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. This form of recognition occurs naturally in complementary nucleic acids, due to an ability to bind their single stranded target through Watson-Crick interactions. The most established nucleic acid based approach to gene suppression at the RNA level is through antisense oligodeoxynucleotides (ODNs). These compounds form heteroduplex with target RNA which are thought to either block its function or mediate its destruction by activation of RNase H. Alternatively, RNA can be targeted by catalytic RNA such as the hammerhead ribozyme. Ribozymes have the advantage of being equipped with their own RNA cleavage apparatus and are therefore independent of host nuclear protein activity. At present, the utility of ribozyme oligonucleotides is restricted by the relative difficulty synthesising active molecules with sufficient resistance to nuclease degradation. Recently the power of in vitro selection has been used to evolve catalytic DNA sequences with RNA cleavage specificity and activity rivalling the very best ribozymes, while maintaining the more robust chemistry of an ODN. These deoxyribozymes or DNAzymes have tremendous potential as gene suppression agents for both target validation and therapeutic applications. A number of studies evaluating the biological activity of these compounds have shown promising results. However, as with other oligonucleotide based strategies, future exploitation of this approach may depend on accessory technology to assist with the accessibility of a target which is folded by its own secondary structure and hidden within the intracellular compartment.

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. However, this pretreatment must be administered within 24 h prior to virus challenge to be protective. Pretreatment with liposome-encapsulated interferon-gamma was found to stimulate cellular immune responses, but the protection is partial. Immunoprophylaxis using liposome-encapsulated double-stranded (ds) RNA poly (ICLC) provided complete and longer-lasting protection against influenza infection. These results suggest liposome-mediated immunoprophylactic approaches are effective in the prevention of respiratory influenza virus infection.

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. Although advances in the development of RNA modifications have improved the biological half-life of synthetic ribozymes, their use is restricted by the mechanistic dependence on conserved 2'OH-moieties. Recently a new class of catalytic nucleic acid made entirely of DNA has emerged through in vitro selection. DNA enzymes or deoxyribozyme with extraordinary RNA cleavage activity has already demonstrated their capacity for gene suppression both in vitro and in vivo. These new molecules, although rivaling the activity and stability of synthetic ribozymes, are limited equally by inefficient delivery to the intracellular target RNA. The challenge of in vivo delivery is being addressed with the assessment of a variety of approaches in animal models with the aim of bringing these compounds closer to the clinic.

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). However, these SPLP exhibit low transfection potencies in vitro as compared to plasmid/cationic lipid complexes formed with liposomes composed of cationic and neutral lipid at a 1:1 lipid ratio. The objective of this study was to construct SPLPs with increased cationic lipid contents that result in maximum transfection levels. A phosphate buffer detergent dialysis technique is described resulting in formation of SPLP containing 7-42.5 mol% DODAC with reproducible encapsulation efficiency of up to 80%. An octanoyl acyl group was used as anchor for the PEG moiety (PEG-CerC8) permitting a quick exchange out of the SPLP to further optimize the in vitro and in vivo transfection. We have demonstrated that this technique can be used to encapsulate either linearized DNA or supercoiled plasmids ranging from 3-20 kb. The SPLP formed could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution of approximately 75 +/- 6 nm as determined by cryo-electron microscopy. The high plasmid-to-lipid ratio observed corresponded to one plasmid per particle. The SPLP consist of a lipid bilayer surrounding the plasmid DNA as visualized by cryo-electron microscopy. SPLP containing a range of DODAC concentrations were tested for in vitro and in vivo transfection. In vitro, in COS-7 cells transfection reached a maximum after 48 h. The transfection efficiency increased when the DODAC concentration in the SPLP was decreased from 42.5 to 24 mol% DODAC. Decreasing the cationic lipid concentration improved transfection in part due to decreased toxicity. In vivo studies using an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP showed maximum transfection activity for SPLP containing 24 mol% DODAC. Gene expression observed in tumor cells was increased by approximately one magnitude as compared to cationic lipid/DNA complexes. The SPLP were stable and upon storage at 4 degrees C no significant change in the transfection activity was observed over a one-year period. Thus this phosphate buffer detergent dialysis technique can be used to generate SPLP formulations containing a wide range of cationic lipid concentrations to determine optimal SPLP composition for high transfection activity and low toxicity.

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. In this work we employed SPLP stabilized by PEG attached to ceramide containing an octanoyl acyl group (PEG-CerC8), which is able to quickly exchange out of the SPLP, to develop systems that give rise to optimized in vitro and in vivo (regional) transfection. A particular objective was to achieve cationic lipid contents that give rise to maximum transfection levels. It is shown that by performing the dialysis procedure in the presence of increasing concentrations of citrate, SPLP containing up to 30 mol% of the cationic lipid dioleoydimethylammonium chloride (DODAC) could be generated. The SPLP produced could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution (62 +/- 8 nm, as determined by freeze-fracture electron microscopy) and a high plasmid-to-lipid ratio of 65 microg/micromol (corresponding to one plasmid per particle) regardless of the DODAC content. It was found that isolated SPLP containing 20-24 mol% DODAC resulted in optimum transfection of COS-7 and HepG2 cells in vitro, with luciferase expression levels comparable to those achieved for plasmid DNA-cationic lipid complexes. In vivo studies employing an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP also demonstrated maximum luciferase expression for DODAC contents of 20-24 mol% and significantly improved gene expression in tumor tissue as compared with complexes. We conclude that SPLP stabilized by PEG-CerC8 and containing 20-24 mol% cationic lipid are attractive alternatives to plasmid DNA-cationic lipid complexes for regional gene therapy applications.

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. Treatment of the cells with aphidicolin had no effect on the rate of internalization of the lipid formulated DNA or on the level of gene expression observable in stably transfected cells. However, cells treated with aphidicolin exhibited 20-fold lower reporter gene activity than asynchronous control cells upon incubation with lipoplexes. When cells arrested in the G1 phase were allowed to proceed through the cell cycle in the presence of the lipoplex or SPLP, transgene expression was found to coincide with the transition of cells from the G2/M phase into the G1 phase of the subsequent cell cycle. In addition, higher levels of reporter gene expression were observed when the cells were incubated with lipoplexes or SPLP during, or just before, mitosis. These results suggest that it may be possible to augment cationic lipid-mediated transfection by manipulating the cell cycle status of the target 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. Rectal temperature of the infected mice was monitored daily, and survival rate was determined at day 14 after infection. The lowest average body temperature recorded in infected mice was approximately 10 degrees C below that in noninfected mice. In mice that developed hypothermia, with body temperature of 32 degrees C or lower, morbidity and mortality inevitably occurred. In this study, the LD50 and the 50% hypothermia-inducing dose (HID50) for mouse-adapted influenza A virus were compared and calculated to be at the same dose. These results suggest that the HID50 model could be used to predict mortality and morbidity associated with influenza virus infection in mice. This model could potentially be used to substantially reduce the time and extent of suffering inflicted on experimental animals due to viral infections, and therefore may serve as a more humane alternative to LD50 determinations.

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. In mice treated with liposome-encapsulated IFN-gamma (LIP-IFN-gamma), both macrophage yield and phagocytic activity further increased by 2-fold over unencapsulated IFN-Y. In addition, the activation of peritoneal macrophages with LIP-IFN-gamma showed enhanced production of NO when the cells were cultured ex vivo. Using a murine respiratory influenza infection model, intranasally administered LIP-IFN-gamma conferred protection to 70% in mice challenged intranasally with 10 LD50 doses of influenza A/PR/8 virus compared with a 20% survival rate using free IFN-gamma. Together these results suggest that liposome-encapsulation increases the immunomodulating and antiviral activities of IFN-gamma. Liposome-encapsulation of IFN-gamma may provide additional therapeutic advantages by reducing IFN-gamma toxicity while prolonging its body retention.

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.LC) (1 mg/kg of body weight per dose) administered intranasally within 12 days prior to infection with 10 50% lethal doses of mouse-adapted influenza A/PR/8 virus fully protected the mice against the infection. Determination of virus titers by hemagglutination and plaque assays showed more than a 2-log10 decrease in virus titers in lung homogenates of pretreated mice compared with those in the lungs of the nonpretreated group. Treatment of infected mice with poly(IC.LC) resulted in a modest (40%) survival rate. These results suggest that poly(IC.LC) provides a highly effective prophylaxis against respiratory influenza A virus infection in mice.

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. For mice given the antiviral antibody intranasally 24 h prior to challenge with 10 LD50 of mouse-adapted influenza A/PR/8 virus, the survival rate at 14 days post-challenge was 60%. However, when mice were given antibody encapsulated within liposomes, the survival rate increased to 100%. In the treatment of mice preinfected with 10 LD50 of the virus, mice were fully protected (100% survival rate) when treated within 8 hr post-infection with free unencapsulated antibody, or within 12 hr with liposome-encapsulated antibody. It is postulated that the improved therapeutic and prophylactic efficacies of the antiviral antibody may be attributed to enhanced delivery as well as retention of antibody molecules in the lungs when liposomes are used as antibody carrier system.

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. Reconstitution experiments with black lipid membranes showed that OprB formed small, cation-selective pores which bound glucose (KS = 110 mM) and other carbohydrates. However, the binding site of OprB appeared to be distinct from that of the maltodextrin-specific porin LamB from Escherichia coli.