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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoo tonic, highly pathogenic virus. The new type of coronavirus with contagious nature spread from Wuhan (China) to the whole world in a very short time and caused the new coronavirus disease (COVID-19). COVID-19 has turned into a global public health crisis due to spreading by close person-to-person contact with high transmission capacity. Thus, research about the treatment of the damages caused by the virus or prevention from infection increases everyday. Besides, there is still no approved and definitive, standardized treatment for COVID-19. However, this disaster experienced by human beings has made us realize the significance of having a system ready for use to prevent humanity from viral attacks without wasting time. As is known, nanocarriers can be targeted to the desired cells in vitro and in vivo . The nano-carrier system targeting a specific protein, containing the enzyme inhibiting the action of the virus can be developed. The system can be used by simple modifications when we encounter another virus epidemic in the future. In this review, we present a potential treatment method consisting of a nanoparticle-ribozyme conjugate, targeting ACE-2 receptors by reviewing the virus-associated ribozymes, their structures, types and working mechanisms.

Survivin is one of the most cancer-specific proteins overexpressed in almost all malignancies, but is nearly undetectable in most normal tissues in adults. Functionally, as a member of the inhibitor of apoptosis family, survivin has been shown to inhibit apoptosis and increase proliferation. The antiapoptotic function of survivin seems to be related to its ability to inhibit caspases directly or indirectly. Furthermore, the role of survivin in cell cycle division control is related to its role in the chromosomal passenger complex. Consistent with its determining role in these processes, survivin plays a crucial role in cancer progression and cancer cell resistance to anticancer drugs and ionizing radiation. On the basis of these findings, recently survivin has been investigated intensively as an ideal tumor biomarker. Thus, multiple molecular approaches such as use of the RNA interfering technique, antisense oligonucleotides, ribozyme, and small molecule inhibitors have been used to downregulate survivin regulation and inhibit its biological function consequently. In this review, all these approaches are explained and other compounds that induced apoptosis in different cell lines through survivin inhibition are also reported.

Hammerhead ribozymes (hhRzs), RNA enzymes capable of site-specific cleavage of arbitrary target mRNAs, have faced significant hurdles in development and optimization as gene therapeutics for clinical translation. Chemical and biological barriers must be overcome to realize an effective therapeutic. A new Facilitated ribozyme has been identified with greatly enhanced kinetic properties that lead new insight on the capacity of ribozymes to target mutant genes to treat inherited retinal degenerations.

Inherited retinal diseases (IRD) encompass a wide spectrum of hereditary blindness with significant genetic heterogeneity. Therapeutics regulating gene expression on an RNA level have significant promise for treating IRD. In this review, we review the molecular basis of oligonucleotide therapeutics such as ribozymes, RNA interference (RNAi), antisense oligonucleotides (ASO), CRISPRi/a, and their applications to treatments of IRD.

Since the breakthrough discovery of catalytic RNAs (ribozymes) in the early 1980s, valuable ribozyme-based gene therapies have been developed for incurable diseases ranging from genetic disorders to viral infections and cancers. Ribozymes can be engineered and used to downregulate or repair pathogenic genes via RNA cleavage mediated by trans-cleaving ribozymes or repair and reprograming mediated by trans-splicing ribozymes, respectively. Uniquely, trans-splicing ribozymes can edit target RNAs via simultaneous destruction and repair (and/or reprograming) to yield the desired therapeutic RNAs, thus selectively inducing therapeutic gene activity in cells expressing the target RNAs. In contrast to traditional gene therapy approaches, such as simple addition of therapeutic transgenes or inhibition of disease-causing genes, the selective repair and/or reprograming abilities of trans-splicing ribozymes in target RNA-expressing cells facilitates the maintenance of endogenous spatial and temporal gene regulation and reduction of disease-associated transcript expression. In molecular imaging technologies, trans-splicing ribozymes can be used to reprogram specific RNAs in living cells and organisms by the 3'-tagging of reporter RNAs. The past two decades have seen progressive improvements in trans-splicing ribozymes and the successful application of these elements in gene therapy and molecular imaging approaches for various pathogenic conditions, such as genetic, infectious, and malignant disease. This review provides an overview of the current status of trans-splicing ribozyme therapeutics, focusing on Tetrahymena group I intron-based ribozymes, and their future prospects. This article is categorized under: RNA in Disease and Development > RNA in Disease., (© 2018 Wiley Periodicals, Inc.)

Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets. To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N = G,C,A,U; H = C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a large set of potential VAI-hhRz expression plasmids against diverse NUH↓ cleavage sites uses cultured human HEK293S cells stably expressing a dicistronic Target-IRES-SEAP target fusion mRNA. Broad utility of this rational RNA drug discovery approach is feasible for any ophthalmological disease-relevant mRNA targets and any disease mRNA targets in general. The approach will permit rank ordering of PTGS agents based on potency to identify a lead therapeutic compound for further optimization., (Published by Elsevier Ltd.)

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are simple linear polymers that have been the subject of considerable research in the last two decades and have now moved into the realm of being stand-alone therapeutic agents. Much of this has stemmed from the appreciation that they carry out myriad functions that go beyond mere storage of genetic information and protein synthesis. Therapy with nucleic acids either uses unmodified DNA or RNA or closely related compounds. From both a development and regulatory perspective, they fall somewhere between small molecules and biologics. Several of these compounds are in clinical development and many have received regulatory approval for human use. This review addresses therapeutic uses of DNA based on antisense oligonucleotides, DNA aptamers and gene therapy; and therapeutic uses of RNA including micro RNAs, short interfering RNAs, ribozymes, RNA decoys and circular RNAs. With their specificity, functional diversity and limited toxicity, therapeutic nucleic acids hold enormous promise. However, challenges that need to be addressed include targeted delivery, mass production at low cost, sustaining efficacy and minimizing off-target toxicity. Technological developments will hold the key to this and help accelerate drug approvals in the years to come., (© 2016 The British Pharmacological Society.)

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.

The vast majority of the human genome is transcribed into RNA molecules that do not code for proteins, which could be small ones approximately 20 nucleotide in length, known as microRNAs, or transcripts longer than 200 bp, defined as long noncoding RNAs. The prevalent deregulation of microRNAs in human cancers prompted immediate interest on the therapeutic value of microRNAs as drugs and drug targets. Many features of microRNAs such as well-defined mechanisms, and straightforward oligonucleotide design further make them attractive candidates for therapeutic development. The intensive efforts of exploring microRNA therapeutics are reflected by the large body of preclinical studies using oligonucleotide-based mimicking and blocking, culminated by the recent entry of microRNA therapeutics in clinical trial for several human diseases including cancer. Meanwhile, microRNA therapeutics faces the challenge of effective and safe delivery of nucleic acid therapeutics into the target site. Various chemical modifications of nucleic acids and delivery systems have been developed to increase targeting specificity and efficacy, and reduce the associated side effects including activation of immune response. Recently, long noncoding RNAs become attractive targets for therapeutic intervention because of their association with complex and delicate phenotypes, and their unconventional pharmaceutical activities such as capacity of increasing output of proteins. Here I discuss the general therapeutic strategies targeting noncoding RNAs, review delivery systems developed to maximize noncoding RNA therapeutic efficacy, and offer perspectives on the future development of noncoding RNA targeting agents for colorectal cancer.

Despite the availability of an effective vaccine against hepatitis B virus (HBV), chronic infection with the virus remains a major global health concern. Current drugs against HBV infection are limited by emergence of resistance and rarely achieve complete viral clearance. This has prompted vigorous research on developing better drugs against chronic HBV infection. Advances in understanding the life cycle of HBV and improvements in gene-disabling technologies have been impressive. This has led to development of better HBV infection models and discovery of new drug candidates. Ideally, a regimen against chronic HBV infection should completely eliminate all viral replicative intermediates, especially covalently closed circular DNA (cccDNA). For the past few decades, nucleic acid-based therapy has emerged as an attractive alternative that may result in complete clearance of HBV in infected patients. Several genetic anti-HBV strategies have been developed. The most studied approaches include the use of antisense oligonucleotides, ribozymes, RNA interference effectors and gene editing tools. This review will summarize recent developments and progress made in the use of gene therapy against HBV.

Introduction: Nucleic acid-based therapeutics (NATs) are proven agents in correcting disorders caused by gene mutations, as treatments against cancer, microbes and viruses, and as vaccine adjuvants. Although many traditional small molecule NATs have been approved for clinical use, commercialization of macromolecular NATs has been considerably slower, and only a few have successfully reached the market. Preclinical and clinical evaluation of macromolecular NATs has revealed many assorted challenges in immunotoxicity, hematotoxicity, pharmacokinetics (PKs), toxicology and formulation. Extensive review has been given to the PK and toxicological concerns of NATs including approaches designed to overcome these issues. Immunological and hematological issues are a commonly reported side effect of NAT treatment; however, literature exploring the mechanistic background of these effects is sparse., Areas Covered: This review focuses on the immunomodulatory properties of various types of therapeutic nucleic acid concepts. The most commonly observed immunological and hematological toxicities are described for various NAT classes, with citations of how to circumvent these toxicities., Expert Opinion: Although some success with overcoming immunological and hematological toxicities of NATs has been achieved in recent years, immunostimulation remains the main dose-limiting factor challenging clinical translation of these promising therapies. Novel delivery vehicles should be considered to overcome this challenge.

Cumulative progress in nanoparticle development has opened a new era of targeted delivery of therapeutics to cancer cells and tissue. However, developing proper detection methods has lagged behind resulting in the lack of precise evaluation and monitoring of the systemically administered nanoparticles. RNA nanoparticles derived from the bacteriophage phi29 DNA packaging motor pRNA have emerged as a new generation of drugs for cancer therapy. Multifunctional RNA nanoparticles can be fabricated by bottom-up self-assembly of engineered RNA fragments harboring targeting (RNA aptamer or chemical ligand), therapeutic (siRNA, miRNA, ribozymes, and small molecule drugs), and imaging (fluorophore, radiolabels) modules. We have recently demonstrated that RNA nanoparticles can reach and target intracranial brain tumors in mice upon systemic injection with little or no accumulation in adjacent healthy brain tissues or in major healthy internal organs. Herein, we describe various functional imaging methods (fluorescence confocal microscopy, flow cytometry, fluorescence whole body imaging, and magnetic resonance imaging) to evaluate and monitor RNA nanoparticle targeting to intracranial brain tumors in mice. Such imaging techniques will allow in-depth evaluation of specifically delivered RNA therapeutics to brain tumors.

Ribozymes are structured RNA molecules that act as catalysts in different biological reactions. From simple genome cleaving activities in satellite RNAs to more complex functions in cellular protein synthesis and gene regulation, ribozymes play important roles in all forms of life. Several naturally existing ribozymes have been modified for use as therapeutics in different conditions, with HIV-1 infection being one of the most studied. This chapter summarizes data from different preclinical and clinical studies conducted to evaluate the potential of ribozymes to be used in HIV-1 therapies. The different ribozyme motifs that have been modified, as well as their target sites and expression strategies, are described. RNA conjugations used to enhance the antiviral effect of ribozymes are also presented and the results from clinical trials conducted to date are summarized. Studies on anti-HIV-1 ribozymes have provided valuable information on the optimal expression strategies and clinical protocols for RNA gene therapy and remain competitive candidates for future therapy.

One of the major concerns with regard to successful cancer gene therapy is to enhance both efficacy and safety. Gene targeting may represent an attractive tool to combat cancer cells without damage to normal cells. Here, we introduce a tumor-targeting approach with the Tetrahymena group I intron-based trans-splicing ribozyme, which cleaves target RNA and trans-ligate an exon tagged at the end of the ribozyme onto the downstream U nucleotide of the cleaved target RNA. We develop a specific trans-splicing ribozyme that can target and reprogram human cytoskeleton-associate protein 2 (hCKAP2)-encoding RNA to trigger therapeutic transgene herpes simplex virus thymidine kinase (HSVtk) selectively in cancer cells that express the RNA. Adenoviral vectors encoding the hCKAP2-specific trans-splicing ribozyme are constructed for in vivo delivery into either subcutaneous tumor xenograft or orthotopically multifocal hepatocarcinoma. We present analyses of the efficacy of the recombinant adenoviral vectors in terms of cancer retardation, target RNA and cell specificity, and in vivo toxicity.

In this review, we discuss recent advances in nucleic acid-based therapeutic technologies that target hepatitis C virus (HCV) infection. Because the HCV genome is present exclusively in RNA form during replication, various nucleic acid-based therapeutic approaches targeting the HCV genome, such as ribozymes, aptamers, siRNAs, and antisense oligonucleotides, have been suggested as potential tools against HCV. Nucleic acids are potentially immunogenic and typically require a delivery tool to be utilized as therapeutics. These limitations have hampered the clinical development of nucleic acid-based therapeutics. However, despite these limitations, nucleic acid-based therapeutics has clinical value due to their great specificity, easy and large-scale synthesis with chemical methods, and pharmaceutical flexibility. Moreover, nucleic acid therapeutics are expected to broaden the range of targetable molecules essential for the HCV replication cycle, and therefore they may prove to be more effective than existing therapeutics, such as interferon-α and ribavirin combination therapy. This review focuses on the current status and future prospects of ribozymes, aptamers, siRNAs, and antisense oligonucleotides as therapeutic reagents against HCV.

Glioblastoma represents one of the most challenging problems in neurooncology. Among key elements driving its behavior is the transmembrane epidermal growth factor receptor family, with the first member epidermal growth factor receptor (EGFR) centered in most studies. Engagement of the extracellular domain with a ligand activates the intracellular tyrosine kinase (TK) domain of EGFR, leading to autophosphorylation and signal transduction that controls proliferation, gene transcription, and apoptosis. Oncogenic missense mutations, deletions, and insertions in the EGFR gene are preferentially located in the extracellular domain in glioblastoma and cause constitutive activation of the receptor. The mutant EGFR may also transactivate other cell surface molecules, such as additional members of the EGFR family and the platelet-derived growth factor receptor, which ignite signaling cascades that synergize with the EGFR-initiated cascade. Because of the cell surface location and increased expression of the receptor along with its important biological function, EGFR has triggered much effort for designing targeted therapy. These approaches include TK inhibition, monoclonal antibody, vaccine, and RNA-based downregulation of the receptor. Treatment success requires that the drug penetrates the blood-brain barrier and has low systemic toxicity but high selectivity for the tumor. While the blockade of EGFR-dependent processes resulted in experimental and clinical treatment success, cells capable of using alternative signaling ultimately escape this strategy. A combination of interventions targeting tumor-specific cell surface regulators along with convergent downstream signaling pathways will likely enhance efficacy. Studies on EGFR in glioblastoma have revealed much information about the complexity of gliomagenesis and also facilitated the development of strategies for targeting drivers of tumor growth and combination therapies with increasing complexity.

Background: Serum and plasma levels of vascular endothelial growth factor (VEGF) correlate with prognosis in patients with metastatic breast cancer (MBC). VEGF binds to 2 receptors on endothelial cells, VEGFR-1 and VEGFR-2. RPI.4610 (Angiozyme0) is an antiangiogenic ribozyme targeting the VEGFR-1 mRNA. Preclinical and phase 1 studies suggested that RPI.4610 is a well-tolerated agent with clinical activity in solid tumors. The authors' trial evaluated the efficacy of RPI.4610 in the treatment of patients with progressive MBC., Methods: This phase 2, multicenter, single-arm study was designed to assess the objective response rate of RPI.4610 in patients with MBC who had experienced disease progression with at least 1 course of chemotherapy for MBC. Patients received daily subcutaneous injections of RPI.4610 100 mg/m(2) for 12 weeks., Results: Most patients (93%) had received at least 2 lines of chemotherapy previously; 69% of patients had received at least 3 lines of chemotherapy. Median follow-up was 2.76 months (range, 0.89-36.6 months). No partial responses nor complete responses were found. Median progression-free survival was 1.41 months (95% confidence interval [CI], 1.35-1.45). The median overall survival from start of treatment was 11.89 months (95% CI, 4.11-23.66). Treatment-related adverse events (AEs) were primarily grade 1 to 2 in intensity. Most common AEs were: injection site reactions, abdominal pain, anorexia, chromaturia, constipation, dyspnea, fatigue, headache, pain at the injection site, nausea, vomiting, and fever., Conclusions: Although RPI.4610 demonstrated a well-tolerated safety profile, its lack of clinical efficacy precludes this drug from further development., (Copyright © 2012 American Cancer Society.)

Introduction: Survivin a multifunctional protein that controls cell division, inhibition of apoptosis and promotion of angiogenesis. It is expressed in most human neoplasm, but is absent in normal and differentiated tissues. The purpose of this article is to overview the expression of survivin, effect of its expression in response to treatment, correlation with other markers and newer advancement in targeting survivin., Methods: A detailed search of Medline was carried out using the following search strategy: "((survivin) OR ((apoptosis) AND (inhibitor OR inhibitors))) AND ((breast) AND (neoplasm OR neoplasms OR tumor OR tumor OR cancer OR carcinoma))". Abstract of all articles thus identified were reviewed to identify the relevant studies, full articles of studies thus identified were then obtained and reviewed. All relevant data was extracted and tabulated., Results: Survivin expression by Immunohistochemistry was identified in 65.3% (55.2-90.0%) of the breast cancer patients among the identified studies while survivin mRNA by RT-PCR was identified in 93.6% (90-97%). Survivin expression has been reported to be associated with over expression of HER 2, vascular endothelial growth factor (VEGF), urokinase plasminogen activator (uPA)/PAI-1., Conclusion: Survivin is over expressed in majority of breast cancers. The over expression of survivin is found to correlate with HER 2 and EGFR expression. Survivin expression has been found to confer resistance to chemotherapy and radiation. Targeting survivin in experimental models improves survival. More studies are needed on the role of survivin in multi drug resistance (MDR) in the presence of Pgp/uPA/PAI-1 and the impact of survivin over expression in triple negative breast cancer., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Recent advances of biological drugs have broadened the scope of therapeutic targets for a variety of human diseases. This holds true for dozens of RNA-based therapeutics currently under clinical investigation for diseases ranging from genetic disorders to HIV infection to various cancers. These emerging drugs, which include therapeutic ribozymes, aptamers, and small interfering RNAs (siRNAs), demonstrate the unprecedented versatility of RNA. However, RNA is inherently unstable, potentially immunogenic, and typically requires a delivery vehicle for efficient transport to the targeted cells. These issues have hindered the clinical progress of some RNA-based drugs and have contributed to mixed results in clinical testing. Nevertheless, promising results from recent clinical trials suggest that these barriers may be overcome with improved synthetic delivery carriers and chemical modifications of the RNA therapeutics. This review focuses on the clinical results of siRNA, RNA aptamer, and ribozyme therapeutics and the prospects for future successes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

RNA nanoparticles have applications in the treatment of cancers and viral infection; however, the instability of RNA nanoparticles has hindered their development for therapeutic applications. The lack of covalent linkage or crosslinking in nanoparticles causes dissociation in vivo. Here we show that the packaging RNA of bacteriophage phi29 DNA packaging motor can be assembled from 3-6 pieces of RNA oligomers without the use of metal salts. Each RNA oligomer contains a functional module that can be a receptor-binding ligand, aptamer, short interfering RNA or ribozyme. When mixed together, they self-assemble into thermodynamically stable tri-star nanoparticles with a three-way junction core. These nanoparticles are resistant to 8 M urea denaturation, are stable in serum and remain intact at extremely low concentrations. The modules remain functional in vitro and in vivo, suggesting that the three-way junction core can be used as a platform for building a variety of multifunctional nanoparticles. We studied 25 different three-way junction motifs in biological RNA and found only one other motif that shares characteristics similar to the three-way junction of phi29 pRNA.

Gene therapy is considered a feasible approach for the treatment and prevention of HIV/AIDS. Targeting both viral genes and host dependency factors can interfere with the viral lifecycle and prevent viral replication. A number of approaches have been taken to target these genes, including ribozymes, aptamers, and RNAi based therapies. A number of these therapies are now beginning to make their way into clinical trials and providing proof of principle that gene therapy is a safe and realistic option for treating HIV. Here, we focus on those therapies that have progressed along the pipeline to preclinical and clinical testing., (Copyright © 2011.)

Based on the natural functions and chemical characteristics of nucleic acids, a variety of novel synthetic drugs and tools to explore biological systems have become available in recent years. To date, a great number of antisense oligonucleotides, RNA interference-based tools, CpG‑containing oligonucleotides, catalytic oligonucleotides, decoys and aptamers has been produced synthetically and applied successfully for understanding and manipulating biological processes and in clinical trials to treat a variety of diseases. Their versatility and potency make them equally suited candidates for fighting viral infections. Here, we describe the different types of nucleic acid-based antivirals, their mechanism of action, their advantages and limitations, and their future prospects.

A brief historical introduction describes early attempts to silence specific genes using the antisense oligonucleotides that flourished in the 1980s. Early aspirations for therapeutic applications were almost extinguished by the unexpected complexity of oligonucleotide pharmacology. Once the biochemistry and molecular biology behind some of the pharmacology was worked out, new approaches became apparent for using oligonucleotides to treat disease. The biochemistry of small nucleic acids is outlined in Section 2. Various approaches employing oligonucleotides to control cellular functions are reviewed in Section 3. These include antisense oligonucleotides and siRNA that bind to RNA, antigene oligonucleotides that bind to DNA, and aptamers, decoys, and CpG oligonucleotides that bind to proteins.

Therapeutic approaches employing RNA as a tool or as a drug target have recently emerged and have been employed for various applications-ranging from cancer treatment to virus infection. Despite the paucity of its molecular groups compared to proteins, RNA has nevertheless proved to be an excellent choice for researchers who have aspired to develop therapeutic tools. Ribozymes and riboswitches are RNA-based therapeutic tools that are most often employed to knockdown gene expression and to inhibit bacterial infections, respectively. The aim of this review is to summarize recent advances observed in ribozyme- and riboswitch-based therapeutic applications that, in some cases, have reached clinical trials., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

Advances in chromosome dynamics have increased our understanding of the significant role of telomeres and telomerase in cancer. Telomerase is expressed in almost all cancer cells but is inactive in most normal somatic cells. Therefore, telomerase is an important target for the design of therapeutic agents that might have minimal side effects. Herein, we evaluate current approaches to telomerase/telomere-targeted therapy, discuss the benefits and disadvantages, and speculate on the future direction of telomerase inhibitors as cancer therapeutics.

The 5'-untranslated region (5'UTR) of the HIV-1 RNA is an attractive target for engineered ribozymes due to its high sequence and structural conservation. This region encodes several conserved structural RNA domains essential in key processes of the viral replication and infection cycles. This paper reports the inhibitory effects of catalytic antisense RNAs composed of two inhibitory RNA domains: an engineered ribozyme targeting the 5' UTR and a decoy or antisense domain of the dimerization initiation site (DIS). These chimeric molecules are able to cleave the HIV-1 5'UTR efficiently and prevent viral genome dimerization in vitro. Furthermore, catalytic antisense RNAs inhibited viral production up to 90% measured as p24 antigen levels in ex vivo assays. The use of chimeric RNA molecules targeting different domains represents an attractive antiviral strategy to be explored for the prevention of side effects from current drugs and of the rapid emergence of escape variants of HIV-1.

The epidermal growth factor receptor (HER1/EGFR) is known to be disregulated in a large subgroup of glioblastoma multiforme cases. Disregulation of HER1/EGFR is related to malignant transformation and tumor growth in various human cancers, including malignant glioma. One mechanism that may lead to disregulated HER1/EGFR signaling is the intrinsic alteration of the receptor structure due to mutational changes. The most common mutant form of HER1/EGFR, named variant III (EGFRvIII), results from an 801 bp in-frame deletion in the DNA sequence encoding the extracellular ligand-binding domain. Independent of ligand-binding, EGFRvIII is constitutively activated and beyond external control. Since its cellular expression was shown to relate enhanced tumorigenicity, various therapeutic strategies were developed to target EGFRvIII, including monoclonal antibodies, vaccination therapies and small-molecule tyrosine kinase inhibitors. In this review, we focus on ribozyme-mediated inhibition of EGFRvIII messenger RNA expression as a gene therapeutic approach for EGFRvIII-expressing glioblastoma multiforme.

With the development of the research on biotechnology and modern pharmacy, the application of enzyme drugs have grown rapidly and enzyme drugs have become an important branch of biopharmaceutics. In this article, some new varieties of therapeutic enzymes, enzyme targets, mechanisms and new technologies of application in therapeutic enzymes were reviewed, and the direction of development of therapeutic enzymes were discussed.

Background: Gene therapy represents a new treatment paradigm for HIV that is potentially delivered by a safe, once-only therapeutic intervention., Methods: Using mathematical modelling, we assessed the possible impact of autologous haematopoietic stem cell (HSC) delivered, anti-HIV gene therapy. The therapy comprises a ribozyme construct (OZ1) directed to a conserved region of HIV-1 delivered by transduced HSC (OZ1+HSC). OZ1+HSC contributes to the CD4+ T lymphocyte and monocyte/macrophage cell pools that preferentially expand under the selective pressure of HIV infection. The model was used to predict the efficacy of OZ1 in a highly active antiretroviral therapy (HAART) naïve individual and a HAART-experienced individual undergoing two structured treatment operations. In the standard scenario, OZ1+HSC was taken as 20% of total body HSC., Results: For a HAART-naïve individual, modelling predicts a reduction of HIV RNA at 1 and 2 years post-OZ1 therapy of 0.5 log(10) and 1 log(10), respectively. Eight years after OZ1 therapy, the CD4+ T-lymphocyte count was 271 cells/mm(3) compared to 96 cells/mm(3) for an untreated individual. In a HAART-experienced individual HIV RNA was reduced by 0.34 log(10) and 0.86 log(10) at 1 and 2 years. The OZ1 effect was maximal when both CD4+ T lymphocytes and monocytes/macrophages were protected from successful, productive infection by OZ1., Conclusions: The modelling indicates a single infusion of HSC cell-delivered gene therapy can impact on HIV viral load and CD4 T-lymphocyte count. Given that gene therapy avoids the complications associated with HAART, there is significant potential for this approach in the treatment of HIV., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Up-to now three variants of antisense technologies are known, namely antisense oligonucleotides, RNA interference, and ribozymes. In spite of different mechanisms of action, all of them are united by the common principle: an antisense preparation works after binding with RNA-target by forming a duplex. Today all three variants are intensively used in vivo. Present posture of affairs in use of antisense technologies for treating various diseases is considered in the review.

A number of studies have identified transforming growth factor-beta (TGF-beta) as a critical factor in renal diseases such as glomerulosclerosis and mesangioproliferative glomerulonephritis. TGF-beta stimulates proliferation of mesangial cells, production of extracellular matrix components and induces epithelial-mesenchymal transformation in renal tissue, which plays a critical role in the pathogenesis of renal injury. Thus, TGF-beta is a treatment target in renal diseases. However, progressive renal diseases cannot be cured with present medical technologies. We have developed ribozymes and a novel gene silencer pyrrole-imidazole polyamide targeted to TGF-beta that effectively ameliorate renal injury in hypertensive rats.

Background: Inhibition of inappropriate tyrosine kinase activity by neoplasms is an attractive strategy for the treatment of malignancy., Objective: We aimed to produce a concise review of the potential role of hepatocyte growth factor (HGF)/Mesenchymal-epithelial transition factor (MET) tyrosine kinase pathway inhibition in the treatment of cancer., Methods: The current literature, abstracts and internet resources related to HGF/MET structure, function and inhibition are summarized. The potential of inhibiting this pathway as a therapy for cancer and remaining hurdles prior to routine clinical use of MET inhibition are discussed., Results/conclusions: Current knowledge suggests that the inhibition of the HGF/MET pathway has significant potential for the treatment of cancer. A number of MET inhibitor molecules are nearing completion of their development for clinical use.

Gene transfer has potential as a once-only treatment that reduces viral load, preserves the immune system and avoids lifetime highly active antiretroviral therapy. This study, which is to our knowledge the first randomized, double-blind, placebo-controlled, phase 2 cell-delivered gene transfer clinical trial, was conducted in 74 HIV-1-infected adults who received a tat-vpr-specific anti-HIV ribozyme (OZ1) or placebo delivered in autologous CD34+ hematopoietic progenitor cells. There were no OZ1-related adverse events. There was no statistically significant difference in viral load between the OZ1 and placebo group at the primary end point (average at weeks 47 and 48), but time-weighted areas under the curve from weeks 40-48 and 40-100 were significantly lower in the OZ1 group. Throughout the 100 weeks, CD4+ lymphocyte counts were higher in the OZ1 group. This study indicates that cell-delivered gene transfer is safe and biologically active in individuals with HIV and can be developed as a conventional therapeutic product.

The use of antisense strategies such as ribozymes, oligodeoxynucleotides (ODNs) and small interfering RNA (siRNA) in gene therapy, in conjunction with the use of stem cells and tissue engineering, has opened up possibilities in curing degenerative diseases and injuries to non-regenerating organs and tissues. With their unique ability to down-regulate or silence gene expression, antisense oligonucleotides are uniquely suited in turning down the production of pathogenic or undesirable proteins and cytokines. Here, we review the antisense strategies and their applications in regenerative medicine with a focus on their efficacies in promoting cell viability, regulating cell functionalities as well as shaping an optimal microenvironment for therapeutic purposes.

A trans-splicing ribozyme that can specifically reprogram human telomerase reverse transcriptase (hTERT) RNA was previously suggested as a useful tool for tumor-targeted gene therapy. In this study, we applied transcriptional targeting with the RNA replacement approach to target liver cancer cells by combining a liver-selective promoter with an hTERT-mediated cancer-specific ribozyme. To validate effects of this system in vivo, we constructed an adenovirus encoding for the hTERT-targeting trans-splicing ribozyme under the control of a liver-selective phosphoenolpyruvate carboxykinase promoter. We observed that intratumoral injection of this virus produced selective and efficient regression of tumors that had been subcutaneously inoculated with hTERT-positive liver cancer cells in mice. Importantly, the trans-splicing reaction worked equally well in a nude mouse model of hepatocarcinoma-derived peritoneal carcinomatosis, inducing the highly specific expression of a transgene, and moreover, the efficient regression of the hTERT-positive liver tumors with minimal liver toxicity when systemically delivered with the adenovirus. In addition to the observed hTERT-dependent therapeutic gene induction, significant reductions in the levels of hTERT RNA (approximately 75%) were also observed. In conclusion, this study demonstrates that a cancer-specific RNA replacement approach using trans-splicing ribozyme with a tissue-selective promoter represents a promising strategy for cancer treatment.

For almost three decades, researchers have studied the possibility to use nucleic acids as antiviral therapeutics. In theory, compounds such as antisense oligonucleotides, ribozymes, DNAzymes, and aptamers can be designed to trigger the sequence-specific inhibition of particular mRNA transcripts, including viral genomes. However, difficulties with their efficiency, off-target effects, toxicity, delivery, and stability halted the development of nucleic acid-based therapeutics that can be used in the clinic. So far, only a single antisense drug, Vitravene for the treatment of CMV-induced retinitis in AIDS patients, has made it to the clinic. Since the discovery of RNA interference (RNAi), there is a renewed interest in the development of nucleic acid-based therapeutics. Antiviral RNAi approaches are highly effective in vitro and in animal models and are currently being tested in clinical trials. Here we give an overview of antiviral nucleic acid-based therapeutics. We focus on antisense and RNAi-based compounds that have been shown to be effective in animal model systems.

Hammerhead ribozymes were designed to target mRNA of several essential herpes simplex virus type 1 (HSV-1) genes. A ribozyme specific for the late gene U(L)20 was packaged in an adenovirus vector (Ad-U(L)20 Rz) and evaluated for its capacity to inhibit the viral replication of several HSV-1 strains, including that of the wild-type HSV-1 (17syn+ and KOS) and several acycloguanosine-resistant strains (PAAr5, tkLTRZ1, and ACGr4) in tissue culture. The Ad-U(L)20 Rz was also tested for its ability to block an HSV-1 infection, using the mouse footpad model. Mouse footpads were treated with either the Ad-U(L)20 Rz or an adenoviral vector expressing green fluorescent protein (Ad-GFP) and then infected immediately thereafter with 10(4) PFU of HSV-1 strain 17syn+. Ad-U(L)20 ribozyme treatment consistently led to a 90% rate of protection for mice from lethal HSV-1 infection, while the survival rate in the control groups was less than 45%. Consistent with this protective effect, treatment with the Ad-U(L)20 Rz reduced the viral DNA load in the feet, the dorsal root ganglia, and the spinal cord relative to that of the Ad-GFP-treated animals. This study suggests that ribozymes targeting essential genes of the late kinetic class may represent a new therapeutic strategy for inhibiting HSV infection.

To improve the efficacy of gene therapy for cancer, we designed four hammerhead ribozyme adenoviruses (R1 to R4) targeting the exposed regions of survivin mRNA. In addition to the in vitro characterization, which included a determination of the sequence specificity of cleavage by primer extension, assays for cell proliferation and for in vivo tumor growth were used to score for ribozyme efficiency. The resulting suppression of survivin expression induced mitotic catastrophe and cell death via the caspase-3-dependent pathway. Importantly, administration of the ribozyme adenoviruses inhibited tumor growth in a hepatocellular carcinoma xenograft mouse model. Co-expression of R1, R3 and R4 ribozymes synergistically suppressed survivin and, as this combination targets all major forms of the survivin transcripts, produced the most potent anti-cancer effects. The adenoviruses carrying the multiple hammerhead ribozymes described in this report offered a robust gene therapy strategy against cancer.

Objective: Telomerase is an attractive molecular target because it is active in most malignant cells but undetectable in most normal somatic cells. Small, interfering ribonucleic acid segments have been shown to be effective tools for inhibiting the expression of a given gene within human cells. In the present study, we examined the effects of short hairpin ribonucleic acid expression vectors on the growth of laryngeal squamous cell carcinoma in nude mice, and we assessed potential side effects in these animals., Methods: Short hairpin ribonucleic acid expression vectors targeting the messenger ribonucleic acid of the telomerase catalytic unit were constructed and transfected into Hep-2 human laryngeal squamous cells carcinoma in nude mice. Apoptosis and telomerase catalytic unit expression within tumour cells were evaluated after treating with short hairpin ribonucleic acid. Peripheral blood was collected for haematological and biochemical analysis., Results: The findings demonstrated that short hairpin ribonucleic acid plasmids could inhibit tumour cell growth by 76.5 per cent, and that many tumour cells underwent necrotic or apoptotic cell death. There were no significant side effects of short hairpin ribonucleic acid on the heart, liver, kidney, spleen or blood system in this experimental model., Conclusion: These results indicated that the short hairpin ribonucleic acid expression vector targeted at the telomerase catalytic unit of messenger ribonucleic acid significantly inhibited the growth of laryngeal carcinoma in nude mice, with no significant side effects on the experimental animals.

The dependence of tumor growth and metastasis on blood vessels makes tumor angiogenesis a rational target for cancer therapy. One of the key mediators of angiogenesis is vascular endothelial growth factor (VEGF), which is an appealing target for anticancer therapy. The development of anti-VEGF/VEGFR agents and the latest clinical data are reviewed, including bevacizumab as a monoclonal antibody to VEGF, sunitinib and sorafenib as VEGFR tyrosine kinase inhibitors, and IMC-1C11 as VEGFR monoclonal antibody.

Chronic deteriorating renal function and renal fibrosis are common features in progressive renal diseases. Renal fibrosis may determine the degree of impairment of renal function and predict long-term prognosis. Advances in cell biology have provided a new understanding of the molecular events underlying renal fibrosis. A central event in tissue repair is the release of cytokines, i.e. transforming growth factor-Beta, in response to injury. The sustained expression of these cytokines underlies the development of renal fibrosis. Understanding the molecular mechanisms of the cytokines and their signaling could lead to the application of clinically useful gene therapy.

The past decade has seen the rapid evolution of small-molecule gene-silencing strategies, driven largely by enhanced understanding of gene function in the pathogenesis of disease. Over this time, many genes have been targeted by specifically engineered agents from different classes of nucleic acid-based drugs in experimental models of disease to probe, dissect, and characterize further the complex processes that underpin molecular signaling. Arising from this, a number of molecules have been examined in the setting of clinical trials, and several have recently made the successful transition from the bench to the clinic, heralding an exciting era of gene-specific treatments. This is particularly important because clear inadequacies in present therapies account for significant morbidity, mortality, and cost. The broad umbrella of gene-silencing therapeutics encompasses a range of agents that include DNA enzymes, short interfering RNA, antisense oligonucleotides, decoys, ribozymes, and aptamers. This review tracks current movements in these technologies, focusing mainly on DNA enzymes and short interfering RNA, because these are poised to play an integral role in antigene therapies in the future.

Oligonucleotides can in a variety of ways inhibit gene expression by interfering with translation. Oligonucleotides that are complementary to a target mRNA, antisense oligonucleotides, can prevent translation either by cleaving the target or by physically blocking the process. Additionally, oligonucleotides can correct the undesired splicing of pre-mRNA. RNA interference using double-stranded oligoribonucleotides also results in cleavage of the target mRNA. Catalytically competent ribozymes and DNAzymes can have the same effect. Even with no RNA as target, oligonucleotides can be selected as aptamers to bind to any protein to inhibit its activity. Moreover, oligonucleotides can act as decoys particularly for transcription factors to prevent binding to the promoter. A different mode of action is the activation of Toll-like receptors to induce an immune response. Several pathways for drug development are still in their infancy, for example microRNAs and antagomirs.

The development of gene-inactivation systems is an active and important field for both functional genomics and gene therapy. Towards this end, ribozymes (i.e. RNA enzymes), that specifically recognize and subsequently catalyze the cleavage of other target RNA molecules, are attractive molecular tools. Ribozymes represent an interesting alternative to the RNA interference (RNAi) approach for gene inactivation, especially given the fact that RNAi seems to trigger an immunological response and has demonstrated off-target effects. However, the design and optimization of a ribozyme-based gene-inactivation system is not a straightforward procedure. Several aspects need to be considered in the experimental design in order to provide a suitable suppression system. In this review we present the advances in this domain made available from work using the hepatitis delta virus (HDV) ribozyme as a cis-acting RNA motif in molecular biology, as well as a trans-acting molecular scissor for the development of a gene-inactivation system. This HDV ribozyme technology possesses several unique features that are all related to the fact that it is the only catalytic cleaving RNA motif that has been discovered in humans.

Influenza viruses are etiological agents of deadly flu that continue to pose global health threats, and have caused global pandemics that killed millions of people worldwide. The global crisis involving the avian H5N1 influenza provides compelling reasons to accelerate fast track development of novel antiviral drugs against the potential pandemic virus. The availability of neuraminidase inhibitors such as oseltamivir (tamiflu) improves our ability to defend against influenza viruses, but the incidences of tamiflu-resistance are rising rapidly. Nucleic acid-based antiviral drugs are promising classes of experimental antiviral drugs that have been shown in pre-clinical studies to be effective against seasonal and avian influenza viruses. The potency and versatility of these drugs make them potential candidates to be used in seasonal and pandemic influenza scenarios. The review will assess the recent patents, research and development of antisense oligonucleotides, small interfering RNA, immunomodulating RNA for the prevention and treatment of influenza infection.

Ribozymes are potential therapeutic agents which suppress specific genes in disease-affected cells. Ribozymes have high substrate cleavage efficiency, yet their medical application has been hindered by RNA degradation, aberrant cell trafficking, or misfolding when fused to a carrier. In this study, we constructed a chimeric ribozyme escorted by the motor pRNA of bacteriophage phi29 to achieve proper folding and enhanced stability. A pRNA molecule contains an interlocking loop domain and a 5'/3' helical domain, which fold independently of one another. When a ribozyme is connected to the helical domain, the chimeric pRNA/ribozyme reorganizes into a circularly permuted form, and the 5'/3' ends are relocated and buried in the original 71'/75' positions. Effective silencing of the anti-apoptotic gene survivin by an appropriately designed chimeric ribozyme, as demonstrated at mRNA and protein levels, led to programmed cell death in various human cancer cell lines, including breast, prostate, cervical, nasopharyngeal, and lung, without causing significant non-specific cytotoxicity. Through the interlocking interaction of right and left loops, monomer pRNA/ribozyme chimeras can be incorporated into multi-functional dimer, trimer and hexamer complexes for specific gene delivery. Using the phi29 motor pRNA as an escort may revive the ribozyme's strength in medical application.

Angiogenesis is vital for numerous physiological and pathological processes, including proliferation, invasion and metastasis in malignancies. Various strategies to suppress angiogenesis are under evaluation in gynecological malignancies, and ovarian cancer--the deadliest of them--has been the focus. Interruption of interaction between vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) with a monoclonal antibody, bevacizumab, has so far been the most promising antiangiogenic strategy in ovarian cancer clinically but is overshadowed by higher than expected frequency of severe toxicities. Interception of VEGF with receptor decoys, such as VEGF-Trap, and inhibiting receptor tyrosine kinases for VEGF and related growth factors with small molecule inhibitors have shown encouraging results in early phase trials of ovarian cancer; validation is ongoing in larger studies. Another approach is targeting pre-mRNA for VEGF receptors with ribozyme (angiozyme). The knowledge gained from developing these different classes of antiangiogenic agents will lay the path to future trials of other types of gynecological cancers.

New blood vessel formation (angiogenesis) is fundamental to the process of tumor growth, invasion, and metastatic dissemination. The vascular endothelial growth factor (VEGF) family of ligands and receptors are well established as key regulators of these processes. VEGF is a glycoprotein with mitogenic activity on vascular endothelial cells. Specifically, VEGF-receptor pathway activation results in signaling cascades that promote endothelial cell growth, migration, differentiation, and survival from pre-existing vasculature. Thus, the role of VEGF has been extensively studied in the pathogenesis and angiogenesis of human cancers. Recent identification of seven VEGF ligand variants (VEGF [A-F], PIGF) and three VEGF tyrosine kinase receptors (VEGFR- [1-3]) has led to the development of several novel inhibitory compounds. Clinical trials have shown inhibitors to this pathway (anti-VEGF therapies) are effective in reducing tumor size, metastasis and blood vessel formation. Clinically, this may result in increased progression free survival, overall patient survival rate and will expand the potential for combinatorial therapies. Having been first described in the 1980s, VEGF patenting activity since then has focused on anti-cancer therapeutics designed to inhibit tumoral vascular formation. This review will focus on patents which target VEGF-[A-F] and/or VEGFR-[1-3] for use in anti-cancer treatment.

Hepatitis C virus (HCV), the major etiological agent of transfusion-associated non-A, non-B hepatitis, is a severe health problem affecting up to 3% of the world population. Since its identification in 1989, enormous efforts have been made to characterize the viral cycle. However, many details regarding the virus' penetration of hepatocytes, its replication and translation, and the assembling of virions remain unknown, mostly because of a lack of an efficient culture system. This has also hampered the development of fully effective antiviral drugs. Current treatments based on the combination of interferon and ribavirin trigger a sustained virological response in only 40% of infected individuals, thus the development of alternative therapeutic strategies is a major research goal. Nucleic acid based therapeutic agents may be of some potential in hepatitis C treatment. In recent years, much effort has gone into the improvement of DNA and RNA molecules as specific gene silencing tools. This review summarizes the state of the art in the development of new HCV therapies, paying special attention to those involving antisense oligonucleotides, aptamers, ribozymes, decoys and siRNA inhibitors. The identification of potential viral targets is also discussed.