Chemosensitization of solid tumors by inhibition of Bcl-xL expression using DNAzyme.

DNAzymes are a novel class of gene suppressors that selectively bind to an RNA substrate by Watson-Crick base pairing and cleave phosphodiester bonds. To explore the potential for therapeutic use of catalytic DNA molecules, active DNAzymes targeting the bcl-xL gene were generated through a multiplex in vitro selection. The DNAzyme-mediated down-regulation of the bcl-xL expression was demonstrated in various cancer cell lines by Western blots.

Broad-spectrum and virus-specific nucleic acid-based antivirals against influenza.

Rapid increase in drug-resistant influenza virus isolates, and pandemic threat posed by highly pathogenic avian influenza A and swine flu viruses provide clear and compelling reasons for fast tracking development of novel antiviral drugs. Nucleic acid-based drugs represent a promising class of novel antiviral agents that can be designed to target various seasonal, pandemic and avian influenza viruses. Nucleic acids can be designed to elicit broad-spectrum antiviral responses in the host, by suppressing viral gene expression, or by inducing cleavage or degradation of viral RNA.

Recent patents in antiviral siRNAs.

The advent of gene silencing siRNA technology has created opportunities to develop therapeutics based on targeting the genomics of the disease state. Amongst the first applications of siRNA technology, antiviral applications have been quickly and extensively exploited allowing emergence of a range of antiviral therapeutic strategies. Patent activity has encompassed a range of the components required to utilize this technology ranging from the identification of susceptible genomic targets through to the development of vector systems to express the siRNA endogenously or the synthesis of stable RNA oligonucleotides for in vivo therapeutics.

Recent Patents on development of nucleic acid-based antiviral drugs against seasonal and pandemic influenza virus infections.

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.

Brothers in arms: DNA enzymes, short interfering RNA, and the emerging wave of small-molecule nucleic acid-based gene-silencing strategies.

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.

Cellular uptake, distribution, and stability of 10-23 deoxyribozymes.

The cellular uptake, intracellular distribution, and stability of 33-mer deoxyribozyme oligonucleotides (DNAzymes) were examined in several cell lines. PAGE analysis revealed that there was a weak association between the DNAzyme and DOTAP or Superfect transfection reagents at charge ratios that were minimally toxic to cultured cells. Cellular uptake was analyzed by cell fractionation of radiolabeled DNAzyme, by FACS, and by fluorescent microscopic analysis of FITC-labeled and TAMRA-labeled DNAzyme.

Catalytic DNA: a novel tool for gene suppression.

RNA, as an intermediate in the production of every gene encoded protein and the genetic material of many pathogenic viruses, presents an attractive target for both biological and therapeutic manipulation. Despite its extensive involvement in living systems, its chemical diversity based on four units is relatively low compared with protein. This provides the opportunity for a generic approach to targeting with specificity based on primary structure rather than complex higher order structures.

Immunoprophylactic strategies against respiratory influenza virus infection.

The objective of this report is to evaluate the prophylactic efficacy of liposome-mediated immunotherapy for prevention of respiratory influenza virus infection in mice. Antiviral antibody, interferon-gamma and poly (ICLC) were encapsulated in liposomes and they were evaluated for their ability to induce protective immunity against lethal influenza infection. Passive immunization using liposome-encapsulated antiviral antibody was found to offer complete protection against the virus challenge.

Effects of liposome-encapsulated ciprofloxacin on phagocytosis, nitric oxide and intracellular killing of Staphylcoccus aureus by murine macrophages.

The effects of liposome-encapsulated ciprofloxacin on phagocytosis, nitric oxide production and intracellular killing of Staphylococcus aureus in murine macrophages were evaluated in this study. Mice were pretreated with three daily doses of liposome-encapsulated ciprofloxacin (45 mg/kg body weight/dose, intraperitoneal injection). At day 3 post drug administration, peritoneal macrophages were harvested by peritoneal lavage, and the phagocytic activity of the macrophages was determined by a chemiluminescence assay using opsonized zymosan particles.

Catalytic nucleic acids: from lab to applications.

Since the discovery of self-cleavage and ligation activity of the group I intron, the expansion of research interest in catalytic nucleic acids has provided a valuable nonprotein resource for manipulating biomolecules. Although a multitude of reactions can be enhanced by this class of catalyst, including trans-splicing activity of the group I intron (which could be applied to gene correction), RNA-cleaving RNA enzymes or "ribozymes" hold center stage because of their tremendous potential for mediating gene inactivation. This application has been driven predominantly by the "hammerhead" and "hairpin" ribozymes as they induce specific RNA cleavage from a very small catalytic domain, allowing delivery either as a transgene expression product or directly as a synthetic oligonucleotide.

Encapsulation of plasmid DNA in stabilized plasmid-lipid particles composed of different cationic lipid concentration for optimal transfection activity.

In previous work (Wheeler et al. (1999) Gene Therapy 6, 271-281) we have shown that plasmid DNA can be entrapped in "stabilized plasmid lipid particles" (SPLP) using low levels (5-10 mol%) of cationic lipid, the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), and a polyethyleneglycol (PEG) coating for stabilization. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG-CerC20).

Stabilized plasmid-lipid particles for regional gene therapy: formulation and transfection properties.

Previous work (Wheeler et al, Gene Therapy 1999; 6: 271-281) has shown that plasmid DNA can be entrapped in 'stabilized plasmid-lipid particles' (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG-CerC20). These SPLP exhibit low transfection potencies in vitro, due in part to the long residence time of the PEG-CerC20 on the SPLP surface.

Cationic lipid-mediated transfection of cells in culture requires mitotic activity.

Cationic lipid-based delivery systems such as lipoplexes or stabilized plasmid-lipid particles (SPLP) represent a safer alternative to viral systems for gene therapy applications. We studied the impact of cell cycle status on the efficiency of transfection of human ovarian carcinoma tumor cells using two cationic-lipid based delivery systems. Cells arrested in the G1 phase of the cell cycle by treatment with aphidicolin were compared with an asynchronous dividing population of cells.

Development of a murine hypothermia model for study of respiratory tract influenza virus infection.

A hypothermia model was developed to predict mortality and morbidity caused by respiratory influenza virus infection in mice. To increase virulence, egg-propagated influenza A/PR/8 virus was adapted for growth in mice by four blind serial passages. The mouse-adapted influenza A virus was then used to infect groups of BALB/c mice via the intranasal route, and the 50% lethal dose (LD50) was determined.

Effect of liposome-encapsulation on immunomodulating and antiviral activities of interferon-gamma 1.

The effect of liposome-encapsulation on the immunomodulating and antiviral activities of interferon-gamma (IFN-gamma) was evaluated in this study. The immunomodulating activity was measured by increases in phagocytic activity and in nitric oxide production by peritoneal macrophages from mice treated with both free and LIP-IFN-gamma (4000 U/mouse, intraperitoneal injection). Resident peritoneal macrophages harvested from mice treated with free unencapsulated IFN-gamma or muramyl dipeptide showed significant increases in macrophage yield, and enhanced ability to phagocytize zymosan particles.

Prophylactic and therapeutic efficacies of poly(IC.LC) against respiratory influenza A virus infection in mice.

Polyriboinosinic-polyribocytidylic acid [poly(IC.LC)] was evaluated for its prophylactic and therapeutic efficacies against respiratory influenza A virus infection in mice. Two doses of poly(IC.

Enhanced protection against respiratory influenza A infection in mice by liposome-encapsulated antibody.

Liposome-mediated passive immunity was evaluated for its efficacy in the prophylaxis and treatment of influenza A/PR/8 virus infection in mice. A mouse LD50 protection model was developed using a polyclonal anti-influenza A antibody which demonstrated strong reactivity against the mouse-adapted virus in a fluorogenic enzyme immunoassay and in an in vitro plaque assay. Using liposomes as an antibody carrier system, the delivery of antibody to the lungs was optimized.

Purification of glucose-inducible outer membrane protein OprB of Pseudomonas putida and reconstitution of glucose-specific pores.

A 43,000 molecular-weight, glucose-inducible, organic acid-repressible protein (OprB) was identified in the outer membrane of Pseudomonas putida. OprB was surface expressed in whole cells, had a high beta-sheet content, and was heat modifiable, as demonstrated by 125I-labeling, circular dichroism spectroscopy, and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. OprB was extracted from outer membrane preparations by using 2% Lubrol PX with 10 mM EDTA and purified by DEAE-Sephacel ion exchange chromatography following ammonium sulfate precipitation.