Cationic liposome-DNA Complexes Research Articles

Effects of herpes simplex virus type 2 glycoprotein vaccines and CLDC adjuvant on genital herpes infection in the guinea pig

Genital herpes simplex virus (HSV) infections are common but results from vaccine trials with HSV-2 glycoprotein D (gD) have been disappointing. We therefore compared a similar HSV gD2 vaccine, to a further truncated gD2 vaccine, to a vaccine with gD2 plus gB2 and gH2/gL2 and to a vaccine with only gB2 and gH2/gL2 in a guinea pig model of genital herpes. All vaccines were administered with cationic liposome-DNA complexes (CLDC) as an adjuvant. All vaccines significantly decreased the severity of acute genital disease and vaginal virus replication compared to the placebo group. The majority of animals in all groups developed at least one episode of recurrent disease but the frequency of recurrent disease was significantly reduced by each vaccine compared to placebo. No vaccine was significantly more protective than gD2 alone for any of the parameters described above. No vaccine decreased recurrent virus shedding. When protection against acute infection of dorsal root ganglia and the spinal cord was evaluated all vaccines decreased the per cent of animal with detectable virus and the quantity of virus but again no vaccine was significantly more protective than another. Improvements in HSV-2 vaccines may require inclusion of more T cell targets, more potent adjuvants or live virus vaccines.

Non-covalent association of folate to lipoplexes: A promising strategy to improve gene delivery in the presence of serum

The success of gene therapy depends on the efficient delivery of therapeutic genes into target cells in vitro and in vivo. Non-viral vectors, such as cationic liposome–DNA complexes (lipoplexes), have been used for numerous gene delivery applications, although their efficacy is still limited, particularly when compared to that of viral vectors. In this work, we assessed the efficacy of a new gene delivery system generated by non-covalent association of folate to lipoplexes (FA-associated lipoplexes) in two different cancer cell lines (SCC-VII and TSA cells). Association of FA with liposomes composed of DOTAP and cholesterol, and subsequent complexation with DNA greatly increased transfection efficiency above that obtained with plain lipoplexes in both cell lines. The addition of 40 μg of FA to lipoplexes was optimal for transfection and allowed to overcome the inhibitory effect induced by the presence of serum. Notably, the biological activity of the FA-associated complexes was even significantly improved under these conditions. Transfection activity mediated by FA-associated lipoplexes was compared with that by FA-conjugated lipoplexes, and the results showed that electrostatic association of FA to the lipoplexes led to considerably higher levels of biological activity than that involving covalent coupling of FA. Moreover, FA-associated lipoplexes confer greater DNA protection than FA-conjugated lipoplexes. To our knowledge, this is the first study reporting the characterization and application of FA-associated lipoplexes in gene delivery and showing their greater efficacy than that of FA-conjugated lipoplexes. Overall, the results obtained in the present work constitute a strong indication that the developed FA-associated lipoplexes are promising candidates for in vivo gene delivery.

Treatment with cationic liposome–DNA complexes (CLDCs) protects mice from lethal Western equine encephalitis virus (WEEV) challenge

Having recently characterized a CD-1 outbred mouse model of pathogenesis for Western equine encephalitis virus, we examined the possible protective effects of cationic liposome–DNA complexes (CLDCs) against encephalitic arboviral infection. In this investigation, mice were pre-treated, co-treated, or post-treated with CLDC then challenged with a subcutaneous or aerosol dose of the highly virulent WEEV-McMillan strain, lethal in mice 4–5 days after inoculation. Pre-treatment with CLDCs provided a significant protective effect in mice, which was reflected in significantly increased survival rates. Further, in some instances a therapeutic effect of CLDC administration up to 12h after WEEV challenge was observed. Mice treated with CLDC had significantly increased serum IFN-γ, TNF-α, and IL-12, suggesting a strong Th1-biased antiviral activation of the innate immune system. In virus-infected animals, large increases in production of IFN-γ, TNF-α, MCP-1, IL-12, and IL-10 in the brain were observed by 72h after infection, consistent with neuroinvasion and viral replication in the CNS. These results indicate that strong non-specific activation of innate immunity with an immune therapeutic such as CLDC is capable of eliciting significant protective immunity against a rapidly lethal strain of WEEV and suggest a possible prophylactic option for exposed individuals.

The adjuvant CLDC increases protection of a herpes simplex type 2 glycoprotein D vaccine in guinea pigs

Herpes simplex virus (HSV) infections are common but there is no vaccine available. We evaluated cationic liposome–DNA complexes (CLDC) as an adjuvant for an HSV gD2 vaccine and compared it to an MPL/Alum adjuvant in a guinea pig model of genital herpes. The addition of CLDC to the gD2 vaccine significantly decreased acute and recurrent disease and most importantly the number of days with recurrent virus shedding compared to gD2 alone. Reductions in these outcomes were also detected when gD2+CLDC was compared to gD2+MPL/Alum. When the vaccine and adjuvants were evaluated as therapeutic vaccines, they were ineffective. CLDC enhanced protection compared to MPL/Alum and is the first vaccine to reduce recurrent virus shedding, a key to decreasing the spread of HSV-2.

Rapid immunity to vaccination with woodchuck hepatitis virus surface antigen using cationic liposome–DNA complexes as adjuvant

Complexes of cationic liposomes and non-coding DNA (CLDC) have shown promise as vaccine adjuvant. Using the woodchuck animal model of hepatitis B virus (HBV) infection, the immunogenic effects of CLDC were evaluated following vaccination with three doses of woodchuck hepatitis virus surface antigen (WHsAg) adjuvanted with either CLDC or conventional alum and administered intramuscularly (im) or subcutaneously (sc). IM vaccination with WHsAg and CLDC elicited antibodies earlier, in more woodchucks, and with higher titers than WHsAg and alum. After two vaccine doses, antibody titers were higher following im than sc administration. Woodchucks administered two vaccine doses sc received the third vaccine dose im, and antibody responses reached titers comparable to those elicited by im administration. Following the second vaccine dose, im vaccination with WHsAg and CLDC induced T cell responses to WHsAg and selected WHs peptides and expression of the leukocyte surface marker CD8 and of the Th1 cytokines interferon-gamma and tumor necrosis factor alpha in woodchucks. T cell responses and CD8/cytokine expression were diminished in woodchucks from the other groups suggesting that this vaccine regimen induced a skew toward Th1 immune responses. The present study in woodchucks demonstrates that CLDC-adjuvanted WHsAg vaccine administered im resulted in a more rapid induction of humoral and cellular immune responses compared to conventional, alum-adjuvanted WHsAg vaccine. While less rapid, the immune responses following sc administration can prime the im immune responses. This adjuvant activity of CLDC over alum may be beneficial for therapeutic vaccination in chronic HBV infection.

Vaccine-Induced CD8+ T Cell-Dependent Suppression of Airway Hyperresponsiveness and Inflammation

Suppressing the abnormalities associated with asthma has been difficult to accomplish using immunotherapy or vaccination once the disease is established. The effector cells necessary for effective immunization/vaccination and immunotherapy of asthma are also not well understood. Therefore, we vaccinated allergen (OVA)-sensitized mice to determine whether therapeutic immunization could suppress airway hyperresponsiveness (AHR) and inflammation and to identify key immune effector cells and cytokines. Mice were immunized with a vaccine comprised of Ag and cationic liposome-DNA complexes (CLDC), a vaccine which has previously been shown to elicit strong CD4(+) and CD8(+) T cell responses and activation of Th1 immunity. We showed that immunization with the OVA-CLDC vaccine significantly suppressed AHR, eosinophilia, goblet cell metaplasia, and Th2 cytokine production. In contrast, immunization with CLDC alone suppressed eosinophilia and Th2 cytokine production, but failed to suppress AHR and goblet cell changes. Using adoptive transfer experiments, we found that suppression of AHR was mediated by Ag-specific CD8(+) T cells and was dependent on IFN-gamma production by the transferred T cells. Thus, we conclude that generation of strong, allergen-specific CD8(+) T cell responses by immunization may be capable of suppressing AHR and allergic airway inflammation, even in previously sensitized and challenged mice.

Mucosal immunotherapy for protection from pneumonic infection with Francisella tularensis

Previous studies have demonstrated that systemically administered immunotherapy can protect mice from systemic challenge with the bacterial pathogen Francisella tularensis. However, for protection from inhalational challenge with this bacterium, we wondered if mucosally administered immunotherapy might be more effective. Therefore, we administered cationic liposome–DNA complexes (CLDC), which are potent activators of innate immunity, intranasally (i.n.) and assessed the effectiveness of protection from lethal inhalational challenge with F. tularensis. We found that pretreatment by i.n. administration of CLDC 24 h prior to bacterial challenge elicited nearly complete protection of BALB/c mice from lethal challenge with F. tularensis LVS strain. We also observed that mucosal CLDC immunotherapy provided a statistically significant increase in survival time in mice challenged with the highly virulent F. tularensis Schu4 strain. Protection was associated with a significant reduction in bacterial burden in the lungs, liver, and spleen. Mucosal administration of CLDC elicited significantly increased expression of IL-12, IFN-γ, TNF-α, IFN-β and IFN-α genes in the lung as detected by real-time quantitative PCR. In vitro treatment of F. tularensis infected macrophages with CLDC-elicited cytokines also significantly suppressed intracellular replication of F. tularensis in infected macrophages. In vivo, depletion of NK cells prior to administration of CLDC completely abolished the protective effects of CLDC immunotherapy. CLDC-elicited protection was also dependent on induction of IFN-γ production in vivo. We conclude therefore that activation of local pulmonary innate immune responses is capable of eliciting significant protection from inhalational exposure to a virulent bacterial pathogen.

Lipid–nucleic acids interactions as base for organization and expression of cellular genome

AbstractAlthough lipid–nucleic acid interactions have been studied, with certain or little progress, for more than 30 years, it is only in recent years that the problem has received particular attention. It should, however, be noted that most studies deal with DNA‐cationic surfactants interactions, whereas DNA‐zwitterionic interactions, which are more complex and close to nature, are poorly investigated. The long‐standing studies of the triple complexes: DNA–phosphatidylcholine liposomes–divalent metal cations allow us to confirm that these complexes are responsible for the formation of not only the structures existing in DNA–cationic liposome complexes but also some other cellular structures. The author proposed hypothesis about the involvement of direct DNA–lipid interactions in the nuclear pore assembly. Only taking into account interactions between DNA and lipids of cellular membrane, one can explain the origin of such structures as nucleoid, nuclear pore, and nuclear matrix. The formation of triple complexes was accompanied by the aggregation and partial fusion of liposomes as was shown by cryo‐TEM technique. The author has presented new data on the structure of triple complexes, which were obtained by phase contrast cryo‐TEM. Biophysical data on the liposomes fusion during triple complex formation and perspective of their computer simulation are also presented. DNA acts as a fusogen in this process and it unwinds in the region of liposomes fusion. The nuclear envelope and pore complexes assembly is provided by membrane vesicles fusion. Author has proposed that the DNA‐induced fusion of zwitterionic liposomes in vitro may suggest the involvement of direct lipids–DNA interaction in nuclear envelope assembly. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010

Cationic lipid/DNA complexes (JVRS-100) combined with influenza vaccine (Fluzone®) increases antibody response, cellular immunity, and antigenically drifted protection

Safe and effective adjuvants for influenza vaccines that could increase both the levels of neutralizing antibody, including against drifted viral subtypes, and T-cell immunity would be a major advance in vaccine design. The JVRS-100 adjuvant, consisting of DOTIM/cholesterol cationic liposome–DNA complexes, is particularly promising for vaccines that require induction of high levels of antibody and T-cell immunity, including CD8+ cytotoxic T lymphocytes (CTL). Inclusion of protein antigens with JVRS-100 results in the induction of enhanced humoral and cell-mediated (i.e., CD4+ and CD8+ T cells) immune responses. The JVRS-100 adjuvant combined with a split trivalent influenza vaccine (Fluzone®—sanofi pasteur) elicited increased antibody and T-cell responses in mice and non-human primates compared to vaccination with Fluzone® alone. Mice vaccinated with JVRS-100–Fluzone® and challenged with antigenically drifted strains of H1N1 (PR/8/34) and influenza B (B/Lee/40) viruses had higher grade protection, as measured by attenuation of weight loss and increased survival, compared to recipients of unadjuvanted vaccine. The results indicate that the JVRS-100 adjuvant substantially increases immunogenicity and protection from drifted-strain challenge using an existing influenza vaccine.

Cationic Liposome-DNA Complex Adjuvant (JVRS-100) Combined with Heat-Inactivated Whole Virion Influenza Vaccine Confers Protection against Homotypic and Heterosubtypic Lethal Challenge (132.1)

Abstract Background: Current vaccine strategies for influenza are highly dependent on matching of hemagglutinin, which is susceptible to periodic antigenic drift. A vaccine that elicits a robust antibody and T-cell response would be less susceptible to primary vaccine failure. Methods: Balb/c mice were vaccinated with heat-inactivated PR/8/34 (H1N1) or HKx31 (H3N2) with or without the JVRS-100 adjuvant. Mice were assessed for antibody and T-cell responses as well as challenged with a lethal dose of PR/8/34. Mice were monitored for clinical signs of infection, changes in body weight, and mortality. Results: Mice vaccinated with heat-inactivated virus combined with JVRS-100 adjuvant, showed increased HAI (450 vs. 30, P=0.007) and T-cell responses based on splenocyte restimulation (7700 vs. 30 pg/ml, P<0.001) when compared with unadjuvanted virus. Mice vaccinated with homologous virus with adjuvant exhibited no weight loss at six days post infection (+5.4% vs. -15.3%) compared with unadjuvanted virus following 6xLD50 challenge with PR/8/34. Mice vaccinated with JVRS-100-adjuvanted heterologous virus also showed less weight loss (-7% vs. -26.7%, P=0.004) compared with unadjuvanted virus following 6xLD50 challenge with PR/8/34. Conclusion: Heat-inactivated whole influenza virus adjuvanted with JVRS-100 showed increased immunogenicity and greater protection from homotypic and heterosubtypic lethal challenge. Adoptive transfer studies suggest that the cross-protection is due at least in part to a cross-reactive antibody response.

A novel vaccine adjuvant for recombinant flu antigens

Many new vaccines under development consist of rationally designed recombinant proteins that are relatively poor immunogens unless combined with potent adjuvants. There is only one adjuvant in common use in the U.S., aluminum phosphate or hydroxide (e.g. alum). This adjuvant, however, has significant limitations, particularly regarding the generation of strong cell-mediated (T-cell) immune responses. A novel adjuvant, JVRS-100, composed of cationic liposome–DNA complexes (CLDC) has been evaluated for immune enhancing activity. The JVRS-100 adjuvant has been shown to elicit robust immune responses compared to CpG oligonucleotides, alum, and MPL adjuvants, and efficiently enhances both humoral and cellular immune responses. Safety has been evaluated in preclinical studies, and the adjuvant is now in early-stage clinical development. One application of this novel adjuvant is to augment the immune responses to recombinant subunit antigens, which are often poorly immunogenic. The JVRS-100 adjuvant, when combined with a recombinant influenza hemagglutinin (H1), elicited increased specific antibody and T-cell responses in mice. Single-dose vaccination and prime/boost vaccinations with JVRS-100-H1 were both shown to be protective (i.e., survival, reduced weight loss) following H1N1 (PR/8/34) virus challenge. Enhanced immunological responses could be critically important for improved efficacy and dose-sparing of a recombinant influenza vaccine.

Potent Adjuvant Activity of Cationic Liposome-DNA Complexes for Genital Herpes Vaccines

Development of a herpes simplex virus (HSV) vaccine is a priority because these infections are common. It appears that potent adjuvants will be required to augment the immune response to subunit HSV vaccines. Therefore, we evaluated cationic liposome-DNA complexes (CLDC) as an adjuvant in a mouse model of genital herpes. Using a whole-virus vaccine (HVAC), we showed that the addition of CLDC improved antibody responses compared to vaccine alone. Most important, CLDC increased survival, reduced symptoms, and decreased vaginal virus replication compared to vaccine alone or vaccine administered with monophosphoryl lipid A (MPL) plus trehalose dicorynomycolate (TDM) following intravaginal challenge of mice. When CLDC was added to an HSV gD2 vaccine, it increased the amount of gamma interferon that was produced from splenocytes stimulated with gD2 compared to the amount produced with gD2 alone or with MPL-alum. The addition of CLDC to the gD2 vaccine also improved the outcome following vaginal HSV type 2 challenge compared to vaccine alone and was equivalent to vaccination with an MPL-alum adjuvant. CLDC appears to be a potent adjuvant for HSV vaccines and should be evaluated further.

Enhanced in vivo immunogenicity of SIV vaccine candidates with cationic liposome-DNA complexes in a rhesus macaque pilot study

This pilot study tested the immunogenicity of a novel cationic liposome-DNA complex (CLDC) immunomodulatory vaccine adjuvant. Combined with a specific antigen, CLDC enhanced anti-SIV immune responses induced by various SIV vaccine candidates. Rhesus macaques immunized in the presence of CLDC developed stronger SIV-specific T and B cell responses compared to animals immunized without CLDC. These differences persisted and resulted in better memory responses after an in-vivo boost of the animals several months later with whole AT-2 inactivated SIVmac239. Thus, CLDC should be explored further as a potential immunomodulatory adjuvant in HIV vaccine design.

Cationic liposome–DNA complexes as gene delivery vectors: Development and behaviour towards bone-like cells

Modulation of the biological pathways responsible for fracture repair and osteogenisis may accelerate regeneration. Gene therapy is an alternative method for the release of osteogenisis-stimulating proteins into tissues. The development of vectors for gene release is still a problem in terms of ethics and techniques. In this work we evaluated whether cationic liposomes constitute a valuable strategy for the release of genetic material into bone tissue cells as non-viral vectors. Liposomes were prepared with 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)–2-dioleoyl-sn-glycero-3-phosphatidylethanolamine and DOTAP–cholesterol, and characterized according to their size, zeta potential, DNA protection capacity and cytotoxicity. Transfection studies were also carried out using pCMVβ-gal plasmid in two osteoblastic cell lines (MG63 and MC3T3-E1) and in the 294T line, varying the charge ratio and the applied DNA dose. Inclusion of transferrin to increase the expression was also tested. The results suggest that there is great dependency between the transfection activity and the lipid formulation, the charge ratios of the complexes, the applied DNA dose and the cell type. There were even some differences concerning both osteoblastic lines under study. The cells of the MC3T3-E1 line present greater expression levels than the cells of the MG-63 line. The conjugation of the transferrin with the complexes contributes to the increase in transfection levels, possibly due to an increase in internalization of complexes. It is thus a good strategy for inducing the expression of specific genes in osteoblast-like cells.

Protection from Pneumonic Infection withBurkholderiaSpecies by Inhalational Immunotherapy

Burkholderia mallei and B. pseudomallei are important human pathogens and cause the diseases glanders and melioidosis, respectively. Both organisms are highly infectious when inhaled and are inherently resistant to many antimicrobials, thus making it difficult to treat pneumonic Burkholderia infections. We investigated whether it was possible to achieve rapid protection against inhaled Burkholderia infection by using inhaled immunotherapy. For this purpose, cationic liposome DNA complexes (CLDC), which are potent activators of innate immunity, were used to elicit the activation of pulmonary innate immune responses. We found that mucosal CLDC administration before or shortly after bacterial challenge could generate complete or nearly complete protection from inhalational challenge with 100% lethal doses of B. mallei and B. pseudomallei. Protection was found to be dependent on the CLDC-mediated induction of gamma interferon responses in lung tissues and was partially dependent on the activation of NK cells. However, CLDC-mediated protection was not dependent on the induction of inducible nitric oxide synthase, as assessed by depletion studies. We concluded that the potent local activation of innate immune responses in the lung could be used to elicit rapid and nonspecific protection from aerosol exposure to both B. mallei and B. pseudomallei.

DNA-Induced Aggregation and Fusion of Phosphatidylcholine Liposomes in the Presence of Multivalent Cations Observed by the Cryo-TEM Technique

By means of cryoelectron transmission microscopy (cryo-TEM), we were able to demonstrate the formation of ternary complexes (TC): DNA-phosphatidylcholine liposome-divalent metal cations. Addition of Ba2+ to TC led to visualization of DNA compacting on the liposome surface. Staining the TC by Tb3+ cations revealed the changed secondary structure of DNA located between fused liposomes. Cryo-TEM and liposome turbidity data were analyzed during TC formation. Liposome aggregation and the liposome fusion induced by DNA in TC were observed. Because TC displayed the property of DNA cationic liposome complexes as well as their own unique properties, we were able to consider cationic lipoplexes as a particular case of TC. The involvement of TC and direct DNA-lipid interactions in the formation nuclear pore complexes were assumed.

Prophylaxis with cationic liposome–DNA complexes protects hamsters from phleboviral disease: Importance of liposomal delivery and CpG motifs

Cationic liposome-DNA complexes (CLDC) are cationic/neutral lipid carriers complexed with plasmid DNA that when administered systemically results in a robust T(H)1 cytokine response. CLDC have been shown to be effective in prophylaxis and therapeutic treatment of animal models of viral disease. To determine the contribution of liposomal delivery and CpG content of the plasmid DNA to the efficacy of CLDC; plasmid, CpG-free plasmid DNA, or CpG-containing oligodeoxynucleotides (ODN) with and without liposomes, as well as poly(I:C(12)U), were evaluated for their ability to elicit protection against lethal Punta Toro virus (PTV, Bunyaviridae, phlebovirus) challenge in hamsters. CLDC-containing plasmid significantly improved survival, decreased systemic and liver viral loads, and reduced liver damage due to progression of viral infection. Mouse-reactive ODNs complexed with liposomes failed to protect hamsters, whereas ODNs known to cross-react with human and mouse (CpG 2006) or non-liposomal poly(I:C(12)U) showed survival benefit but did not limit liver injury. Liposomes complexed with a non-CpG motif-containing plasmid reduced liver viral load and tissue damage, but did not protect hamsters from death. To evaluate the mechanisms of the enhanced activity of CLDC, microarray experiments examined differences in the gene expression profile. The results suggest a broad T(H)1 response elicited by liposomal delivery of a diverse sequence containing CpG and non-CpG elements may be a more effective antiviral treatment than other nucleic acid based immunotherapeutics.

Radiation improves gene delivery by a novel transferrin-lipoplex nanoparticle selectively in cancer cells

Selective gene transfer to tumor is critical in cancer gene therapy. We previously used ionizing radiation to improve adenovirus uptake in intrahepatic tumors but liver cytotoxicity associated with the viral administration still occurred. Here, we explore the potential of radiation for improving gene delivery by a virus-mimicking nanoparticle, transferrin (Tf)-cationic liposome-DNA complex (Tf-lipoplex). Transduction by Tf-lipoplex was highly efficient in various cell lines and further increased by radiation in a dose- and time-dependent manner. This radiation induction, which was associated with an increase in Tf-lipoplex uptake (3- to 4-folds in hepatocytes WB and lung cancer cells, LLC1), was absent when a Tf-deficient complex was used or abolished by the presence of free Tf, suggesting that Tf receptor (TfR) interaction is required for radiation induction. Radiation (10-20 Gy) markedly induced transgene (LacZ) expression in LLC1 xenografts (3.5- to 7.4-folds), correlating with increased plasmid content and TfR expression in irradiated tumors. Moreover, Tf-lipoplex-mediated gene expression was not observed in the liver or other normal tissues regardless of radiation treatment. We conclude that radiation improves Tf-lipoplex gene delivery selectively to tumor cells both in vitro and in vivo. Our findings may provide insight in developing ligand-specific lipoplex for molecularly targeted cancer gene therapy.

Immunotherapy of Acute Leukemia with Cationic Lipid DNA Complexes (JVRS‐100)

Acute leukemias have long been recognized as suitable targets for immunotherapy. Administration of cationic liposome‐DNA complexes (JVRS‐100) elicits cytokines of the TH1 subset, especially IL‐12, IFN‐gamma and IFN‐alpha. These cytokines are particularly important in mediating host defense against cancer. In multiple experiments using the 32Dp210‐GFP model, intravenous dosing of JVRS‐100 prevented or significantly delayed death from leukemia when delivered between 7 and 15 days following leukemic challenge. Specifically, control animals died at ∼19 days of leukemia whereas 80% of similarly leukemia challenged but JVRS‐100 treated animals (1 ug) were alive >40 days with weekly treatment beginning at day 15 (4 days prior to expected mortality). Animals dying of leukemia had splenomegaly and leukemic infiltration of spleen and liver. The GFP expression allowed quantification of leukemia in peripheral blood (PB). At day 12 following challenge PB leukocytes were ∼30% GFP positive with either no treatment, or 0.1 ug JVRS‐100, <0.1% with 1.0 ug or 5.0 ug JVRS‐100 treatment at days 2 and 9. Furthermore there is a similar cell activation profile and cytokine response in vitro following exposure of human PBMCs to JVRS‐100 as is observed following in vivo administration of JVRS‐100 to mice, making this immunostimulation technology a promising candidate for product development and eventual clinical evaluation.

Transferrin-Associated Lipoplexes as Gene Delivery Systems: Relevance of Mode of Preparation and Biophysical Properties

The successful application of gene therapy depends highly on understanding the properties of gene carriers and their correlation with the ability to mediate transfection. An important parameter that has been described to improve transfection mediated by cationic liposomes involves association of ligands to cationic liposome-DNA complexes (lipoplexes). In this study, ternary complexes composed of 1,2-dioleoyl-3-(trimethylammonium) propane:cholesterol, plasmid DNA and transferrin (Tf, selected as a paradigm of a ligand) were prepared under various conditions, namely, in medium with different ionic strengths (HEPES-buffered saline [HBS] or dextrose), at different lipid/DNA (+/-) charge ratios and using different modes for component addition. We investigated the effect of these formulation parameters on transfection (in the absence and presence of serum), size of the complexes, degree of DNA protection and extent of their association with cells (in terms of both lipid and DNA). Our results show that all the tested parameters influenced to some extent the size of the complexes and their capacity to protect the carried genetic material, as well as the levels of cell association and transfection. The best transfection profile was observed for ternary complexes (Tf-complexes) prepared in high ionic strength solution (HBS), at charge ratios close to neutrality and according to the following order of component addition: cationic liposomes-Tf-DNA. Interestingly, in contrast to what was found for dextrose-Tf-complexes, transfection mediated by HBS-Tf-complexes in the presence of serum was highly enhanced.