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Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo . However, the relationship between the features of the lipid-DNA complexes (`lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin Nterminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/ DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo , such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo. [ABSTRACT FROM AUTHOR]

It has already been shown that influenza virus binds unspecifically to liposomes containing ganglioside GM1 whereas with gangliosides GD1b and GT1b binding occurs in a specific and saturable manner [Slepushkin et al. (1986) Biol. Membr. 3, 229-235]. In the present study the mode of interaction between influenza virus and various gangliosides or phospholipid liposomes containing cholesterol and gangliosides has been investigated. The influence of exogenous gangliosides on the structure of the viral envelope was studied using fluorescent and photoactivatable phospholipids incorporated into the viral membrane. With both types of probes maximal effects of gangliosides were caused by GT1b. Addition of that ganglioside resulted in a marked decrease in the fluorescence polarization (P) of fluorescent labeled virus as well as in substantial changes in the binding of photoactivatable analogues of sphingomyelin and phosphatidylcholine to virus proteins, mainly hemagglutinin. The effects of GT1b and GD1b on P value were comparable, whereas gangliosides with other oligosaccharide chains mused much smaller changes in P. Furthermore GT1b but not GMI, influenced phospholipid-hemagglutinin crosslinking. Interaction of the virus with large unilamellar liposomes was monitored by two fluorescence assays based on resonance-energy transfer from the tryptophans and tyrosines of viral proteins to vesicles labeled with a triacylglycerol (anthrylvinyldioleoylglycerol) or from these labeled vesicles to virions labeled with a perylenoyl derivative of galactosylcerebroside (PGalSph). A third fluorescence assay was based on relief of self-quenching in PGalSph-labeled virions, upon low-pH-induced virus-liposome fusion. With all three fusion assays the changes of fluorescence mused by GT1b were more pronounced than those induced by GM1. On the other hand, virus-induced release of [14C]glucose from multilameltar liposomes was enhanced by GMI but not by GT1b or GD1b. It is concluded that the interaction of GT1b or GD1b with virus hemagglutinin induces a rearrangement of the viral lipids rendering lipid bilayer areas of the viral envelope significantly fluid, which in turn promotes fusion of the virus with target membranes. Probably virus-liposome fusion and virus-induced liposome leakage are brought about by different mechanisms depending on specific or unspecific binding of the virions to the target. [ABSTRACT FROM AUTHOR]

Abstract: We examined the ability of a HIV-1-based vector (VRX494) encoding a 937-bp antisense HIV-1 envelope sequence to inhibit the replication of chimeric SIV/HIV-1 viruses encoding the HIV-1 envelope. Challenge of VRX494-transduced CEMx174 cells resulted in potent inhibition of HIV-1 and several SHIV strains. To evaluate the potential efficacy of the VRX494 vector for stem cell gene therapy, rhesus CD34+ bone marrow cells were transduced with VRX494 and then cultured on thymus stroma to induce T cell differentiation. Transduction conditions for CD34+ cells were optimized to yield high transduction efficiency with minimal effective multiplicity of infection. Purified CD4+ GFP+ T cells derived from VRX494-transduced CD34+ cells strongly inhibited SHIV HXBC2P 3.2 and SHIV 89.6P replication compared to controls. Southern blot analysis of VRX494-transduced T cell clones revealed a subset of cells with multiple proviral copies per cell. Expression of GFP and the antisense inhibitor in VRX494-transduced cells was upregulated by Tat. Analysis of HIV-1 envelope sequences in VRX494-transduced cells revealed modifications consistent with those mediated by double-stranded RNA-dependent adenosine deaminase. These results indicate that the macaque/SHIV model should serve as a useful preclinical model to evaluate this lentiviral vector expressing an HIV-1 antisense inhibitor for stem cell gene therapy for AIDS. [Copyright &y& Elsevier]

We present preclinical studies that demonstrate in vitro the feasibility and efficacy of lentivirus-based vector antisense gene therapy for control of HIV replication in primary T lymphocytes isolated from HIV-infected patients discordant for clinical status. VRX496 is a VSV-G-pseudotyped HIV-based vector that encodes an antisense payload against the HIV envelope gene. The antisense payload is under the control of the native LTR promoter, which is highly transactivated by tat upon HIV infection in the cell. Transfer of autologous CD4+ T lymphocytes genetically modified with VRX496 (VRX496T) into HIV-infected patients is intended to provide a reservoir of cells capable of controlling HIV, potentially delaying AIDS onset. To determine the patient population likely to respond to VRX496 for optimal efficacy, we examined the ability of our research vector, VRX494, to modify and suppress HIV in vitro in lymphocytes isolated from 20 study subjects discordant for CD4 count and viral load. VRX494 is analogous to the clinical vector VRX496, except that it contains GFP as a marker gene instead of the 186-tag marker in the clinical vector. To transfer VRX494 to target cells we developed a novel scalable two-step transduction procedure that has been translated to the clinic in an ongoing clinical trial. This procedure achieved unprecedented transduction efficiencies of 94 ± 5% in HIV+ study subject cells. In addition the vector inhibited HIV replication ≥93% in culture regardless of the viral load or CD4 count of the subject or tropism of the virus strain with which they were infected. These findings demonstrate that VRX496T therapy is expected to be beneficial to patients that differ in their status in term of CD4 count and viral load. The methods described represent significant technical advances facilitating execution of lentivirus vector-mediated gene therapy for treatment of HIV and are currently being employed in the first trial evaluating lentivirus vector safety in humans. [Copyright &y& Elsevier]

An abstract of the article "Phase I Clinical Trial Demonstrates Safety and Feasibility of Autologous Cellular Therapy with Lentiviral Modified CD4 T Cells Expressing Anti-HIV Antisense in Patients with HAART Resistant HIV-1 Infection," by Boro Dropulic and colleagues is presented.

An abstract of the article "Unexpected Immune Enhancement by Autologous Lentiviral Modified CD4 T Cells: Results from a Phase I Gene Transfer Trial of Lentiviral Vector-Modified T Cells Expressing Antisense Envelope," by Rob R. MacGregor and colleagues is presented.

An abstract of the article "Development of SIV Based Vectors with Efficient Delivery for Vector Dynamic Studies in the Simian System," by Christopher Chen, Jiamo Lu, Tatiana Slepushkina, Andrew Worden, Vladimir Slepushkin and Laurent M. Humeau is presented.