps. After successful digestion of the XT-1 mRNA shown by qPCR we observed an overall behavioral improvement of DNAXT-1as treated rats at 8, 10 and 14 weeks after insult to the spine compared to the control animals. This is accompanied by the growth of the cortical spinal tract (CST) in DNAXT-1as treated animals after a 19 week survival period. Furthermore, after evaluating the lesion size tissue-protective effects in the DNAXT-1as treated animals compared to DNAXT-1mb and PBS treated rats are revealed. The results yield new insights into the regeneration processes and provide confirmation to involve DNA enzyme administration in future therapeutic strategies to medicate SCI.

Enhancing the pharmacokinetic/pharmacodynamic properties of therapeutic nucleotides using lipid nanoparticle systems.

Although activity has been reported in vivo, free nucleic acid-based drugs are rapidly degraded and cleared following systemic administration. To address these challenges and improve the potency and bioavailability of genetic drugs, significant efforts have been made to develop effective delivery systems of which lipid nanoparticles (LNP) represent the most advanced technology currently available. In this review, we will describe and discuss the improvements to the pharmacokinetic and pharmacodynamic properties of nucleic acid-based drugs mediated by LNP delivery. It is envisioned that the significant improvements in potency and safety, largely driven by the development of LNP encapsulated siRNA drugs, will be translatable to other types of genetic drugs and enable the rapid development of potent molecular tools and drugs.

Development of a protective dermal drug delivery system for therapeutic DNAzymes.

RNA-cleaving DNAzymes are a potential novel class of nucleic acid-based active pharmaceutical ingredients (API). However, developing an appropriate drug delivery system (DDS) that achieves high bioavailability is challenging. Especially in a dermal application, DNAzymes have to overcome physiological barriers composed of penetration barriers and degrading enzymes. The focus of the present study was the development of a protective and penetration-enhanced dermal DDS that was tailor made for DNAzymes. DNAzyme Dz13 was used as a potential API for topical therapy against actinic keratosis. In the progress of development and selection, different preservatives, submicron emulsions (SMEs) and the physiological pH range were validated with respect to the API's integrity. A physicochemical stable SME of a pharmaceutical grade along with a high API integrity was achieved. Additionally, two developed protective systems, consisting of a liposomal formulation or chitosan-polyplexes, reduced the degradation of Dz13 in vitro. A combination of SME and polyplexes was finally validated at the skin and cellular level by in vitro model systems. Properties of penetration, degradation and distribution were determined. The result was enhanced skin penetration efficiency and increased cellular uptake with a high protective efficiency for DNAzymes due to the developed protective DDS.

DCE-MRI assessment of the effect of Epstein-Barr virus-encoded latent membrane protein-1 targeted DNAzyme on tumor vasculature in patients with nasopharyngeal carcinomas.

BACKGROUND: EBV-encoded latent membrane protein 1 (EBV-LMP1) is an important oncogenic protein for nasopharyngeal carcinoma (NPC) and has been shown to engage a plethora of signaling pathways. Correspondingly, an LMP1-targeted DNAzyme was found to inhibit the growth of NPC cells both in vivo and in vitro by suppressing cell proliferation and inducing apoptosis. However, it remains unknown whether an LMP1-targeted DNAzyme would affect the vasculature of NPC. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been applied in the clinical trials of anti-angiogenic drugs for more than ten years, and Ktrans has been recommended as a primary endpoint. Therefore, the objective of the current study was to use DCE-MRI to longitudinally study the effect of an EBV-LMP1-targeted DNAzyme on the vasculature of patients with NPC. METHODS: Twenty-four patients were randomly divided into two groups: a combined treatment group (radiotherapy + LMP1-targeted DNAzyme) and a radiotherapy alone group (radiotherapy + normal saline). DCE-MRI scans were conducted 1 ~ 2 days before radiotherapy (Pre-RT), during radiotherapy (RT 50 Gy), upon completion of radiotherapy (RT 70 Gy), and three months after radiotherapy (3 months post-RT). Parameters of vascular permeability and intra- and extravascular volumes were subsequently obtained (e.g., Ktrans, kep, ve) using nordicICE software. RESULTS: Both Ktrans and kep values for NPC tumor tissues decreased for both groups after treatment. Moreover, a statistically significant difference in Ktrans values at the pre-therapy and post-therapy timepoints emerged earlier for the combined treatment group (RT 50 Gy, P =0.045) compared to the radiotherapy alone group (3 months post-RT, P = 0.032). For the kep values, the downward trend observed for both the combined treatment group and the radiotherapy alone group were similar. In contrast, ve values for all of the tumor tissues increased following therapy. CONCLUSIONS: The EBV-LMP1-targeted DNAzyme that was tested was found to accelerate the decline of Ktrans values for patients with NPC. Correspondingly, the LMP1-targeted DNAzyme treatments were found to affect the angiogenesis and microvascular permeability of NPC. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01449942. Registered 6 October 2011.

Therapeutic evaluation of Epstein-Barr virus-encoded latent membrane protein-1 targeted DNAzyme for treating of nasopharyngeal carcinomas.

The ability of the 10-23 DNAzyme to specifically cleave RNA with high efficiency has fuelled expectation that this agent may have useful applications for targeted therapy. Here, we, for the first time, investigated the antitumor and radiosensitizing effects of a DNAzyme (DZ1) targeted to the Epstein-Barr virus (EBV)-LMP1 mRNA of nasopharyngeal carcinoma (NPC) in patients. Preclinical studies indicated that the DNAzyme was safe and well tolerated. A randomized and double-blind clinical study was conducted in 40 NPC patients who received DZ1 or saline intratumorally, in conjunction with radiation therapy. Tumor regression, patient survival, EBV DNA copy number and tumor microvascular permeability were assessed in a 3-month follow-up. The mean tumor regression rate at week 12 was significantly higher in DZ1 treated group than in the saline control group. Molecular imaging analysis showed that DZ1 impacted on tumor microvascular permeability as evidenced by a faster decline of the K(trans) in DZ1-treated patients. The percentage of the samples with undetectable level of EBV DNA copy in the DZ1 group was significantly higher than that in the control group. No adverse events that could be attributed to the DZ1 injection were observed in patients.

The distribution, clearance, and safety of an anti-MMP-9 DNAzyme in normal and MMTV-PyMT transgenic mice.

Catalytic oligonucleotides, known as DNAzymes, are a new class of nucleic acid-based gene therapy that have recently been used in preclinical animal studies to treat various cancers. In this study the systemic distribution, pharmacokinetics, and safety of intravenously administered anti-MMP (matrix metalloproteinase)-9 DNAzyme (AM9D) were determined in healthy FVB and in MMTV-polyoma virus middle T (PyMT) transgenic mice bearing mammary tumors. MMP-9 is known to be involved in tumor cell development, angiogenesis, invasion, and metastasis. Sulfur-35 ((35)S) labeled ([(35)S]-AM9D) administered intravenously, without the use of carrier molecules, to healthy and mammary tumor bearing MMTV-PyMT transgenic mice distributed to all major organs. The order of percentages of [(35)S]-AM9D accumulation in different organs of healthy and MMTV-PyMT mice were blood>liver>kidney>lung>spleen>heart and mammary tumor>blood≈liver>kidney>spleen>lung>heart, respectively. The amount of AM9D accumulated in mammary tumors 2 hours post injection was 0.6% and 0.2% higher than in either blood or liver, respectively, and its rate of initial clearance from mammary tissue was at least 50% slower than the other organs. Approximately 43% of the delivered dosage of [(35)S]-AM9D was cleared from the system via feces and urine over a period of 72 hours. No evidence of acute or chronic cytotoxicity, local or widespread, associated with AM9D treatment (up to 75 mg AM9D /kg of body weight) was observed in the organs examined. These data suggest that DNAzyme in general and AM9D in particular can be used systemically as a therapeutic agent to treat patients with breast cancer or other metastatic and surgically inaccessible tumors.

Selective inhibition of the master regulator transcription factor Egr-1 with catalytic oligonucleotides reduces myocardial injury and improves left ventricular systolic function in a preclinical model of myocardial infarction.

BACKGROUND: Egr-1 is implicated in the pathogenesis of myocardial ischemia-reperfusion injury. The aim of this study was to ascertain the effectiveness of intracoronary delivery of DNAzyme targeting the transcription factor Egr-1 at reperfusion following experimental myocardial ischemia. METHODS AND RESULTS: Functional DNAzyme targeting Egr-1 or a size-matched scrambled control were delivered via the intracoronary route immediately on reperfusion after 60 minutes' balloon occlusion of the left anterior descending coronary artery in a pig model of myocardial I/R injury (n=7 per treatment group). Heart function and extent of myocardial infarction were determined following intervention by echocardiography and cardiac magnetic resonance imaging, respectively. Hearts were removed and examined for molecular and histological markers of inflammation and apoptosis. Administration of functional DNAzyme led to an overall decrease in the expression of inflammatory markers including intracellular adhesion molecule-1, tissue factor, and complement 3, with associated decreases in the extent of neutrophil infiltration, oxidative damage, and subsequent apoptosis within the infarct border zone. Functional significance was indicated by an increase in salvaged left ventricular myocardium (P=0.012), ejection fraction (P=0.002), and fractional area change (P=0.039) in the functional DNAzyme-treated group compared with the control. CONCLUSIONS: Egr-1 silencing through intracoronary delivery of a targeting DNAzyme at the time of reperfusion following acute myocardial ischemia decreases myocardial inflammation and apoptosis leading to improved cardiac function.

Biodistribution of the GATA-3-specific DNAzyme hgd40 after inhalative exposure in mice, rats and dogs.

The DNAzyme hgd40 was shown to effectively reduce expression of the transcription factor GATA-3 RNA which plays an important role in the regulation of Th2-mediated immune mechanisms such as in allergic bronchial asthma. However, uptake, biodistribution and pharmacokinetics of hgd40 have not been investigated yet. We examined local and systemic distribution of hgd40 in naive mice and mice suffering from experimental asthma. Furthermore, we evaluated the pharmacokinetics as a function of dose following single and repeated administration in rats and dogs. Using intranasal administration of fluorescently labeled hgd40 we demonstrated that the DNAzyme was evenly distributed in inflamed asthmatic mouse lungs within minutes after single dose application. Systemic distribution was investigated in mice using radioactive labeled hgd40. After intratracheal application, highest amounts of hgd40 were detected in the lungs. High amounts were also detected in the bladder indicating urinary excretion as a major elimination pathway. In serum, low systemic hgd40 levels were detected already at 5 min post application (p.a.), subsequently decreasing over time to non-detectable levels at 2h p.a. As revealed by Single Photon Emission Computed Tomography, trace amounts of hgd40 were detectable in lungs up to 7 days p.a. Also in the toxicologically relevant rats and dogs, hgd40 was detectable in blood only shortly after inhalative application. The plasma pharmacokinetic profile was dose and time dependent. Repeated administration did not lead to drug accumulation in plasma of dogs and rats. These pharmacokinetic of hgd40 provide guidance for clinical development, and support an infrequent and convenient dose administration regimen.

Inhibition of vein graft stenosis with a c-jun targeting DNAzyme in a cationic liposomal formulation containing 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

BACKGROUND/OBJECTIVES: Coronary artery bypass grafting (CABG) is among the most commonly performed heart surgical procedures. Saphenous vein graft failure due to stenosis impedes the longer-term success of CABG. A key cellular event in the process of vein graft stenosis is smooth muscle cell hyperplasia. In this study, we evaluated the effect of a DNAzyme (Dz13) targeting the transcription factor c-Jun in a rabbit model of vein graft stenosis in a cationic liposomal formulation containing 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Dz13 in DOTAP/DOPE has undergone preclinical toxicological testing, and a Phase I clinical trial we recently conducted in basal cell carcinoma cancer patients demonstrates that it is safe and well tolerated after local administration. METHODS: Effects of Dz13 in a formulation containing DOTAP/DOPE on smooth muscle cell (SMC) growth and c-Jun expression were assessed. Dz13 transfection was determined by cellular uptake of carboxyfluorescein-labeled Dz13. Autologous jugular vein to carotid artery transplantation was performed in New Zealand White rabbits to investigate the effect of the Dz13 in DOTAP/DOPE formulation on intimal hyperplasia. RESULTS: Dz13/DOTAP/DOPE reduced SMC proliferation and c-Jun protein expression in vitro compared with an impotent form of Dz13 bearing a point mutation in its catalytic domain (Dz13.G>C). The Dz13(500 µg)/DOTAP/DOPE formed lipoplexes that were colloidally stable for up to 1h on ice (0°C) and 30 min at 37°C, allowing sufficient uptake by the veins. Dz13 (500 µg) inhibited neointima formation 28 d after end-to-side transplantation. CONCLUSIONS: This formulation applied to veins prior to transplantation may potentially be useful in efforts to reduce graft failure.

Therapeutic potential of nucleic acid-based drugs in coronary hyper- proliferative vascular diseases.

The thickening of the vessel wall (intimal hyperplasia) is a pathological process which often follows revascularization approaches such as transluminal angioplasty and artery bypass graft, procedures used to re-vascularize stenotic artery. Despite the significant improvements in the treatment of intimal hyperplasia obtained in the last years, the problem has not completely solved. Nucleic acid based-drugs (NABDs) represent an emergent class of molecules with potential therapeutic value for the treatment of intimal hyperplasia. NABDs of interest in the field of intimal hyperplasia are: ribozymes, DNAzymes, antisense oligonucleotides, decoy oligonucleotides, small interfering RNAs and micro interfering RNAs. These molecules can recognize, in a sequencespecific fashion, a target which, depending on the different NABDs, can be represented by a nucleic acid or a protein. Upon binding, NABDs can down-modulate the functions of the target (mRNA/proteins) and thus they are used to impair the functions of disease-causing biological molecules.In spite of the great therapeutic potential demonstrated by NABDs in many experimental model of intima hyperplasia, their practical use is hindered by the necessity to identify optimal delivery systems to the vasculature. In the first part of this review a brief description of the clinical problem related to intima hyperplasia formation after revascularization procedures is reported. In the second part, the attention is focused on the experimental evidences of NABD therapeutic potential in the prevention of intimal hyperplasia. Finally, in the third part, we will describe the strategies developed to optimize NABD delivery to the diseased vessel.